Drosophila gene families: Visual signal transduction

Drosophila visual signal transduction, the process by which incoming light is converted to neural signals that can be passed to the brain, provides an ideal system for the molecular dissection the process by which extracellular signals are transduced across the plasma membrane leading to neuron activation. In this signal transduction pathway, light stimulates rhodopsin, which activates an eye-specific G protein (Galphaq). Six genes have been identified that code for different Rhodopsins expressed in different sets of photoreceptor cells in the visual system (FlyBase, 1998). The phototransduction cascade is one of the fastest known G protein receptor coupling systems. In Drosophila, the high temporal resolution is evidenced by the less than 20 milli-second latency between photon excitation and photoreceptor cell depolarization (Ranganathan, 1991). Activated Galphaq triggers NorpA (no receptor potential A), a phospholipase C-beta to catalyze the breakdown of phospholipids to generate inositol trisphosphate (IP₃) and diacylglycerol. Both Trp (transient receptor potential) and Trp-like are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. The rise in IP₃ is thought to result in the release of Ca²⁺ from the internal Ca²⁺ stores. However, the release of Ca²⁺ has been shown not to involved the Inositol 1,4,5,-tris-phosphate receptor, leaving unanswered questions as to the source and regulation of the initial Ca²⁺ current (Acharya, 1997). It has now been shown, however, that polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999). The opening of Ca²⁺ channels leads to depolarization of photoreceptors. This depolarization leads to the activation of the the Trp store-operated Ca²⁺ channel. The store-operated calcium channel in the plasma membrane is responsible for replenishing the internal calcium stores depleted during phosphoinositide-mediated process (Shieh, 1997 and references).

Among the proteins of critical importance in visual phototransduction is Inactivation-no-afterpotential D, a photoreceptor-specific protein containing five repeated protein interaction motifs known as PDZ repeats. InaD is an adaptor protein that homomultimultimerizes and has the ability to interacts with multiple components of the signal transduction pathway, including Rhodopsin, NorpA, PKC, Calmodulin, Trp and Trp-like (Shieh, 1997 and references and Xu, 1998a and references). The view that signaling through G protein-coupled cascades occurs via random stochastic collisions between membrane receptors and effector molecules has been widely held for many years. However, the alternative proposal suggesting that signaling cascades are comprised of components that are physically coupled seems to better reflect reality. Work of several research groups have shown that Drosophila vision is mediated by a massive supramolecular complex and that assembly of such a complex is facilitated by homomultimerization of the scaffold protein InaD. Thus, most of the proteins critical in phototransduction appear to couple directly to InaD. The InaD supramolecular complex may not be a particle, consisting of a single InaD monomer to which a maximum of five target proteins bind. Instead, the visual cascade appears to be mediated through a more complicated higher order signaling web or complex (signalplex) consisting of an extended network of InaD homomultimers to which more than five targets bind. Most of these targets appear to bind to more than one PDZ module and several targets appear to associate with INAD via the same PDZ domains. Thus, the nature of the InaD signalplex appears to be more complicated than a single particle held together by a scaffolding protein (Xu, 1998a and references).

An important Ca2+ sensor in Drosophila vision appears to be Calmodulin, since a reduction in levels of retinal Calmodulin causes defects in adaptation and termination of the photoresponse. These functions of Calmodulin appear to be mediated, at least in part, by four calmodulin-binding proteins: the Trp and Trp-like ion channels, NinaC and InaD. Eight additional Calmodulin-binding proteins have been found to be expressed in the Drosophila retina. These included six targets that were related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Both UNC13 and CRAG are implicated in the regulation of synaptic vesicle exocytosis. Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. The full-length sequences corresponding to three of the calmodulin-binding proteins have been described previously. These corresponded to two Calmodulin-dependent protein kinases, MLCK and CaM kinase II, as well as to one of the two previously described Calcineurin proteins. A third calmodulin-dependent protein kinase is expressed in the Drosophila retina, CaM kinase I. No CaM kinase I had previously been reported from any invertebrate raising the possibility that this protein kinase was specific to vertebrates. Nevertheless, the Drosophila CaM kinase I was highly related, ~60% identical over 349 amino acids, to vertebrate CaM kinase I. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission, and others directly in phototransduction (Xu, 1998b).

Photomechanical responses in Drosophila photoreceptors

Phototransduction in Drosophila microvillar photoreceptor cells is mediated by a G protein-activated phospholipase C (PLC). PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), leading by an unknown mechanism to activation of the prototypical transient receptor potential (TRP) and TRP-like (TRPL) channels. This study found that light exposure evokes rapid PLC-mediated contractions of the photoreceptor cells and modulates the activity of mechanosensitive channels introduced into photoreceptor cells. Furthermore, photoreceptor light responses are facilitated by membrane stretch and are inhibited by amphipaths, which alter lipid bilayer properties. These results indicate that, by cleaving PIP₂, PLC generates rapid physical changes in the lipid bilayer that lead to contractions of the microvilli, and suggest that the resultant mechanical forces contribute to gating the light-sensitive channels (Hardie, 2012).

In most invertebrate photoreceptor cells, the visual pigment (rhodopsin) and other components of the phototransduction cascade are localized within tightly packed microvilli (tubular membranous protrusions), together forming a light-guiding rod-like stack (rhabdomere). After photoisomerization, rhodopsin activates a heterotrimeric guanine nucleotide-binding protein (Gq protein), releasing its guanosine triphosphate-bound α subunit, which in turn activates phospholipase C (PLC). How PLC activity leads to gating of the light-sensitive transient receptor potential channels (TRP and TRPL) in the microvilli is unresolved. PLC hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), yielding soluble inositol 1,4,5-trisphosphate (InsP₃), diacylglycerol (DAG, which remains in the inner leaflet of the microvillar lipid bilayer), and a proton. The light-sensitive channels in Drosophila photoreceptors can be activated by a combination of PIP₂ depletion and protons, but it remains unclear how PIP₂ depletion might contribute to channel gating. It has been speculated that changes in membrane properties play a role, and members of the TRP ion channel family have been repeatedly, although controversially, implicated as mechanosensitive channels. This led the current study to ask whether cleavage of the bulky, charged inositol head group of PIP₂ from the inner leaflet might alter the physical properties of the lipid bilayer in the microvilli, resulting in mechanical forces that contribute to channel gating (Hardie, 2012).

Remarkably, Drosophila photoreceptors responded to light flashes with small (<1 μm) but rapid contractions that are directly visible in dissociated cells via bright-field microscopy. To obtain improved temporal and spatial resolution, these photomechanical responses were recorded with an atomic force microscope (AFM), positioning the AFM cantilever on the distal tips of photoreceptors in a whole excised retina glued to a coverslip. Contact force (~100 pN) was maintained constant, so that changes in sample height resulted in immediate, matching changes in the cantilever's z-position. Contractions were elicited indefinitely by repeated brief flashes of modest intensity, with kinetics similar to those of electrical responses recorded from dissociated photoreceptors. The latencies of contractions induced by the brightest stimuli (4.9 ± 0.9 ms) were significantly shorter than the latencies of voltage responses to the same stimuli (6.6 ± 0.6 ms). Only a few hundred effectively absorbed photons per photoreceptor are required to elicit detectable contractions, which saturate with flashes containing ~10⁶ photons, corresponding to ~30 effectively absorbed photons per microvillus. This intensity dependence overlapped with that of the electrical response. Like the electrical responses, the contractions were eliminated in mutants lacking PLC, which shows that they too required PLC activity (Hardie, 2012).

Because PLC activity is normally terminated by Ca²⁺ influx through the light-sensitive channels, net PIP₂ hydrolysis is enhanced when the light-sensitive current is blocked. Therefore contractions were measured in trpl mutants expressing only TRP light-sensitive channels before and after blocking TRP channel activity with La³⁺ and ruthenium red (RR). Indeed, after blocking the light-sensitive channels the contractions are enhanced, more sensitive to light, and saturate at lower intensities, corresponding to only ~1 to 5 effectively absorbed photons per microvillus. In the absence of Ca²⁺ influx, such intensities deplete virtually all microvillar PIP₂, resulting in temporary loss of sensitivity to light. After such saturating flashes, the photomechanical response is also temporarily refractory, recovering sensitivity with a time course (t_1/2 ~ 40 s) similar to that of PIP₂ resynthesis. By contrast, without channel blockers, sensitivity recovers within ~10 s (Hardie, 2012).

Blockade of all light-sensitive current in these experiments also shows that the contractions cannot result from any downstream effects of Ca²⁺ influx or osmotic changes caused by ion fluxes associated with the light response. Therefore, these results indicate that the contractions result from hydrolysis of PIP₂. Although other downstream effects of PLC cannot be excluded, the speed of the contractions supports a simple and direct mechanism. Cleavage of the bulky head groups from PIP₂ molecules, which represent 1% to 2% of lipids in the plasma membrane, leaves DAG in the membrane, which occupies a substantially smaller area than PIP₂. This should increase membrane tension, leading to shrinkage of the microvillar diameter, as reported for the action of PLC on the diameter of artificial liposomes. Integrated over the stack of ~30,000 microvilli, such a mechanism seems capable of accounting for the observed macroscopic contractions, which represent at most ~0.5% of the rhabdomere length. Within each microvillus, it is suggested that the alteration to the mechanical properties of the lipid bilayer may contribute to channel gating (Hardie, 2012).

To test whether phototransduction generates sufficient mechanical forces to gate mechanosensitive channels (MSCs), whole-cell patch-clamp recordings were made from dissociated photoreceptors lacking all light-sensitive channels (trpl;trp double mutants, or trpl mutants exposed to La³⁺ and RR). The photoreceptors were then perfused with gramicidin, a monovalent (Ca²⁺-impermeable) cation channel and one of the best-characterized MSCs, which is known to be regulated by changes in bilayer physical properties. Incorporation of gramicidin channels into the membrane generated a constitutive inward current that stabilized after a few minutes. Despite having replaced the native light-sensitive channels with MSCs, the photoreceptors still responded to light, with a rapid increase in the gramicidin-mediated current. Like the photomechanical responses recorded after blocking Ca²⁺ influx through the light-sensitive channels, these gramicidin-mediated responses inactivate slowly, are temporarily refractory to further stimulation and have a similar intensity dependence (Hardie, 2012).

To test whether the light-sensitive channels were mechanically sensitive, membrane tension was manipulated osmotically. Channels were not directly activated by perfusing cells with hyper- or hypo-osmotic solutions; however, it was reasoned that it would be impossible to mimic the exact physical effects of PIP₂ hydrolysis, which would include a specific combination of changes in membrane tension, curvature, thickness, lateral pressure profile, charge, and pH. Therefore whether osmotic manipulation could enhance or suppress light-sensitive channel activity was tested. In wild-type photoreceptors, light-induced currents are rapidly and reversibly facilitated by ~50% after perfusion with hypo-osmotic solutions (200 mOsm), which, like PIP₂ depletion, would be expected to alleviate crowding between phospholipids, increase tension, and reduce membrane thickness. Conversely, responses in hyperosmotic (400 mOsm) solutions were about half those in control solutions. Analysis of single-photon responses (quantum bumps) indicated that modulation resulted from changes in both quantum efficiency (fraction of rhodopsin photoisomerizations generating a quantum bump) and bump amplitude. Recordings from trp and trpl mutants showed that both TRP and TRPL channels were modulated, although facilitation of currents mediated by TRPL channels (in trp mutants) was more pronounced. Modulation of the light response by osmotic manipulation was at least as pronounced in Ca²⁺-free bath, indicating that facilitation by membrane stretch did not result from leakage of Ca²⁺ into the cell from the extracellular space (Hardie, 2012).

To test whether modulation might have been mediated by effects on upstream components of the cascade such as PLC, the activity of spontaneously active TRP channels was measured in recordings made with pipettes lacking adenosine triphosphate. Under these conditions, PIP₂ becomes depleted (thereby removing PLC’s substrate), sensitivity to light is lost, and the TRP channels enter a constitutively active ('rundown') state uncoupled from the phototransduction cascade. Nonetheless, the channels were still similarly modulated by osmotic manipulation, whereas single-channel conductance, estimated by noise analysis, was unaffected. These results indicate that osmotic pressure directly modulates the open probability of both TRP and TRPL channels (Hardie, 2012).

MSCs such as gramicidin are sensitive to amphiphilic compounds, which insert into the lipid bilayer. Because they are attracted to anionic phospholipids, cationic amphipaths insert preferentially into the inner leaflet, where they increase crowding, promote negative (concave) curvature, and decrease membrane stiffness. This study found that four structurally unrelated cationic amphipaths were all effective, reversible inhibitors of the light-induced current. Neither light-induced PLC activity (measured using a genetically targeted PIP₂-sensitive biosensor to monitor PIP₂ hydrolysis) nor single-channel conductance were substantially affected. The 50% inhibitory concentrations (IC₅₀ values) were much higher than those of their traditional drug targets and ranged over approximately three orders of magnitude. However, after correcting for pK_a and partitioning, the effective concentration of the compounds in the membrane was similar (~5 mM) in each case. Thus, their mode of action is likely related to their physicochemical properties rather than conventional drug-receptor interactions. Because cationic amphipaths are also lipophilic weak bases, and because it is proposed that protons are also critical for activating the light-sensitive channels, an alternative but not mutually exclusive possible mechanism of action is as lipophilic pH buffers of the membrane environment. It was also noted that polyunsaturated fatty acids (PUFAs) such as arachidonic and linolenic acid (effective activators of both TRP and TRPL) are not only anionic amphipaths (predicted to have opposite effects to cationic amphipaths) but also, as weak acids, natural protonophores; such a dual action could account for their agonist effect (Hardie, 2012).

The mechanism of activation of the light-sensitive channels in invertebrate microvillar photoreceptors has long remained an enigma. Neither InsP₃ nor DAG -- the two obvious products of PIP₂ hydrolysis -- are reliable agonists for the light-sensitive channels. Although PUFAs are effective agonists and might be generated from DAG, a DAG lipase with the appropriate specificity has not been found in the photoreceptors. By contrast, two neglected consequences of PLC activity -- the depletion of its substrate (PIP₂) together with protons released by PIP₂ hydrolysis -- were recently shown to potently activate the light-sensitive channels in a combinatorial manner (Huang, 2010). The current results support the hypothesis that the effect of PIP₂ depletion is mediated mechanically by changes to the physical properties of the lipid bilayer, thereby introducing the concept of mechanical force as an intermediate or 'second messenger' in metabotropic signal transduction (Hardie, 2012).

A dPIP5K dependent pool of Phosphatidylinositol 4,5 Bisphosphate (PIP2) is required for G-Protein coupled signal transduction in Drosophila photoreceptors

Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. This study identified dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function, and are probably carried out by Skittles. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. These results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes (Chakrabarti, 2015).

The detection and conversion of external stimuli into physiological outputs is a fundamental property of neurons and depends on intracellular signal transduction pathways. Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol are key signalling molecules and of these the most abundant PIP2 has multiple roles in neurons. Several neuronal receptors (such as the metabotropic glutamate, growth factor and sensory receptors) transduce stimuli into cellular information using the hydrolysis of PIP2 by phospholipase C enzymes. Additionally, within the context of neuronal cell biology PIP2 has several roles including cytoskeletal function and several ion channels and transporters require PIP2 for their activity. At the pre-synaptic terminal, a regulated cycle of PIP2 turnover is essential to regulate synaptic vesicle cycling. Thus PIP2 plays multiple roles at the plasma membrane of neurons; hence not surprisingly, changes in phosphoinositide metabolism have been linked to several inherited diseases of the human nervous system. Finally, one of the molecular targets of lithium, used in the treatment of bipolar disorders, is inositol monophosphatase a key regulator of PIP2 turnover in neurons (Chakrabarti, 2015).

Given the multiple functions of PIP2 at the plasma membrane, it is unclear if a common pool of PIP2 supports all these functions. Alternatively, if there are distinct pools, it is unclear how these are generated and sequestered on the nanoscale structure of the membrane. In principle, PIP2 can be generated by the activity of two classes of phosphatidylinositol phosphate kinase (PIPK) enzymes, designated PIP5K and PIP4K; PIP5K phosphorylates PI4-P at position 5 of the inositol ring, whereas PIP4K phosphorylates PI5-P at position 4. Although PIP4K and PIP5K synthesize the same end product, they are not functionally redundant and studies of the mammalian enzymes has defined the molecular basis of substrate specificity. Genes encoding PIP5K are present in all sequenced eukaryotes; however PIP4K appears to be a feature of metazoans; mammalian genomes contain three distinct genes for each of these two activities. However, the functional importance of these two classes of enzymes in generating plasma membrane PIP2 has remained unclear (Chakrabarti, 2015).

Drosophila photoreceptors are a well-established model for analyzing phosophoinositide signaling in-vivo. In these cells, the absorption of photons is transduced into neuronal activity by G-protein coupled, phospholipase Cβ (PLCβ) mediated PIP2 hydrolysis (NorpA). Thus, during phototransduction, PIP2 needs to be resynthesized to match consumption by ongoing PLCβ activity. PIP2 turnover is tightly regulated in photoreceptors; mutants in molecules that regulate PIP2 turnover show defects in phototransduction. However the role of PIPK enzymes in regulating PIP2 synthesis during phototransduction is unknown. This study analyzed each of the three PIPK encoded in the Drosophila genome that could generate PIP2 in the context of phototransduction. This analysis defines three pools of PIP2 supporting distinct molecular processes in photoreceptors (Chakrabarti, 2015).

The hydrolysis of PIP2 by PLC in response to receptor activation is a widespread mechanism of signalling at the plasma membrane. In some cells such as neurons, activation of cell surface receptors by neurotransmitter ligands (e.g glutamate, Ach) or sensory stimuli triggers high rates of PLC activation and rapid consumption of PIP2. Under these conditions, it is essential that levels of PIP2, the substrate for PLC are maintained as failure to do so would likely result in desensitization. In mammalian cells, multiple classes of PIPK, the enzymes that resynthesize PIP2 have been described; yet the contribution of these enzymes to PIP2 resynthesis following PLC activation during cell signalling in vivo remains unclear. Broadly two classes of PIPK can synthesize PIP2 have been described; PIP5K that phosphorylates PI4P at position 5 or PIP4K that can phosphorylate PI5P at position 4. This study has analyzed the consequence of loss of each of these two classes of PIPK to resynthesis following PLC mediated PIP2 depletion during Drosophila phototransduction (Chakrabarti, 2015).

Loss of dPIP5K function results in profound defects in the light activated electrical response as well as slower recovery of plasma membrane PIP2 levels. Conversely overexpression of dPIP5K was able to substantially accelerate the recovery of PIP2 levels following stimulation with a bright flash of light. dPIP5K is localized to the microvillar plasma membrane, the site at which PIP2 needs to be produced to support ongoing light induced PLC activity. Finally, this study found that loss of dPIP5K enhances the ERG defect in a hypomorphic allele of rdgB, a gene with a well-established defect in the response to light. Collectively these observations strongly suggest that dPIP5K activity underlies the conversion of PI4P to PIP2 at the microvillar membrane where it is then available as a substrate for light induced PLCβ activity. By contrast loss of the only PIP4K enzyme in the Drosophila genome has minimal effects on phototransduction and this enzyme is not targeted to the microvillar plasma membrane. These findings also imply that dPIP4K activity (and hence the conversion of PI5P into PIP2) is dispensable for maintaining PIP2 levels during Drosophila phototransduction. This is consistent with a previous study which found no reduction in the levels of PIP2 in flies lacking dPIP4K function. These observations validate the conclusion from biochemical studies in mammalian cells that the levels of PI5P are substantially lower than those of PIP2 and hence it is unlikely to be the source of the majority of PIP2 in cells. The identity of the PI4K isoform that generates the substrate, PI4P used by dPIP5K remains unknown although a recent study in mammalian systems suggests that PI4KIIIα is likely to be the relevant isoform (Chakrabarti, 2015).

Although the ERG response is severely affected in the dPIP5^K18 variant, it is not abolished as seen in null mutants of PLCβ (norpA) that are not able to hydrolyse PIP2. Additionally, the resting levels of PIP2 as detected by the PIP2 biosensor are comparable to wild type and following a bright flash of light that depletes PIP2, its levels do recover albeit at a slower rate than in wild type photoreceptors. Given that dPIP5^K18 is a protein null allele, these observations imply that there must be a second pool of PIP2 in dPIP5^K18 cells that is able to support phototransduction and microvillar PIP2 re-synthesis albeit with lower efficiency. This second pool of PIP2 is likely available with low efficiency for PLC activity in the absence of the dPIP5K dependent pool thus accounting for the residual light response and observed PIP2 dynamics in dPIP5^K18 photoreceptors (Chakrabarti, 2015).

The ultrastructure of dPIP5^K18 photoreceptors was essentially normal. This was particularly surprising given that in addition to phototransduction, PIP2 at the microvillar membrane is also expected to regulate multiple processes required to maintain normal microvillar structure including dynamin dependent endocytosis as well as cytoskeletal function. However, using multiple readout, molecular readouts of endocytosis and cytoskeletal function were found to be unaffected in dPIP5^K18 photoreceptors. These observations imply that the PIP2 required for these processes is not dependent on dPIP5K activity; rather PIP2 generated by a separate PIPK supports these processes. Thus far, dPIP4K has not been detected on the microvillar plasma membrane, dPIP4^K29 photoreceptors show normal ultrastructure on eclosion and do not undergo light dependent microvillar degeneration; thus dPIP4K is unlikely to be the critical enzyme that generates the PIP2 required to support dynamin dependent endocytosis, p-Moesin localization, or phototransduction. The Drosophila genome encodes an additional PIP5K activity, sktl that is expressed at low levels in the adult retina but is localized to both the microvillar and basolateral membrane and hence could synthesize PIP2 at both these locations. Complete loss of sktl function is cell lethal and overexpression of sktl in developing photoreceptors results in a severe block in rhabdomere biogenesis whereas overexpression of sktl results in light dependent retinal degeneration in post-development photoreceptors. These findings presumably reflect an essential and non-redundant role for SKTL in supporting fundamental PIP2 dependent cellular processes such as endocytosis and cytoskeletal function that are not dependent on PIP2 hydrolysis by PLC. This model is consistent with the cell-lethal phenotype of photoreceptors that are null for sktl and previous studies showing a role for sktl in supporting cytoskeletal function and endocytosis in other Drosophila tissues and processes such as spermiogenesis and oogenesis (Chakrabarti, 2015).

Collectively, these observations imply that there are at least two pools of PIP2 in photoreceptors; one generated by dPIP5K that is required to support a normal electrical response to light but is dispensable for non-PLC dependent functions of PIP2 in photoreceptors and another that is generated by enzymes other than dPIP5K (most likely SKTL) that is also capable of supporting PIP2 synthesis during the light response albeit with reduced efficiency. In summary the PIP2 pool synthesized by dPIP5K is unique in that it is required for a normal light response and apparently dispensable for other PIP2 dependent functions/processes. It also reflects the existence of distinct/segregated pools of PIP2 on the same microvillar plasma membrane that are maintained by distinct kinases (Chakrabarti, 2015).

A number of previous studies have shown that in multiple eukaryotic cell types, plasma membrane PIP2 levels are remarkably stable, undergoing transient fluctuations despite ongoing PLC mediated PIP2 hydrolysis. However the reasons for this remarkable finding have remained unclear although pharmacological studies have suggested the importance of PIP2 resynthesis in this process. One potential explanation for this idea is the existence at the plasma membrane of two pools of PIP2, a larger but less dynamic pool of that is not normally accessed by PLC and supporting non-PLC dependent functions of this lipid and a second, quantitatively smaller but more dynamic pool that is the substrate for PLC activity. What underpins such pools of PIP2? The existence of separate enzymes that generate unique pools of PIP2 has been previously suggested but there have been limited experimental studies to support this model. In murine platelets where thrombin induced PIP2 hydrolysis appears to be dependent on PIP5K1β but not PIP5Kγ; since both these enzymes are expressed in platelets this implies the existence of two pools of PIP2 in these cells of which the PIP5K1β dependent pool is available for thrombin dependent PIP2 turnover (Chakrabarti, 2015).

This finding together with this study in Drosophila photoreceptors implies that the plasma membrane in general may contain a specific pool of PIP2 dedicated for the use of receptor dependent PLC signalling and synthesized by a specific PIPK. It is possible that given the high rates of PLC activated PIP2 turnover at the plasma membrane (such as the microvillar membrane in photoreceptors) eukaryotic cells have evolved a mechanism to generate distinct PIP2 pool for this purpose so that other PIP2 dependent functions at the plasma membrane remain unaffected by ongoing receptor activated PIP2 hydrolysis. It is likely that dPIP5K and mammalian PIP5K1β represent PIP5K enzymatic activities required to support such a pool of PIP2 at the plasma membrane (Chakrabarti, 2015).

It is presently unclear what properties might make dPIP5K more suitable for generating PIP2 in the context of receptor triggered PLC activity. One possibility is that the kinetic properties of the enzyme encoded by dPIP5K is distinct from that encoded by sktl allowing it to function in the context of high rates of PIP2 turnover. Alternatively (or additionally) within the nanoscale organization of the microvillar plasma membrane, it is possible that dPIP5K is segregated such that PIP2 generated by this enzyme is available within molecular distances of the phototransduction machinery. Interestingly, Drosophila photoreceptors contain within their microvillar membrane a macromolecular signalling complex organized by the PDZ domain protein INAD. It is presently not known if dPIP5K is part of a similar complex but the existence of such mechanisms has been previously shown for mammalian PIP5K1γ in the context of focal adhesion function. Interestingly, it has been reported that the INAD protein complex that includes PLCβ is recruited to detergent resistant membranes during light stimulation which themselves have been previously implicated in the formation of PIP2 microdomains and receptor activated PIP2 turnover. It is possible that the two PIPKs, SKTL and dPIP5K show differential localization to such domains thus generating and segregating such pools of PIP2 and further studies in this direction are likely to provide insight into this issue. Nevertheless this study has provided evidence for the concept of distinct PIPK enzymes as the basis for functionally distinct pools of PIP2 at the plasma membrane. Further analysis in this system is likely to reveal the molecular basis for the organization of PIP2 pools at cellular membranes (Chakrabarti, 2015).

Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved this study predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, the study used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38% to ~5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ~10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ~2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade (Randall, 2015).

The ability to manipulate fatty acid composition of phospholipids in flies by diet has been reported previously. However, there are no convincing reports of physiological consequences. This study found that the proportionof polyunsaturated phospholipids in flies reared on yeast was reduced ~7-fold, and that this was associated with pronounced effects on visual performance, with photoreceptor responses slowed 2- to 3-fold,without significantly affecting quantum bump waveform, Na⁺/Ca²⁺ exchange, or PLC activity. Although absolute sensitivity was little affected by diet in wild-type photoreceptors, this could be attributed to compensation by Ca²⁺-dependent positive feedback acting on TRP channels. In the absence of Ca²⁺ influx or in trp mutants expressing only TRPL channels, sensitivity was reduced up to ~10-fold, and essentially abolished in hypomorphic Gαq¹ mutants. The results indicate that these effects, all of which were rescued by the addition of a single species of PUFA tothe yeast diet, were mediated predominantly downstream of PLC and were associated with slowing of photomechanical responses (Randall, 2015).

The macroscopic light response in Drosophila reflects the summation of quantum bumps, each arising from activation of most of the ~20 TRP channels in a single microvillus. Briefly, a single activated rhodopsin is believed to activate ~5 or so G_q-proteins by random diffusional encounters, and each released G_q α-subunit diffuses further before binding and activating PLC. Each PLC molecule rapidly hydrolyzes PIP₂, building up sufficient excitatory 'messenger' to overcome a finite threshold required to activate the first TRP channel with a stochastically variable latency of ~15-100 ms. The resulting Ca²⁺ influx raises Ca²⁺ throughout the microvillus into the micromolar range within milliseconds, facilitating activation of the remaining channels, and generating an 'all-or-none' quantum bump localized to a single microvillus. This raises Ca²⁺ within the affected microvillus to ~1 μm, terminating the bump by Ca²⁺-dependent inactivation of the channels and preceding steps of the cascade. This highly nonlinear positive and negative feedback cycle, which shapes the quantum bump waveform, was apparently not influenced by diet. However, bump latency, i.e., the time taken to activate the first channel, was clearly profoundly delayed in flies reared on the YF diet (Randall, 2015).

Which products of PLC activity are responsible for this initial gating remains controversial. InsP₃ and Ca²⁺ stores apparently play no role, because mutants of the InsP₃ receptor have normal phototransduction. Alternative candidates include DAG or PUFAs, which might be released from DAG by an appropriate lipase. Of these, DAG has generally proved ineffective as an agonist when exogenously applied; however, there is genetic evidence, based on mutants of DAG kinase (rdgA), for DAG as an excitatory messenger. One lab has also reported that DAG can activate TRP channels in excised patches from dissociated rhabdomeres; however, activation was very sluggish with delays of up to 60 s, in a preparation that is in a physiologically severely compromised state (Randall, 2015).

In contrast, there is universal consensus that PUFAs are very effective agonists when exogenously applied to both native channels in the photoreceptors and heterologously expressed TRPL channels. Apparent support for endogenous PUFAs came from the reduced light response found in a Drosophila DAG lipase mutant, inaE. However, inaE encodes an sn-1 DAG lipase, which rather than PUFAs, releases mono-acyl glycerols (MAGs), which are at best weak and slowly acting channel agonists when applied exogenously (Hardie, unpublished results). For PUFA generation, either an sn-2 DAG lipase or an additional enzyme (MAG lipase) would be required, but there is no evidence for either in photoreceptors. Furthermore, the inaE gene product immunolocalizes to the cell body with, at most, occasional puncta in the rhabdomere, and there is no evidence that PUFAs are generated in response to illumination (Randall, 2015).

An alternative hypothesis comes from evidence showing that the channels can be activated by the strict combination of two further consequences of PLC action, namely PIP₂ depletion and proton release. Furthermore it was suggested that PIP₂'s role is mediated not by ligand binding/unbinding but by the physical effects of PIP₂ depletion on the lipid bilayer. Thus, removal of PIP₂'s inositol headgroup from the inner leaflet effectively reduces membrane area, and it was proposed that the resulting mechanical effects (on, e.g., membrane tension, curvature, or lateral pressure) lead to mechanical gating of the channels, in combination with protons. Evidence supporting this included: (1) the ability of light to activate ectopic mechanosensitive channels (gramicidin); (2) facilitation of light responses by hypotonic solutions; and (3) rapid contractions of the photoreceptors in response to light, interpreted as the concerted contraction of microvilli due to PIP₂ hydrolysis in the inner leaflet (Randall, 2015).

The present study used diet to increase the degree of saturation of the phospholipids, which should make membranes stiffer and less flexible, and it ws predicted that this might suppress mechanosensitive channel activity. Lipidomic analysis confirmed a drastic sevenfold reduction in PUFA content across all phospholipid species in YF-reared flies. This was associated with a 2- to 3-fold longer latency and a marked reduction in open probability once Ca²⁺-dependent positive feedback was eliminated. In principle PLC activity might be suppressed by the YF diet, because membrane fluidity, and hence diffusion (e.g., of G-proteins, or PIP₂), might be slower in a membrane dominated by saturated phospholipids. However, molecular dynamic simulations predict that lateral diffusion coefficients in fact depend only weakly on the degree of phospholipid saturation. Importantly, the rate of PIP₂ hydrolysis monitored by proton release appeared unaffected by diet suggesting that diet was acting primarily downstream of PLC. That dietary manipulation had indeed resulted in alteration to the mechanical properties of the membrane was supported by AFM measurements of the light-induced contractions and responses mediated by ectopic mechanosensitive gramicidin channels. These indicated that, despite the lack of effect on PLC activity, YF-reared flies generated slower photomechanical responses (Randall, 2015).

Although consistent with the mechanical gating hypothesis, by themselves these data do not exclude the alternative suggestions, that the excitatory messenger is DAG or a PUFA. According to a recent study (Delgado, 2014), DAG generated by light-induced PIP₂ hydrolysis in Drosophila photoreceptor membrane includes both monounsaturated and polyunsaturated species (32:1, 32:2, 34:1, 34:2, 36:3, and 36:4) suggesting at least some derive from polyunsaturated PIP₂ species. In turn, should any fatty acids be released from DAG, they would most likely include PUFAs. However, the near absence of polyunsaturated PIP₂ in flies reared on YF diets implies that DAG generated in response to light would now be predominantly saturated or monounsaturated. If different species of DAG and/or PUFA had different potencies for activating the channels, then in principle this might also explain the results. In this respect, the otherwise equivocal evidence for DAG and PUFAs as messengers notwithstanding, it should be noted that heterologously expressed TRPL channels are activated more potently by linolenic than by oleic acid and that polyunsaturated DAG species have been reported to be more effective activators of PKC in mammalian cells (Randall, 2015).

In conclusion, these results confirm that fatty acid composition of phospholipids is strongly influenced by diet in Drosophila. Increasing the degree of saturation leads to pronounced slowing of the electrical light response, which is associated with slower photomechanical responses. These findings are consistent with the suggestion that TRP and TRPL channel activation in Drosophila photoreceptors are mediated, together with protons, by mechanical forces in the membrane induced by PIP₂ hydrolysis by PLC. To what extent visual performance in flies might be affected by dietary fatty acids under natural conditions is unknown. Although Drosophila thrive on a yeast diet in the laboratory, their typical food is rotting plant material, which will contain PUFAs to varying degrees. Interestingly, in food-preference studies, Drosophila larvae show preference for unsaturated over saturated fatty acids, but this preference is reversed in adult flies. The fatty acid composition of mammalian phospholipids is also influenced by diet, and the effects of fatty acid intak on health and disease are extensively documented. Certain PUFAs such as ω-3 (e.g., α-linolenic acid) and ω-6 (e.g., linoleic acid) can only be supplied by dietary intake in mammals. Of these, α-linolenic acid is also precursor for docosahexaenoic acid (22:6), which is the most abundant ω-3 fatty acid in mammalian brain and retina (though absent in flies). ω-3 and ω-6 deficiency is associated with cardiovascular disease and diabetes, can lead to cognitive defects in rodents, and within the mammalian retina leads to a reduced a-wave in the ERG. However, the underlying mechanisms are poorly understood, and in this respect, the current results provide a striking and novel example of how dietary fatty acids can profoundly and specifically influence in vivo performance and behavior via a defined step within the context of a classical G-protein-coupled signaling cascade. Interestingly, the importance of the mechanical properties of membranes containing polyunsaturated phospholipids has also been highlighted in recent studies of mechanosensation and rapid endocytosis (Randall, 2015).

Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. Recent studies have shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLC activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. This study shows that in IP3 receptor (IP₃R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP₃R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP₃R knockdown with reduced PLCbeta catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP₃R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP₃R does not participate in light excitation, this study shows that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP₃R found here may represent a common feature of the inositol-lipid signaling (Kohn, 2015).

In this study, in vivo light-response suppression was accompanied by reduced Ca2+ release from IP3-sensitive stores. In addition, the rate of spontaneously produced dark bumps, which is highly sensitive to Gqα-dependent PLCβ catalytic activity and cellular Ca2+ level, was virtually abolished in IP₃R-deficient photoreceptors. This dark-bump elimination indicates that the suppressed Ca2+ release from IP3-sensitive stores underlies the suppressed catalytic activity of PLCβ, leading to suppressed light response in IP₃R-deficient photoreceptors. Further evidence that the suppressed light response arises from inhibition of Ca2+ release from IP3-sensitive stores came from blocking the Ca2+ pump by Tg, which mimicked the phenotype of the IP₃R-deficient photoreceptors in WT flies. The above findings indicate that IP₃R-mediated Ca2+ release has a critical role in light excitation of Drosophila photoreceptors. The combination of the PLCβ mutant norpAH43 with IP₃R-deficient photoreceptors, which synergistically suppressed the light response, strongly suggests that there is functional cooperation between the IP₃R and PLCβ in generation of the light response (Kohn, 2015).

It has been previously shown that an increase in cytosolic Ca2+ participates in light excitation as evidenced by enhancement of the light response following photo release of caged Ca2+ at the rising phase of the light response. The target of Ca2+ action has not been entirely resolved. PLCβ is an important target for Ca2+ action and the regulation of its catalytic activity by Ca2+ has been thoroughly investigated. These studies showed that the positive charge of Ca2+ is used to counterbalance local negative charges formed in the active site during the course of the catalytic reaction. Accordingly, Ca2+ performs electrostatic stabilization of both the substrate and the transition state, thus providing a twofold contribution to lower the activation energy of the enzyme reaction (Kohn, 2015).

The following model explains how functional cooperation between the IP₃R and PLCβ via the released Ca2+ operates and secures quantum-bump production: absorption of a single photon, which induces activation of several PLCβ molecules, is initially insufficient at resting Ca2+ levels to reach the critical level of PLCβ activity required for TRP/TRPL channel activation. Nevertheless, the IP3 molecules produced by the given PLCβ activity are able to activate the nearby IP₃Rs, mobilize Ca2+ from the stores, and elevate PLCβ activity above the threshold required for TRP/TRPL channel activation. In addition, the released Ca2+ may also reduce the threshold of TRP/TRPL channel activation and allow bump generation. According to this model, the following enzymatic reactions may explain the current findings. Each Gqα-activated PLCβ has low catalytic activity due to the relatively low (<160 nM) resting Ca2+ concentration in the cytosol. In addition, each activated PLCβ remains active for only a short (approximately several tens of milliseconds) time due to the GTPase-activating protein activity of PLCβ that causes a rapid hydrolysis of Gqα-GTP followed by inactivation of PLCβ. The initial low catalytic activity of PLCβ is apparently below the threshold required for activation of the TRP and TRPL channels, but this low activity still results in hydrolysis of PIP2 producing IP3. Since there are no IP3 buffers in the microvilli and the IP3 degradation time is relatively slow (~1 s), the produced IP3 molecules diffuse fast along the microvillus at an estimated time of ~1 ms along 1 microm long microvillus and bind to IP₃R located at the nearby submicrovillar cisternae (SMC; the photoreceptors' extensions of smooth ER). IP₃R channels residing at the SMC, which are large channels with high sensitivity for IP3 and thus can be activated at low PLCβ activity, open and release Ca2+ juxtaposed to the base of the microvillus. The released Ca2+ steeply raises the local Ca2+ concentration, probably to the microM range, because of the very small aqueous volume of the microvillus and the relatively large local Ca2+ elevation via the release mechanism (Kohn, 2015).

Accordingly, a single IP₃R channel can release ∼104 Ca2+ ions in 1 ms channel opening and Ca2+-induced Ca2+ release mechanism is a property of the IP₃R channels and of the ryanodine receptors, which reside in the ER. Ca2+ released via IP₃R of the WT SMC diffuse back toward the activated PLCβ and the TRP/TRPL channels in the microvillus. Although Ca2+ diffuses ∼20-fold slower than IP3 due to strong buffering, the diffusion constant strongly depends on Ca2+ concentration. Accordingly, at ~250 μM the Ca2+ diffusion coefficient is as large as that of IP3. Once a single TRP channel is activated, the large Ca2+ influx through this channel is sufficient to facilitate the rest of the active PLCβ molecules or reduce the threshold for TRP/TRPL channel activation in this microvillus and produce a bump that reflects activation of the entire microvillus . When there is abnormally low Ca2+ release via the IP₃R because of low IP₃R expression levels (IP₃R-RNAi), there is not enough Ca2+ to increase PLC activity or to reduce TRP activation threshold, and activated PLC in this microvillus does not produce a bump, leading to abnormally low frequency of dark bumps (Kohn, 2015).

The invasive whole-cell recording technique, which was used in previous studies and avoided Ca2+ buffering of the pipette solution, most likely resulted in abnormally elevated cytosolic Ca2+ concentration, which also allowed the Ca2+ pump to keep the stores full. This artificially elevated cytosolic [Ca2+] together with the constitutive Ca2+ leak from the full stores, bypassed the need to mobilize Ca2+ via functional IP₃R to facilitate PLCβ activity and reach its critical catalytic activity level needed to activate the TRP/TRPL channels. In the present study in the intact eye, a significant reduction in light-response amplitude was observed when the IP₃R level was reduced. Furthermore, when cellular [Ca2+] was reduced by prolonged extracellular EGTA application, the light response of the IP₃R-deficient flies was further suppressed. Moreover, when using invasive patch-clamp whole-cell recordings without Ca2+ buffering of the pipette solution, no significant difference between WT and IP₃R-deficient flies was observed, as found in the previous study. However, when pipette Ca2+ was reduced with EGTA, the phenotype of reduced light excitation was observed in both reduced quantum-bump frequency as well as in macroscopic light-response suppression. Unlike quantum bumps, dark bumps were virtually eliminated even without buffering the pipette Ca2+ in IP₃R-deficient flies, indicating that in the dark the IP₃R-deficiency led to abnormally low cytosolic [Ca2+], possibly due to reduced Ca2+ leak from stores leading to cellular [Ca2+] below the critical level required for PLC activation observed in WT flies. Alternatively, the positive feedback between the released Ca2+ and PLC may function at single PLC molecules. Hence the nominal pipette Ca2+ is not sufficient to allow PLC activity to pass the threshold of channel activation, but the released Ca2+ via IP₃R activation together with pipette Ca2+ allows PLC activity to pass this threshold and generate dark-bump (Kohn, 2015).

There is a striking functional similarity between, the cerebellar Purkinje cell (PC) proteins of the IP signaling and Drosophila photoreceptors, but the link of cerebellar mGluR1 receptor to TRPC3 activation is not clear. Interestingly, in PC neurons, stromal interaction molecule 1 (STIM1) was proved an essential regulator of Ca2+ level in neuronal endoplasmic reticulum Ca2+ stores. Accordingly, STIM1-specific deletion caused impairments in slow synaptic current and cerebellar motor behavior. Strikingly, refilling empty Ca2+ stores through increased Ca2+ level in the cytosol partially rescued the phenotype of the stim1 knock-out mice, reminiscent of the rescue of the phenotype of the IP₃R-deficient fly by artificially elevated cytosolic Ca2+. Thus, the facilitatory role of released Ca2+ on PLC in light excitation of Drosophila photoreceptors represents an essential mechanism that operates in other PI systems (Kohn, 2015).

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. This study reports that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function (Basak, 2021).

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca2+ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, the Drosophila protein, Calphotin, has been characterized as a cytosolic immobile Ca2+ buffer, which participates in Ca2+ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca2+ influx into photoreceptor cells, results in severe Ca2+-dependent degeneration. This study shows that this degeneration is autophagy and ER-stress related and thus provides a new model in which genetic manipulations trigger changes in cellular Ca2+ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca2+, ER-stress and autophagy in human disorders and diseases (Weiss, 2015).

Autophagy and ER-stress are highly conserved during evolution as many other essential cell maintenance pathways. Autophagy has been implicated in several pathologies, including protein aggregate disorders, neurodegeneration and cancer. Both autophagy and ER-stress are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate. Many human genes, implicated in degenerative diseases, have orthologs in Drosophila. Studies in fly models have identified novel factors that regulate these diseases; therefore, Drosophila is a powerful model system for studying these genes (Weiss, 2015).

An illuminating example is the Drosophila model for class III autosomal dominant retinitis pigmentosa (ADRP), in which ER-stress has been shown to play a protective role against retinal degeneration. Moreover, it has been shown that when Drosophila cells (both photoreceptors and S2 cells) are pre-exposed to a mild ER-stress, they are protected from treatments with various cell death agents. It has also been shown recently that activation of mild ER stress is neuroprotective, both in Drosophila and mouse models of Parkinson disease, and this protection is a consequence of autophagy activation. Accordingly, it is well established that activation of the ER-stress response by accumulation of misfolded proteins (unfolded protein response, UPR) can trigger autophagy (Weiss, 2015).

It is well established that elevation of intracellular Ca2+ can trigger autophagy. The stimulation of autophagy by elevated cytosolic [Ca2+] has been studied mainly in tissue culture cells, in which mobilization of Ca2+ is triggered by application of various Ca2+ mobilizing agents (e.g. thapsigargin, ionomycin, vitamin D). However, since increased cytosolic [Ca2+] also activates ER-stress which induces autophagy, a question arises as to whether the activation of autophagy by Ca2+ is induced directly or indirectly via activation of ER-stress. The fact that thapsigargin-induced autophagy occurs in UPR-deficient cells, suggests a direct involvement of Ca2+ in the induction of autophagy. Although Ca2+ regulation of autophagy has been found to be mediated through the activation of Bcl-2 and calmodulin-dependent kinase kinase-β, the target by which Ca2+ activates autophagy directly is yet to be elucidated. These unsolved issues can be conveniently addressed in the new model of Ca2+ induced autophagy in Drosophila photoreceptor cells (Weiss, 2015).

Neurodegenerative diseases, such as Parkinson, Alzheimer and Huntington’s diseases are accompanied by the accumulation of large aggregates of mutant proteins and are autophagy associated diseases. These autophagy associated diseases, which are characterized by abnormal protein aggregations, have highly developed model systems of Drosophila. The increased autophagosome formation observed in these diseases, may play a pro- tective role by degrading misfolded proteins. Many of the above diseases, including Parkinson and Alzheimer diseases, are also associated with reduced cellular Ca2+ buffering and impairments in cellular Ca2+ homeostasis reminiscent of Drosophila photoreceptors with reduced calphotin. Therefore, combining the Drosophila models of autophagy associated diseases with calphotin hypomorph can be very useful for studying the possible link between reduced cellular Ca2+ buffering and neurodegeneration (Weiss, 2015).

In the genetic fly model of calphotin hypomorph, reduced calphotin levels, which trigger changes in cellular Ca2+ homeostasis allows induction of Ca2+ induced autophagy, simply by changing the illumination conditions of the flies’ environment. Accordingly, both the severity and progression of the observed degeneration can be highly controlled and manipulated. The degeneration process of this experimental model is sufficiently slow, allowing monitoring the progression of the degeneration process and elucidation of the involved proteins. Thus, the Drosophila Cpn hypomorph photoreceptor cells constitutes a powerful model, in which genetic manipulation combined with illumination determine the level of sustained cellular Ca2+ that trigger the induction of Ca2+ dependent autophagy, ER- stress and cell death. This model can provide a framework for further investigations into the link between cytosolic Ca2C, ER-stress and autophagy in human disorders and diseases (Weiss, 2015).

The GTP- and phospholipid-binding protein TTD14 regulates trafficking of the TRPL ion channel in Drosophila photoreceptor cells

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels Transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. This study identified the gene mutated in trpl translocation defective 14 (ttd14) (CG30118), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane (Cerny, 2015).

Drosophila vision depends on carcinine uptake by an organic cation transporter

Recycling of neurotransmitters is essential for sustained neuronal signaling, yet recycling pathways for various transmitters, including histamine, remain poorly understood. In the first visual ganglion (lamina) of Drosophila, photoreceptor-released histamine is taken up into perisynaptic glia, converted to carcinine, and delivered back to the photoreceptor for histamine regeneration. This study identified an organic cation transporter, CarT (carcinine transporter), that transports carcinine into photoreceptors during histamine recycling. CarT mediated in vitro uptake of carcinine. Deletion of the CarT gene caused an accumulation of carcinine in laminar glia accompanied by a reduction in histamine, resulting in abolished photoreceptor signal transmission and blindness in behavioral assays. These defects were rescued by expression of CarT cDNA in photoreceptors, and they were reproduced by photoreceptor-specific CarT knockdown. These findings suggest a common role for the conserved family of CarT-like transporters in maintaining histamine homeostasis in both mammalian and fly brains (Chaturvedi, 2016).

Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine>

Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. This study investigated this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the Hdc^JK910 mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that Hdc^JK910 photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected Hdc^JK910 photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to Hdc^JK910 R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in Hdc^JK910 mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons (Dau, 2016).

Tadr is an axonal histidine transporter required for visual neurotransmission in Drosophila

Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. This study identified a specific histidine transporter, TADR (torn and diminished rhabdomeres), which is required for visual transmission in Drosophila. Both TADR and Hdc localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission (Han, 2022).

Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling

During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. This study found that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition (Thakur, 2016).

CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila, the major light sensing rhodopsin, Rh1, and the downstream ion channel, Transient Receptor Potential Like (TRPL), are endocytosed in response to light, but the mechanism is unclear. Using an RNA-Sequencing approach, this study discovered CULD (CG17352), a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1- or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1 but not TRPL suppressed retinal degeneration in culd null mutant flies. These data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity (Xu, 2016)

G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors and, therefore, transduce signals from a wide variety of hormones, cytokines, neurotransmitters, as well as sensory stimuli. Each of these interactions triggers distinct intracellular responses through heterotrimeric G proteins. Upon continuous stimulation, GPCRs are deactivated by arrestins, and internalized through dynamin-dependent endocytosis. Many internalized GPCRs undergo lysosomal degradation and/or recycling, leading to downregulation of receptor levels, which is important for reducing the strength and duration of cellular responsiveness following various stimuli (Xu, 2016).

The Drosophila phototransduction cascade is a model pathway for the dissection of GPCR signaling and associated regulatory processes. Proteins of the visual signal transduction cascade are found within rhabdomeres, which are specialized compartments within photoreceptor cells that contain tightly packed microvilli. Light-induced activation of rhodopsin triggers the phototransduction cascade by stimulating the vision protein phospholipase C, which is encoded by the no receptor potential A (norpA) gene, through the α subunit of the heterotrimeric G protein DGq. This opens the transient receptor potential (TRP) channel and the TRP-like (TRPL) Ca²⁺/cation channel, and depolarizes the photoreceptor neurons. Meanwhile, activated rhodopsin, which is referred to as metarhodopsin, is immediately bound by arrestin and deactivated. After inactivation, metarhodopsin is either photoconverted back into rhodopsin or internalized for degradation. Although the majority of internalized metarhodopsin is degraded, with newly synthesized rhodopsin replenishing the pool, it has recently been reported that internalized rhodopsin (Rh1; encoded by ninaE in Drosophila melanogaster) can be recycled upon stimulation with light. The principle arrestin, Arr2, plays a pivotal role in deactivating rhodopsin, whereas Arr1 binds and internalizes rhodopsin (Xu, 2016).

Long-term adaptation to light stimuli also involves the dynamic activity-dependent translocation of signaling proteins that are not GPCRs. As seen with mammalian Rod photoreceptors, light induces the movement of Arr2 and Arr1 into the rhabdomeres. In Drosophila, TRP and TRPL function as the primary light-activated channels. TRP stably localizes to the rhabdomeres by forming a multiprotein signaling complex, the signalplex with inactivation-no-after-potential D protein (INAD), a protein that contains five PDZ domains. In contrast, illumination results in TRPL translocating from the rhabdomeres to an intracellular storage compartment within the cell body. However, the mechanisms that underlie light-induced translocation and trafficking of rhodopsin and TRPL are not yet fully understood. Furthermore, it is unclear whether this endocytic trafficking of TRPL plays a physiological role in maintaining the integrity of photoreceptor cells (Xu, 2016).

By using an RNA-Sequencing (RNA-Seq) approach, this study identified a so-far-unknown gene that is enriched in photoreceptors, and encodes a transmembrane protein with both a CUB and an LDLa domain. This protein was named CULD (CUB- and LDLa-domain protein). CULD mainly localized to the endocytic TRPL- or Rh1-positive vesicles. Mutations in culd led to endosomal accumulation of Rh1 and TRPL, which disrupted the light sensitivity of photoreceptors; blocking of Arr1-mediated endocytosis eliminated the intracellular accumulation of Rh1. Moreover, culd mutants underwent light-dependent retinal degeneration, and resulted in a phenotype that could be rescued by reducing the levels of Rh1. These data indicate that CULD is essential for the function and survival of photoreceptor cells by promoting the endocytic turnover of Rh1 and TRPL (Xu, 2016).

A microarray analysis has previously been used to compare the genes expressed in wild-type heads with heads from a mutant fly that lacked eyes in order to identify eye-enriched genes, which led to the further identification of some genes functioning in phototransduction. However, owing to multiple cell types in the compound eye, many genes identified in this analysis might not function in photoreceptor cells. This study describes an RNA-Seq screen to identify genes expressed predominantly in photoreceptors. Among the 58 genes identified, 36 genes were known to function in photoreceptor cells, representing most of the genes that play major roles in phototransduction or retinal degeneration. However, 22 genes had not been described as being enriched in photoreceptor cells previously. Among them, cg9935 (Eye-enriched kainate receptor: Ekar) has been recently reported to regulate the retrograde glutamate signal in photoreceptor cells and contribute to light-evoked depolarization during phototransduction (Hu, 2015). This study further characterized the new photoreceptor cell-enriched gene culd as being required for turnover of Rh1 and TRPL. Although culd had also been identified as an eye-enriched gene in the earlier microarray analysis that compared RNA expression in wild-type and eyeless heads, 93 other eye-enriched candidates prevented focusing on CULD. In this RNA-Seq screen, only photoreceptor-cell-enriched genes can be identified, and a reasonable number of candidates might represent new factors functioning in phototransduction. However, some eye-enriched genes important for phototransduction might be missed in this screen. For example, recently identified polyglutamine-binding protein 1 (PQBP1) was not found as a photoreceptor-cell-specific gene in the RNA-Seq screen. This might be because PQBP1 is also expressed in other non-photoreceptor retinal cells. Overall, this screen for photoreceptor-enriched genes sheds a light on further understanding of phototransduction and mechanisms of retinal degeneration (Xu, 2016).

Appropriate signals cause arrestins to translocate to the plasma membrane where they bind to activated GPCRs, thereby inhibiting G-protein-dependent signaling and regulating GPCR endocytosis and trafficking. In Drosophila there are two arrestins within photoreceptors, Arr1 and Arr2. Although Arr2 binds to Rh1, it is Arr1 that primarily colocalizes with Rh1 in internalized vesicles. Therefore, Arr1 might mediate light-dependent endocytosis of Rh1, whereas Arr2 functions to quench activated Rh1. In culd mutant flies, Rh1 was immobilized within endocytic vesicles and Arr1 colocalized with the endocytic Rh1; blocking the Arr1-medicated endocytosis in culd mutant cells eliminated the abnormal intracellular accumulation of Rh1. These data strongly suggest that CULD functions downstream of Arr1-mediated endocytosis of Rh1 (Xu, 2016).

Early endosomes containing Rab5 serve as a focal point of the endocytic pathway. Sorting events initiated in early endosomes determine the subsequent fate of internalized proteins, that is, whether they will be recycled to the plasma membrane or degraded within lysosomes. Rh1 and TRPL share the same internalization pathway, and during light stimulation Rab5 initially mediates this vesicular transport pathway. In wild-type photoreceptors, however, Rh1 and TRPL have different fates from common Rab5-positive early endosomes. The majority of Rh1 is eventually delivered to lysosomes for degradation, whereas most internalized TRPL tends to be stored. In wild-type cells, the photoreceptor-enriched protein CULD colocalized with the endocytic TRPL or Rh1 vesicles, and the majority of CULD-positive vesicles were also Rab5-positive. This spatial pattern indicates that CULD is required for the endocytic trafficking of TRPL and Rh1 after they are internalized (Xu, 2016).

CULD functions during the early steps of endocytosis that immediately follow internalization, which is a pathway involved in rhodopsin and TRPL endocytic turnover. Eliminating CULD had profound effects on the photoreceptor physiology. In both vertebrates and invertebrates, the light sensitivity of photoreceptor cells is primarily determined by functional rhodopsin. The culd mutant flies exhibited a gradual reduction in light sensitivity, which suggests that the amount of functional rhodopsin is reduced in culd mutant flies. As the amount of the monomer form of Rh1 was not affected and a large fraction of Rh1 accumulated within intracellular vesicles in culd mutant photoreceptor cells, the rhabdomeral Rh1 levels might be reduced. It is also likely that the endocytic degradation of Rh1 scavenges damaged Rh1 molecules, and blocking this process might lead to the accumulation of dysfunctional Rh1 in rhabdomeres (Xu, 2016).

TRPL has been reported to translocate from rhabdomeres to intracellular compartments for storage during prolonged light stimulation. However, a recent study suggests that some endocytic TRPL proteins are also delivered to lysosomes for degradation. Mutations in culd impaired TRPL endocytic trafficking upon light stimulation, leading to the retention of TRPL in Rab7-positive vesicles. TRPL protein levels were increased in culd mutants and this is probably due to decreased TRPL degradation (Xu, 2016).

As a major light sensor within photoreceptor cells, a small amount of activated Rh1 is internalized and degraded upon light stimulation. This is followed by replenishment of the rhabdomeric Rh1 pool. Therefore, a balance between Rh1 endocytosis and replenishment is required for Rh1 homeostasis under light conditions. Prolonged exposure to blue light triggers massive endocytosis of Rh1 and leads to a gradual loss of Rh1. Mutations in culd blocked Rh1 degradation during prolonged light treatment, indicating that the loss of CULD inhibited the Rh1-degradation pathway. Unlike TRPL, Rh1 levels were not increased, which suggests that Rh1 replenishment is strictly controlled. Given that, in Drosophila, rhodopsin levels are regulated by both the synthesis of the opsin and the chromophore subunits, it might be reasonable that in culd mutant cells, the chromophore is not released from the accumulated rhodopsin, and the reduction of free retinal pool might limit the synthesis of new rhodopsin (Xu, 2016).

Both vertebrates and invertebrates have a family of transmembrane proteins that contain both CUB- and LDLa- domains. However, only a few CUB/LDLa proteins have been functionally characterized. Among these proteins, NETO1 and NETO2 (see Drosophila Neto) have been intensively studied. NETO1 functions as an auxiliary subunit of ionotropic glutamate receptors, N-methyl-D-aspartate receptors and kainate receptors, modulating the channel properties of these glutamate receptors. NETO2 maintains normal levels of the neuron-specific K+-Cl- co-transporter KCC2 (also known as SLC12A5), and loss of NETO2-KCC2 interactions reduces KCC2-mediated Cl- extrusion, and decreases synaptic inhibition in hippocampal neurons. Moreover, in both Drosophila and C. elegans, the CUB/LDLa proteins NETO and SOL-2 are required for the clustering and functioning of glutamine receptors, thereby contributing to neuronal signaling pathways. This study cloned a new gene culd, which encodes a member of the CUB/LDLa family proteins specifically expressed in the Drosophila photoreceptor cell; this protein containing a CUB domain, an LDLa domain and one predicted transmembrane motif. CULD was not directly required for the activity of receptors or channels, but instead mediated the endocytic trafficking of Rh1 and TRPL. Loss of CULD led to the accumulation of Rh1 and TRPL in endocytic vesicles, and subsequent retinal degeneration. Therefore, this study revealed a new function of the CUB/LDLa family proteins, namely the endocytic turnover of receptors and channels (Xu, 2016).

It has been proposed that the accumulation of Rh1-Arr2 complexes in late endosomes triggers cell death of photoreceptor cells. Internalized Rh1-Arr2 complexes are not degraded but instead accumulate in late endosomes of norpA mutant photoreceptor cells. Similar rhodopsin accumulations are seen in mutations that affect the trafficking of late endosomes to lysosomes, which causes light-dependent retinal degeneration. Toxic Rh1-Arr2 complexes also induce retinal degeneration in rdgC, rdgB and fatp mutant flies. The culd mutations caused the accumulation of Rh1-Arr1 complexes and TRPL in endocytic vesicles and light-dependent retinal degeneration, suggesting that endosomal accumulation of either channels or receptors induced cell death. In addition, the evidence that ninaE^P332 but not trpl³⁰² rescued photoreceptor degeneration of the culd¹ mutants suggests that abnormal Rh1-Arr1 accumulation induces cell degeneration, whereas intracellular accumulation of TRPL does not contribute to the neuronal degeneration (Xu, 2016).

Functions of neuronal Synaptobrevin in the post-Golgi transport of Rhodopsin in Drosophila photoreceptors

Polarized transport is essential for constructing multiple plasma membrane domains in the cell. Drosophila photoreceptors are an excellent model system to study the mechanisms of polarized transport. Rab11 is the key factor regulating the post-Golgi transport of rhodopsin 1 (Rh1; also known as NinaE), a photoreceptive protein, to the rhabdomere, a photoreceptive plasma membrane. This study found that neuronal Synaptobrevin (nSyb) colocalizes with Rab11 on the trans-side of Golgi stacks and post-Golgi vesicles at the rhabdomere base, and nSyb deficiency impairs rhabdomeric transport and induces accumulation of Rh1 and vesicles in the cytoplasm; this is similar to the effects of Rab11 loss. These results indicate that nSyb acts as a post-Golgi SNARE toward rhabdomeres. Surprisingly, in Rab11-, Rip11- and nSyb-deficient photoreceptors, illumination enhances cytoplasmic accumulation of Rh1, which colocalizes with Rab11, Rabenosyn5, nSyb and Arrestin 1 (Arr1). Arr1 loss, but not Rab5 dominant negative (Rab5DN) protein expression, inhibits the light-enhanced cytoplasmic Rh1 accumulation. Rab5DN inhibits the generation of Rh1-containing multivesicular bodies rather than Rh1 internalization. Overall, these results indicate that exocytic Rh1 mingles with endocytosed Rh1 and is then transported together to rhabdomeres (Yamashita, 2022).

Dedicated photoreceptor pathways in Drosophila larvae mediate navigation by processing either spatial or temporal cues

To integrate changing environmental cues with high spatial and temporal resolution is critical for animals to orient themselves. Drosophila larvae show an effective motor program to navigate away from light sources. How the larval visual circuit processes light stimuli to control navigational decision remains unknown. The larval visual system is composed of two sensory input channels, Rhodopsin5 (Rh5) and Rhodopsin6 (Rh6) expressing photoreceptors (PRs). This study characterized how spatial and temporal information are used to control navigation. Rh6-PRs are required to perceive temporal changes of light intensity during head casts, while Rh5-PRs are required to control behaviors that allow navigation in response to spatial cues. This study characterized how distinct behaviors are modulated and identify parallel acting and converging features of the visual circuit. Functional features of the larval visual circuit highlight the principle of how early in a sensory circuit distinct behaviors may be computed by partly overlapping sensory pathways (Humberg, 2018).

The beta-alanine transporter BalaT is required for visual neurotransmission in Drosophila

The recycling of neurotransmitters is essential for sustained synaptic transmission. In Drosophila, histamine recycling is required for visual synaptic transmission. Synaptic histamine is rapidly taken up by laminar glia, and is converted to carcinine. After delivered back to photoreceptors, carcinine is hydrolyzed to release histamine and β-alanine. This histamine is repackaged into synaptic vesicles, but it is unclear how the β-alanine is returned to the laminar glial cells. This study identified a new beta-alanine transporter, which has been named BalaT (Beta-alanine Transporter). Null balat mutants exhibited lower levels of β-alanine, as well as less β-alanine accumulation in the retina. Moreover, BalaT is expressed and required in retinal pigment cells for maintaining visual synaptic transmission and phototaxis behavior. These results provide the first genetic evidence that retinal pigment cells play a critical role in visual neurotransmission, and suggest that a BalaT-dependent beta-alanine trafficking pathway is required for histamine homeostasis and visual neurotransmission (Han, 2017).

A single residue mutation in the Galphaq subunit of the G protein complex causes blindness in Drosophila

Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Galphaq is not clear since animals with strong loss of function mutations previously reported remain responsive to light stimuli. This study recovered a new allele of Galphaq in Drosophila that abolishes light response in a conventional ERG assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least 5 orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. The new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Galphaq and its downstream effector PLC. This study thus uncovered possibly the first point mutation that specifically affects this interaction in vivo (Cao, 2017).

Genetic dissection of the phosphoinositide cycle in Drosophila photoreceptors

Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2-, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. This study used transgenic flies expressing fluorescently tagged probes (P4M and Tb(R332H)), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, Drosophila PI4KIIIalpha (CG10260) was identified as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. These results also indicate that PI4KIIIalpha activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIalpha activity in yeast and mammals. However, the evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (Liu, 2018).

Blue light induces a neuroprotective gene expression program in Drosophila photoreceptors

Light exposure induces oxidative stress, which contributes to ocular diseases of aging. In contrast to mature adults, which undergo retinal degeneration when exposed to prolonged blue light, newly-eclosed flies are resistant to blue light-induced retinal degeneration. This study sought to characterize the gene expression programs induced by blue light in flies of different ages by profiling the nuclear transcriptome of Drosophila photoreceptors. Flies were exposed to 3 h blue light, which increases levels of reactive oxygen species but does not cause retinal degeneration. Substantial gene expression changes were identified in response to blue light only in six-day-old flies. In six-day-old flies, blue light induced a neuroprotective gene expression program that included upregulation of stress response pathways and downregulation of genes involved in light response, calcium influx and ion transport. An intact phototransduction pathway and calcium influx were required for upregulation, but not downregulation, of genes in response to blue light, suggesting that distinct pathways mediate the blue light-associated transcriptional response. These data demonstrate that under phototoxic conditions, Drosophila photoreceptors upregulate stress response pathways and simultaneously, downregulate expression of phototransduction components, ion transporters, and calcium channels. Together, this gene expression program both counteracts the calcium influx resulting from prolonged light exposure, and ameliorates the oxidative stress resulting from this calcium influx.Developmental transitions during the first week of adult Drosophila life lead to an altered gene expression program in photoreceptors that includes reduced expression of genes that maintain redox and calcium homeostasis, reducing the capacity of six-day-old flies to cope with longer periods (8 h) of light exposure. Together, these data provide insight into the neuroprotective gene regulatory mechanisms that enable photoreceptors to withstand light-induced oxidative stress (Hall, 2018).

The eye is susceptible to light-induced oxidative stress, which has been implicated in photoreceptor damage in a variety of eye diseases. To characterize the light stress response in Drosophila photoreceptors, the transcriptome was profiled of photoreceptors exposed to high intensities of blue light. Although longer durations of blue light induce severe retinal degeneration in white-eyed flies, shorter exposures to blue light induced major gene expression changes in photoreceptors but did not cause retinal degeneration. Instead, blue light induced expression of a broad range of genes involved in stress response, together with a concomitant reduction in expression of genes required for the light response including voltage-gated calcium, potassium and chloride ion channels. It is expected that these transcriptional changes would result in altered protein levels; however, this has not been tested in this study. Previous studies showed that very young flies (1 day post-eclosion) were resistant to blue light-induced retinal degeneration, and the current work revealed that the blue light-induced transcriptional changes differed according to the age of the fly; mature flies (6 days post-eclosion) showed substantially more differentially expressed genes in response to blue light exposure than very young flies (1 day post-eclosion). The increase in susceptibility to blue light between day one and six correlated with developmental transitions in photoreceptor gene expression, which included reduced expression of genes that function in redox and calcium homeostasis. Together, these data support a model in which mature adult flies upregulate stress response pathways in an effort to deal with light-induced oxidative stress, and concomitantly quench the light response to diminish phototransduction-associated calcium influx. Newly-eclosed flies might be able to withstand blue light exposure better because of an increased capacity to buffer the calcium influx and oxidative stress resulting from prolonged phototransduction. Indeed, relatively young, yet mature, flies (day six) can withstand moderate blue light exposure without significant retinal degeneration but lose the ability to resist longer durations of light exposure. Recent work demonstrated that white-eyed flies (w1118), but not their pigmented counterparts, undergo age-associated retinal degeneration under normal light/dark cycles by 30 days. Thus, the acute blue light paradigm used in this study may reveal insight into mechanisms associated with age-associated retinal degeneration (Hall, 2018).

The transient, blue light-dependent downregulation of the calcium channel gene, trp, in day six flies corresponds well with previous observations that mutations in trp suppress blue light-induced retinal degeneration. However, many voltage-gated potassium and chloride channels were also downregulated in response to blue light. Could decreasing activity of potassium or chloride channels ameliorate phototoxicity in flies? Excessive calcium influx is associated with brain ischemia-induced neuronal death, and potassium channel blockers reduced hypoxia-induced neuronal apoptosis in rodent models of ischemia. However, eye-specific knockdown of ATPα, a subunit of a sodium/potassium channel, using the longGMR-Gal4 driver caused age-dependent retinal degeneration in flies. It is currently unclear whether transient repression of other voltage-gated ion channels in photoreceptors could attenuate retinal degeneration under phototoxic conditions (Hall, 2018).

How could exposure to blue light downregulate expression of genes, independent of phototransduction or calcium influx? In Drosophila, the blue light receptor Cryptochrome (Cry) entrains circadian rhythms to light-dark cycles via light-activated degradation of the clock protein Timeless (Tim). Fly photoreceptors possess a functional circadian clock and express PAR-domain protein 1 (Pdp1), tim, and cry. An enrichment of genes involved in circadian rhythm was observed among the blue light-downregulated genes. Regulators of the circadian clock including tim, Pdp1, and vrille (vri) were downregulated in response to blue light in day six, but not day one flies. When the blue light-regulated genes in six-day-old flies were compared with genes showing rhythmic expression patterns in fly heads, it was found that 14 and 24 of the blue light up- and downregulated genes respectively (including trp) overlapped with the 331 genes showing rhythmic expression profiles in heads. While in flies Cry is thought to mainly function by mediating light-dependent degradation of Timeless, some data suggest that Cry also acts as a transcriptional repressor in peripheral circadian clocks because loss of cry and period (per) in the eye leads to ectopic expression of tim. However, increased, rather than decreased, tim levels would be expected following blue light exposure if Cry-mediated transcriptional repression was involved because blue light causes degradation of Cry. Thus, its is proposed that some unknown part of the circadian gene regulatory machinery regulates a light-dependent gene expression program in photoreceptors that attenuates the light response under strong illumination. Other transcription factors such as Kayak, which has a promoter motif in the blue light-upregulated genes, have been shown to affect expression of circadian-regulated genes in pacemaker neurons. It is noted that the design of the current study presents some difficulty in teasing out a potential role for circadian pathway components because it is not possible to readily distinguish between gene expression changes that occur in response to blue light and expression changes that occur in response to dark incubation, which was used as a control for these experiments. The data suggest that the dark incubation does not itself cause major changes in gene expression because day one flies showed very few gene expression changes in response to blue light relative to dark control. Further, the subsets of genes tested by qPCR in dissected eyes showed similar directions of change to the RNA-seq analysis when normalized to a pre-treatment sample. Thus, it is speculated that some components of the circadian machinery are coopted in Drosophila photoreceptors to repress the expression of light response pathway genes in response to strong illumination (Hall, 2018).

Although light is essential for vision, it also poses a stress to photoreceptor cells within the eye. Young flies at 6 days post-eclosion undergo retinal degeneration when exposed to prolonged blue light exposure. This study shows that exposure to blue light induces substantial gene expression changes in photoreceptors from six-day-old flies. In these flies, blue light upregulates stress response pathways and downregulates light response genes to mitigate oxidative stress, and quench the light response. Newly-eclosed flies, which are resilient to blue light-induced retinal degeneration, show no such changes in gene expression. The data suggest that newly-eclosed flies express higher levels of genes that help withstand light stress because of their recent transition from the developing pupal to early adult stage. Together, the results from this study provide insight into neuroprotective pathways utilized by photoreceptors to resist light-induced oxidative stress (Hall, 2018).

Regulation of PI4P levels by PI4KIIIalpha during G-protein coupled PLC signaling in Drosophila photoreceptors

The activation of phospholipase C (PLC) is a conserved mechanism of receptor activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and continued signaling requires the resynthesis and availability of PI(4,5)P2 at the plasma membrane. PI(4,5)P2 is synthesized by the phosphorylation of phosphatidylinositol-4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. This study shows that in Drosophila photoreceptors, PI4KIIIalpha activity is required to support signaling during G-protein coupled PLC activation. Depletion of PI4KIIIalpha results in impaired electrical responses to light and reduced plasma membrane levels of PI4P and PI(4,5)P2. Depletion of conserved proteins Efr3 and TTC7 that assemble PI4KIIIalpha at the plasma membrane also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P2 levels. Thus PI4KIIIalpha activity at the plasma membrane generates PI4P and supports PI(4,5)P2 levels during receptor activated PLC signaling (Balakrishnan, 2018).

LOVIT is a putative vesicular histamine transporter required in Drosophila for vision

Classical fast neurotransmitters are loaded into synaptic vesicles and concentrated by the action of a specific vesicular transporter before being released from the presynaptic neuron. In Drosophila, histamine is distributed mainly in photoreceptors, where it serves as the main neurotransmitter for visual input. In a targeted RNAi screen for neurotransmitter transporters involved in concentrating photoreceptor synaptic histamine, thus study identified an SLC45 transporter protein, LOVIT (loss of visual transmission). LOVIT is prominently expressed in photoreceptor synaptic vesicles and is required for Drosophila visual neurotransmission. Null mutations of lovit severely reduced the concentration of histamine in photoreceptor terminals. These results demonstrate a LOVIT-dependent mechanism, maintaining the synaptic concentration of histamine, and provide evidence for a histamine vesicular transporter besides the vesicular monoamine transporter (VMAT) family (Xu, 2019).

Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq¹ isoform protein playing a major role in fly phototransduction. However, Gαq¹ null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. This study isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, it was found that mutations in the Gαq gene disrupt light responses, and the Gαq3 isoform protein was demonstrated to be responsible for the residual light response in Gαq¹ null mutants. Moreover, this study reports that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation of stable Rh1-arrestin 2 (Arr2) complexes. These findings clarify essential roles for several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis (Gu, 2020).

Heterotrimeric G proteins and G protein-coupled receptors play pivotal roles in mediating a variety of extracellular signals to intracellular signaling pathways, such as hormones, neurotransmitters, peptides, and sensory stimuli. In the Drosophila visual system, light stimulation activates the major rhodopsin (Rh1) to form metarhodopsin, which in turn activates heterotrimeric G proteins and norpA gene-encoded phospholipase C (PLCβ). Activated PLC catalyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 induces the release of Ca2+ from intracellular Ca2+ stores, whereas both DAG and IP3 may trigger extracellular Ca2+ influx by opening transient receptor potential (Trp) and transient receptor potential-like (TrpL) channels on the cell membrane. The Gαq gene encodes several Gαq splice variants, among which the Gαq-RD variant generates Gαq¹ isoform protein, and other splice variants generate Gαq3 isoform protein. Although both strong alleles of norpA and trpl;trp double mutants show completely abolished photoresponses, the Gαq¹ null mutant allele (Gαq⁹⁶¹) still displays a residual light response. These data indicate that other Gαq splice variants, or the Gq α subunits encoded by additional genes, contribute to the residual light responses in Gαq¹ null mutants (Gu, 2020).

Intracellular Ca2+ homeostasis controlled by Gq signaling is also essential for photoreceptor cell survival. Mutations in phototransduction cascade components, such as those in trp and norpA, prevent normal light-induced Ca2+ influx, resulting in stable Rh1/Arr2 complex formation and severe rapid light-dependent retinal degeneration. Disruption of stable Rh1/Arr2 complexes by genetic removal of Arr2 or suppression of Rh1 endocytosis can suppress the retinal degeneration either in norpA or trp mutant flies. Rh1/Arr2 complex formation is thought to contribute to impaired Ca2+ influx-activated CaM kinase II, which usually phosphorylates Arr2 to release Arr2 from Rh1. However, neither Gαq¹ nor Gαq⁹⁶¹ mutants undergo rapid retinal degeneration, exhibiting only slight retinal degeneration after keeping them in 12-h light/12-h dark cycles for 21 days. The disparate retinal degeneration phenotype between Gαq and norpA mutant is therefore unclear (Gu, 2020).

This study isolated a mutant fly with no detectable light responses and revealed that mutations in the Gαq gene cause the defective light responses. Gαq3 is responsible for the residual light response in Gαq¹ null mutants, and depletion of all Gαq splice variants results in rapid light-dependent retinal degeneration due to formation of stable Rh1/Arr2 complexes. In addition, this study revealed that Gαq3 plays essential roles in Rh1 synthesis. This study clarifies the essential role of different Gαq splice variants in fly phototransduction, retinal degeneration, and rhodopsin synthesis (Gu, 2020).

In Drosophila photoreceptors, G proteins are essential to activate the phototransduction cascade. The Gαq gene encodes several Gαq splice variants, and Gαq¹ has been shown to function as the predominant G protein in fly phototransduction. This study identified a mutation (5501T/A) in the Gαq gene, which specifically mutates Val to Asp at residue 303 in Gαq¹ but not Gαq3 isoforms. Although Val is replaced with Ile at residue 303 in vertebrate Gαq proteins, the hydrophobicity at this position is evolutionally conserved. Structural analyses have shown that the V303 region localizes to the interface between Gα proteins and its downstream effector PLC. The change of a hydrophobic residue to a polar one may affect the interaction between these two proteins. A recent study has shown that Gαq^V303D mutant protein is unable to activate PLC in vivo (Gu, 2020).

Although the 5501T/A Gαq gene mutation largely contributes to abolished light responses, this mutation is not fully responsible for the abolished light responses in no detectable light response (nlr) mutants because both nlr/Gαq¹ and nlr/Gαq⁹⁶¹ flies still exhibited a residual light response similar to Gαq¹ and Gαq⁹⁶¹ mutants. These data also excluded the possibility that Gαq^V303D mutant protein dominantly suppresses the function of Gαq protein. Gαq¹ expression in nlr mutants largely recovers the light response, further excluding the possibility that abolished light responses in nlr mutants are due to the dominant suppression of Gαq^V303D mutant protein (Gu, 2020).

The Gαq gene encodes several Gαq splice variants, and Gαq^221c mutants disrupt the expression of all Gαq splice variants (21). An ERG recording revealed that Gαq^221c null mutant clones showed no light responses. Previous whole-cell voltage-clamp recordings showed that the photoresponse of Gαq¹ homozygous cells is larger than that of Gαq¹ heterozygous cells. These results indicate that other Gαq splice variants might contribute to the residual light response in Gαq¹ null mutants. This study demonstrates that Gαq3 contributes to the residual light response in Gαq¹ null mutants (Gu, 2020).

The Gαq gene encodes several Gαq splice variants. Originally, two cDNAs resulting from different Gαq gene splicing were isolated. These two cDNAs encode Gαq1 and Gαq2 isoform proteins, respectively. Functional studies demonstrated that Gαq1 mediates the light response, whereas Gαq2 has no effect on phototransduction. Subsequently, two additional Gαq splice variants were isolated. To date, seven total Gαq splice variants have been annotated in Flybase, and these splice variants encode three different isoform proteins, including Gαq1, Gαq3, and Gαq4. This study has demonstrated that Gαq3 also mediates phototransduction. Overexpression of Gαq3 in nlr mutants induced detectable light responses but failed to fully restore the light response. Interestingly, the rescue flies exhibited comparable ERG trace amplitude and dynamics as those of Gαq¹ and Gαq⁹⁶¹ flies. These results indicate that different Gαq isoform proteins play different roles in phototransduction. Gαq mediates retinal degeneration (Gu, 2020).

Mutations in most genes encoding components of the phototransduction cascade result in rapid retinal degeneration, except for Gαq hypomorphic allele Gαq¹ and Gαq¹ isoform null mutant allele Gαq⁹⁶¹. Previous studies have shown that both Gαq¹ and Gαq⁹⁶¹ mutants undergo slow light-dependent retinal degeneration due to slow accumulation of stable Rh1/Arr2 complexes. In these Gαq mutants, the small residual photoresponse may reduce Ca2+ influx, which partially activates CaM kinase II and leads to the slow release of Arr2 from Rh1. This study shows that nlr mutants undergo rapid light-dependent retinal degeneration similar to that observed in norpA mutants. Disruption of stable Rh1/Arr2 complexes formation prevented retinal degeneration in the mutants. Under normal conditions, the interaction between Arr2 and Rh1 is transient, because light-triggered Ca2+ influx may activate CaM kinase II, which subsequently phosphorylates Arr2 to release Arr2 from Rh1. In nlr mutants, photoresponses were completely abolished so that the normal rise in Ca2+ after light stimulation was blocked, causing stable Rh1/Arr2 complex formation and retinal degeneration. These observations and explanations are consistent with mutations such as trp and norpA (Gu, 2020).

This study has shown the first evidence that Gαq3 regulates Rh1 synthesis. Rh1 is transported to the plasma membrane by vesicular transport mechanisms regulated by a large number of trafficking proteins. Previous studies have shown that Gαq homologue CG30054 regulates inositol 1,4,5,-tris-phosphate receptor (IP3R) to mediate calcium mobilization from intracellular stores and promote calcium-regulated secretory vesicle exocytosis. Given that Gαq3 shows high sequence identity to CG30054, they may regulate Rh1 synthesis through promoting calcium-regulated secretory vesicle exocytosis (Gu, 2020).

The spectral sensitivity of Drosophila photoreceptors

Drosophila melanogaster has long been a popular model insect species, due in large part to the availability of genetic tools and is fast becoming the model for insect colour vision. Key to understanding colour reception in Drosophila is in-depth knowledge of spectral inputs and downstream neural processing. While recent studies have sparked renewed interest in colour processing in Drosophila, photoreceptor spectral sensitivity measurements have yet to be carried out in vivo. This study has fully characterised the spectral input to the motion and colour vision pathways, and directly measured the effects of spectral modulating factors, screening pigment density and carotenoid-based ocular pigments. All receptor sensitivities had significant shifts in spectral sensitivity compared to previous measurements. Notably, the spectral range of the Rh6 visual pigment is substantially broadened and its peak sensitivity is shifted by 92 nm from 508 to 600 nm. This deviation can be explained by transmission of long wavelengths through the red screening pigment and by the presence of the blue-absorbing filter in the R7y receptors. Further, direct interactions between inner and outer photoreceptors were tested using selective recovery of activity in photoreceptor pairs (Sharkey, 2020).

Calmodulin binds to Drosophila TRP with an unexpected mode

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca(2+) mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca(2+) modulated CaM/TRP interaction is poorly understood. This study discovered an unexpected, Ca(2+)-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca(2+)-mediated feedback on the channel activity. Finally, TRPC4, the closest mammalian paralog of Drosophila TRP, was found to adopt a similar CaM binding mode (Chen, 2020).

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca²⁺-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca²⁺ stores in this important model for Ca²⁺ signaling remains obscure. A low affinity Ca²⁺ indicator (ER-GCaMP6-150) was expressed in the ER, and its fluorescence was measured both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau approximately 0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca²⁺ stores, followed by a slower decay, typically reaching approximately 50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP₃ receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na⁺ and in mutants of the Na⁺/Ca²⁺ exchanger, CalX, which was immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na⁺/Ca²⁺ exchange across the ER membrane induced by Na⁺ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na⁺/Ca²⁺ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca²⁺ in photoreceptors exposed to Ca²⁺-free solutions (Liu, 2020).

Phototransduction in microvillar photoreceptors is mediated by a G-protein-coupled phospholipase C (PLC), which hydrolyzes phosphatidyl inositol (4,5) bisphosphate (PIP₂) to generate diacylglycerol and inositol (1,4,5) trisphosphate (InsP₃). In Drosophila photoreceptors, activation of PLC leads to opening of two related Ca²⁺-permeable nonselective cation channels: TRP (transient receptor potential) and TRP-like (TRPL) in the microvillar membrane. TRP is the founding member of the TRP ion channel superfamily, so named because the light response in trp mutants is transient, decaying rapidly to baseline during maintained illumination. Because the most familiar product of PLC activity is InsP₃, it was initially thought that activation of the TRP/TRPL channels required release of Ca²⁺ from endoplasmic reticulum (ER) stores via InsP₃ receptors (InsP₃Rs) and that in the absence of Ca²⁺ influx via TRP channels the stores depleted leading to the response decay. However, it was subsequently found that phototransduction was intact in InsP₃R mutants, whereas response decay in trp mutants was associated with severe depletion of PIP₂. This suggested an alternative explanation of the trp decay phenotype, namely failure of Ca²⁺-dependent inhibition of PLC and the consequent runaway consumption of its substrate, PIP₂. Nevertheless, a role for InsP₃ and Ca²⁺ stores in Drosophila phototransduction remains debated. For example, a recent study reported that sensitivity to light was attenuated by RNAi knockdown of InsP₃R , although this study was unable to confirm this using either RNAi or null InsP₃R mutants (Bollepalli, 2017; Liu, 2020).

Relevant to this debate, Ca²⁺ imaging reveals a small, but significant light-induced rise in cytosolic Ca²⁺ in photoreceptors bathed in Ca²⁺-free solutions. Although some have attributed this to InsP₃-induced Ca²⁺ release from the ER, it was found that the rise was unaffected in InsP₃R mutants but was dependent on Na+/Ca²⁺ exchange (Hardie, 1996; Asteriti, 2017; Bollepalli, 2017). This suggested that the Ca²⁺ rise was due to Na+/Ca²⁺exchange following Na+ influx associated with the light response. However, it is difficult to understand how such a Ca²⁺ rise could be achieved by Na+/Ca²⁺ exchange across the plasma membrane when extracellular Ca²⁺ was buffered to low nanomolar levels. The source of the Ca²⁺ rise in Ca²⁺-free bath thus remains unresolved, and to date there have been no measurements of ER store Ca²⁺ levels in Drosophila photoreceptors. To address this, flies were generated expressing a low-affinity GCaMP6 variant in the ER lumen. Using this probe, a rapid light-induced depletion of ER Ca²⁺ was demonstrated and characterized, which, like the cytosolic Ca²⁺ signal in Ca²⁺-free bath, was unaffected by InsP₃R mutations, but dependent on Na+ influx and the CalX Na+/Ca²⁺ exchanger. These results indicate that the exchanger is also expressed on the ER membrane, that the Na+ influx associated with the light-induced current leads to Ca²⁺ extrusion from the ER by Na+/Ca²⁺exchange and that this accounts for the rise in cytosolic Ca²⁺ observed in Ca²⁺-free solutions (Liu, 2020).

This study measured ER Ca²⁺ levels using a low affinity GCaMP6 variant targeted to the photoreceptor ER lumen, where it generated bright fluorescence throughout the ER network. The probe (ER-GCaMP6-150), originally developed and expressed in mammalian neurons, has a 45-fold dynamic range, which was confirmed in situ, and allows measurements of ER luminal [Ca²⁺] with excellent signal-to-noise ratio. Not only could ER Ca²⁺ levels be monitored in dissociated ommatidia, it was also straightforward to make in vivo measurements from the eyes of completely intact flies. The results demonstrate rapid light-induced, PLC-dependent depletion of the ER Ca²⁺ stores, which refilled in the dark over a time course of 100-200 s (Liu, 2020).

Strikingly the results indicate that the rapid light-induced store depletion was mediated by Na+/Ca²⁺ exchange. Drosophila CalX belongs to the NCX family of Na+/Ca²⁺ exchangers, which are generally considered to act only at the plasma membrane. Although Drosophila CalX clearly does function at the plasma membrane, the results now provide compelling evidence that it also operates across the ER membrane. NCX activity has not previously been reported on the ER; however, Na+/Ca²⁺ exchange on internal membranes is not without precedent: for example NCX has been reported on the inner nuclear membrane providing a route for Ca²⁺ transfer between nucleoplasm and the nuclear envelope and hence ultimately the ER network with which it is continuous. In addition a dedicated mitochondrial Na+/Ca²⁺ exchanger (NCLX) plays important roles in uptake and release of mitochondrial Ca²⁺ (Liu, 2020).

The time course of the Na+/Ca²⁺-dependent rapid store depletion in Ca²⁺-free solutions appeared very similar to the rise in cytosolic Ca²⁺ reported from dissociated ommatidia in Ca²⁺-free bath, the source of which has been a subject of debate for over 20 years. It had recently been claimed that this 'Ca²⁺-free rise' was due to InsP₃-mediated release from ER Ca²⁺ stores; however, it was found that it was unaffected in null mutants of the InsP₃R. Instead, it was found that the Ca²⁺-free cytosolic rise was dependent on Na+/Ca²⁺ exchange, but it was difficult to understand how this could be mediated by a plasma membrane exchanger when extracellular Ca²⁺ was buffered with EGTA to low nanomolar levels. The demonstration of rapid Na+/Ca²⁺-dependent release of Ca²⁺ from ER with a very similar time course now provides an obvious mechanism for this Ca²⁺-free rise and seems finally to have resolved this long-standing enigma. Interestingly the Na+/Ca²⁺-dependent rapid store depletion signal was most pronounced in very young flies around the time of eclosion. Also of note, it was found that trp mutants were very resistant to depletion, both in vivo and in dissociated ommatidia. This argues strongly and directly against the hypothesis that the trp decay phenotype reflects depletion of the ER Ca²⁺ stores (Liu, 2020).

Although up to ~80% rapid store depletion could be observed in newly eclosed adults, even in 1-d-old flies the rapid store depletion signal in vivo was much reduced (to ~10%). However, a much slower depletion was observed in mature adults in vivo, and in dissociated ommatidia after Na+/Ca²⁺ exchange was blocked. The origin of this slow phase depletion remains uncertain: in dissociated ommatidia from young flies this slower depletion was ~50% attenuated, but not blocked in null InsP₃R mutants (itpr), whereas in vivo measurements of the slow depletion phase in adult itpr mutants appeared similar to wild-type. This suggests that although Ca²⁺ release via InsP₃ receptors may contribute to the slow depletion in young flies, some other mechanism(s), such as Ca²⁺ release via ryanodine receptors, is largely responsible (Liu, 2020).

This evidence strongly suggests a novel role for NCX exchangers in mediating Na+/Ca²⁺ exchange across the ER membrane, but its physiological significance is unclear. Although rapid store depletion was routinely observed under experimental conditions used in this study, the Ca²⁺ released into the cytosol from the ER seems unlikely to play a direct role in phototransduction. First, it has a latency of ~100 ms (cf. ~10 ms for the light-induced current), and second it will in any case be swamped by the much more rapid Ca²⁺ influx via the light-sensitive channels. Thus measurements of cytosolic Ca²⁺ in 0 Ca²⁺ bath indicated a rise to only ~200-300 nm. This compares with much faster rises in the high micromolar range due to direct Ca²⁺ influx via the light-sensitive TRP channels. One possible role for an ER Na+/Ca²⁺ exchanger would be that it normally operates as a Ca²⁺ uptake mechanism and only briefly giving Ca²⁺ extrusion (and store depletion) following the extreme, and unnatural conditions of many of the current experiments. This the sudden onset of bright illumination from a dark-adapted state, which results in a massive transient surge of Na+ influx. Rapid Ca²⁺ uptake (store refilling), presumably via re-equilibration of the exchanger as the initial Na+ level subsided during the peak-to-plateau transition, was in fact routinely observed during maintained blue illumination. Furthermore, it is perhaps significant, that despite lacking the rapid depletion phase, the final level of store Ca²⁺ (i.e., after 30 s illumination) in calx^A mutants was if anything lower than that in wild-type backgrounds, although the cytosolic Ca²⁺ levels experienced in calx^A mutants are much higher because of the failure to extrude Ca²⁺ across the plasma membrane (Liu, 2020).

Although store depletion seems unlikely to contribute to activation of the phototransduction cascade, the possibility cannot be excluded that it may play some role in long-term light adaptation. Maintenance of ER Ca²⁺ levels is also important for many other cellular functions including protein folding and maturation in which Ca²⁺ is a cofactor for optimal chaperone activity. With conspicuously high cytosolic Ca²⁺ levels in the presence of light, photoreceptors face unusual homeostatic challenges and Na+/Ca²⁺ exchange across the ER may provide an important additional mechanism. In principle the balance between forward and reverse Na+/Ca²⁺ exchange (i.e., uptake vs release) by an ER Na+/Ca²⁺ exchanger will depend on the Na+ gradient across the ER membrane and whether this is actively regulated. There is no information on ER Na+ levels, although luminal Na+ in the nuclear envelope (which is continuous with the ER) has been reported to be concentrated (84 mm) in nuclei from hepatocytes by Na/K-ATPase expressed on nuclear membranes. The possibility that Na+/Ca²⁺ exchange across the ER might play only a minor physiological role cannot be excluded, but is an unavoidable consequence of the presence of functional CalX protein in ER membranes during protein synthesis and targeting. At least this may account for the enhanced depletion signal measured around the time of eclosion when there may be a rapid final phase of protein synthesis for the developing rhabdomere (Liu, 2020).

These results provide unique insight into ER Ca²⁺ stores in Drosophila photoreceptors. The ER-GCaMP6-150 probe lights up an extensive ER network and indicates a high luminal Ca²⁺ concentration probably in excess of 0.5 mm. The results reveal a rapid, and uniform light-induced depletion of the ER stores mediated by the CalX Na+/Ca²⁺ exchanger expressed on the ER membrane. The resulting extrusion of Ca²⁺ into the cytosol can readily account for the rise in cytosolic Ca²⁺ observed in dissociated ommatidia in Ca²⁺-free solutions), thus resolving this decades old mystery. In addition to the rapid depletion, a much slower depletion was also resolved that appears to be independent of Na+/Ca²⁺ exchange and also largely independent of InsP₃-induced Ca²⁺ release. The physiological significance of the ER Na+/Ca²⁺ exchange activity remains uncertain. It is perhaps more likely that it serves as a low affinity Ca²⁺ uptake mechanism supplementing the SERCA pump, and that rapid depletion is only seen during unnatural abrupt bright stimulation from dark-adapted backgrounds leading to massive Na+ influx and reverse exchange. Ultimately, to resolve the physiological significance of Na+/Ca²⁺ exchange across the ER membrane it will probably be necessary to selectively disrupt Na+/Ca²⁺ exchange on the ER without affecting the exchanger on the plasma membrane, which is known to play very important roles in Ca²⁺ homeostasis in the photoreceptors with direct consequences for channel activation and adaptation (Liu, 2020).

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. This study generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with approximately 1200 effectively absorbed photons and saturated (DeltaF/F0 approximately 10-20) with 10000-30000 photons. In Ca²⁺ free bath, smaller (DeltaF/F0 approximately 4), long latency (~ 200ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10nM (DeltaF/F0 approximately 0.1), and unlikely to play any role in phototransduction (Asteriti, 2017).

Although Ca²⁺ signals in Drosophila photoreceptors were first studied over 20 years ago using Ca²⁺ indicator dyes, only one, recent study had used genetically encoded Ca²⁺ indicators. That study measured signals from dissociated ommatidia using the Gal4-UAS system, combining UAS-GCaMP6f with GMR-Gal4, which drives expression throughout the retina including all photoreceptor classes as well as accessory cells such as pigment and cone cells. GMR-Gal4 expression also causes significant abnormalities in photoreceptor structure and physiology. In the present study, flies were generated in which GCaMP6f expression was driven directly via the Rh1 (ninaE) promoter ensuring exclusive expression in R1-6 photoreceptors with wild-type morphology and physiology. The excellent signal-to-noise ratio of recordings in ninaE-GCaMP6f flies was distinctly superior to that in GMR-Gal4/UAS-GCaMP6f flies, and in many cases the maximum Δ/F0 ratio approached or exceeded 20 (cf ~3 using GMR-Gal4/UAS-GCaMP6f). This is close to the maximum value (23.5) determined by in situ calibrations or in vitro. Although the blue excitation light used for measuring GCaMP6f fluorescence is a super-saturating stimulus, 2-pulse paradigms allowed sensitive and accurate measurements of intensity and time dependence of signals in response to stimuli in the physiological range. Recordings in vivo from the deep pseudopupil (DPP) of intact flies are simple to perform and can be readily maintained over many hours, making this approach a valuable, and completely non-invasive tool for assessing in vivo photoreceptor performance. Even in the more vulnerable dissociated ommatidia preparation, multiple repeatable measurements could be made for up to at least an hour from the same ommatidium as long as metarhodopsin was reconverted to rhodopsin by long wavelength light after each measurement (Asteriti, 2017).

In vivo (DPP) or in dissociated ommatidia bathed in physiological solutions, the GCaMP6f signal reached 50% Fmax at intensities equivalent to ~1000-2500 effectively absorbed photons. It is believed that the elementary single photon response (quantum bump) is generated by activation of Ca²⁺ permeable channels (TRP and TRPL) within a single microvillus and that the consequent Ca²⁺ rise in the affected microvillus reaches near mM levels. Because such levels inevitably saturate GCaMP6f (Kd 290 nM, saturating at 1-2 μM), to a first approximation the Δ/F0 values under physiological conditions are probably best interpreted as the proportion of microvilli 'flooded' with Ca²⁺. In total, the rhabdomere contains ~30000 microvilli, meaning that 50% Fmax is reached when only ~3-8% of the microvilli have been activated by a photon. This implies that the Ca²⁺ influx into a single microvillus must spread to at least the immediately neighbouring microvilli within the timeframe of the response. In ninaE-calx flies over-expressing the Na+/Ca²⁺ exchanger, or in trp mutants lacking the major Ca²⁺ permeable channel, 50% Fmax was only obtained with flashes containing ~12000-15000 effective photons. This should activate ~50% of the microvilli, suggesting that in these flies Ca²⁺ is largely prevented from spreading to neighbouring microvilli under the same conditions (Asteriti, 2017).

The dark-adapted 'pedestal' level can be used to gain an estimate of the resting Ca²⁺ concentration in dissociated ommatidia (in physiological solutions) assuming in vitro calibration data. With reference to F0 measured in Ca²⁺ free solution in the same ommatidia, the mean dark-adapted value in normal bath was 0.77 ± 0.14 (mean ± S.E.M. n = 11). This would be equivalent to ~80 nM (assuming Kd = 290 nM and Fmax 23.5). This value was significantly lower in ninaE-calx flies over-expressing the exchanger (0.19 ± 0.04 n = 11 equivalent to ~50 nM) and higher in calx¹ mutants (1.94 ± 0.24 n = 14 equivalent to ~120 nM) (Asteriti, 2017).

The recovery of GCaMP6f fluorescence to baseline is likely to be a reasonably accurate reflection of the falling Ca²⁺ levels during response recovery, although the initial decrease (from initial ~mM levels to low μM levels) will still be subject to saturation effects. With relatively dim flashes (up to ~1000 effectively absorbed photons) the GCaMP6f signal in wild-type backgrounds fell to near baseline within ~2-3 s with a half time (t 1/2) of ~1 s. This is slower than the GCaMP6f off-rate (~200 ms), and thus likely to approximate the true time-course of Ca²⁺ recovery. The recovery was significantly accelerated in ninaE-calx flies (~500 ms), and slowed in calx¹ mutants (~2 s increasing to >10 s following brighter flashes), consistent with a dominant role of the Na+/Ca²⁺ exchanger in Ca²⁺ extrusion. Nevertheless, even after bright flashes, given sufficient dark-adaptation time (~30-60 s), resting [Ca²⁺] in calx¹ mutants fell to levels close to those in dark-adapted wild-type photoreceptors, reflecting either residual function of the exchanger in this hypomorphic mutant and/or alternative extrusion mechanism(s) (Asteriti, 2017).

The smaller signals recorded in Ca²⁺ free bath fall within the dynamic range of GCaMP6f and allow estimates of the absolute Ca²⁺ levels reached under these conditions (e.g., Δ/F0 of 6 corresponds to ~200 nM). These signals were used to investigate the long disputed origin of the light-induced rise in cytosolic Ca²⁺ in Ca²⁺ free solutions. Originally, using INDO-1, it was found that this Ca²⁺ free rise was dependent upon extracellular Na+ and suggested that the rise might be due to re-equilibration of Na+/Ca²⁺ exchange in response to the massive light-induced Na+ influx that persists under these conditions. This was challenged by by a study that confirmed the requirement of external Na+ for a significant Ca²⁺ rise in Ca²⁺ free solutions, Na²⁺, but reported that a rise still occurred in Ca²⁺ free bath in the presence of Na+ when the photoreceptors were voltage clamped at the Na+ equilibrium potential to prevent Na+ influx. It was concluded that a Na+ gradient − but not influx − was required, that the Ca²⁺ free rise reflected release from internal stores, and that the requirement of extracellular Na+ reflected involvement of some other Na+ dependent process, such as Na/H transport. But how this might affect release of Ca²⁺ from intracellular stores is far from clear. A more recent study reported that the Ca²⁺ free rise was attenuated following RNAi knockdown of the IP3R. However, this is difficult to reconcile with an earlier study using INDO-1, where the rise was found to be unaffected in null IP3R mosaic eyes and confirmed again in this study using GCaMP6f (Asteriti, 2017).

This study used a variety of approaches to investigate the source of this Ca²⁺ free signal further. It was first confirmed that it was all but abolished in the absence of external Na+, whether substituted for Li+, Cs+, K+ or NMDG+. Importantly, it was found that the rise was also effectively eliminated in trpl;trp double mutants both in vivo and in dissociated ommatidia despite the presence of normal extracellular solutions containing both Na+ and Ca²⁺. Although it might be argued that, for some reason, PLC activity (and hence InsP3 generation) was compromised in trpl;trp mutants, convincing evidence indicates that net PLC activity is in fact greatly enhanced in trpl;trp due to the lack of Ca²⁺ and PKC dependent inhibition of PLC. Thus the rate and intensity dependence of PIP2 hydrolysis, measured using GFP-tagged PIP2 binding probes are greatly enhanced in trpl;trp mutants, as are the PLC-induced photomechanical contractions, and the acidification due to the protons released by the PLC reaction. Overall, therefore these results strongly suggest that Na+ influx is indeed required for the Ca²⁺ free rise. Crucially, the involvement of the Na+/Ca²⁺ exchanger in this rise was confirmed by finding that it was essentially eliminated in an exchanger mutant (calx¹), but greatly accelerated in ninaE-calx photoreceptors over-expressing the exchanger (Asteriti, 2017).

The question remains, how Na+/Ca²⁺ exchanger activity could generate such a sizeable Ca²⁺ signal (~100-200 nM) in cells perfused with EGTA buffered solutions, when free Ca²⁺ in the bath should be reduced to low nM levels. There is no unequivocal answer to this, and assuming the standard equation for the Na+/Ca²⁺ exchange equilibrium it would seem difficult for reverse Na+/Ca²⁺ exchange to raise Ca²⁺ into the range that was observed. However, at least three, not mutually exclusive factors might result in higher cytosolic Cai levels than predicted. Firstly, external Ca²⁺ might be relatively resistant to buffering in the intra-ommatidial space, and specifically the extremely narrow spaces between the microvilli or their bases, where the exchanger is believed to be localised. For example, with 500 nM Ca_o remaining, it is predicted that 130 nM Cai would be reached with 70 mM Na_i, 110 mM Na_o and the cell depolarised to 0 mV (values that could realistically be reached with the huge inward Na+ currents flowing under these conditions). Although one might also expect Ca²⁺ influx via the light-sensitive channels at such Ca_o concentrations, experiments buffering external Ca²⁺ at different concentrations with EGTA showed that direct Ca²⁺ influx signals could only be detected once external Ca²⁺ was raised above ~400 nM. Secondly, resting cytosolic Ca²⁺ concentration is determined not only by the exchanger, but also by any other Ca²⁺ fluxes, which might include tonic leakage from intracellular compartments such as endoplasmic reticulum (ER) or mitochondria. Massive Na+ influx would compromise the ability of the exchanger to counter any such fluxes. A third possibility is that, contrary to conventional dogma, the exchanger might also be expressed on intracellular membranes of endoplasmic reticulum or other Ca²⁺ containing compartments and that Na+ influx leads to re-equilibration of Na+/Ca²⁺ exchange across these (Asteriti, 2017).

Whatever the exact mechanism, the results indicate that the Ca²⁺ rise in Ca²⁺ free bath is strictly dependent upon both Na+ influx and the activity level of the Na+/Ca²⁺ exchanger, but unaffected in null IP3R mutants. Its time-course, with no detectable rise for ~200 ms, also appears much too slow to play any role in initiating the light response, which has a latency of ~10 ms and peaks within ~100-200 ms in response to bright illumination even under Ca²⁺ free conditions. The residual GCaMP6f signal remaining in the absence of Na+ influx and/or in the absence of Na+/Ca²⁺ exchanger activity − whether achieved by Na+ substitution, trpl;trp or calx mutants − was also still observed in IP3R mutants and was so small that it is questionable whether it reflects a Ca²⁺ signal. Because of the rapid inhibition of PLC by Ca²⁺ influx under physiological conditions any presumptive PLC-mediated Ca²⁺ release under physiological conditions would be even less. Together with a study in which no phototransduction defects were found in null IP3R mutants, these results suggest that InsP3-induced Ca²⁺ release plays no significant role in Drosophila phototransduction (Asteriti, 2017).

Suppression of motion vision during course-changing, but not course-stabilizing, navigational turns

From mammals to insects, locomotion has been shown to strongly modulate visual-system physiology. Does the manner in which a locomotor act is initiated change the modulation observed? Patch-clamp recordings were performed from motion-sensitive visual neurons in tethered, flying Drosophila. This study observed motor-related signals in flies performing flight turns in rapid response to looming discs and also during spontaneous turns, but motor-related signals were weak or non-existent in the context of turns made in response to brief pulses of unidirectional visual motion (i.e., optomotor responses). Thus, the act of a locomotor turn is variably associated with modulation of visual processing. These results can be understood via the following principle: suppress visual responses during course-changing, but not course-stabilizing, navigational turns. This principle is likely to apply broadly-even to mammals-whenever visual cells whose activity helps to stabilize a locomotor trajectory or the visual gaze angle are targeted for motor modulation (Fenk, 2021).

Different spectral sensitivities of ON- and OFF-motion pathways enhance the detection of approaching color objects in Drosophila

Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. This study demonstrates an unexpected, additional contribution of color to motion vision in Drosophila. Behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and cellular pathways were identified connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and it was shown how this could generalize for systems with ON- and OFF-motion pathways. The results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects (Longden, 2023).

Color and motion are two visual cues used by many species to identify salient moving objects. They are processed largely independently, for example along the ventral and dorsal pathways, respectively, in primates. However, color contributes to motion perception in humans and other primates, as indicated by psychophysical experiments using chromatic stimuli lacking luminance contrast allowing the edge motion of objects to be detected even when the background illumination changes to match the luminance of the object. By identifying cellular pathways for color contributing to motion processing in Drosophila, this study demonstrates how color can additionally boost the motion detection of specifically colored objects, without, surprisingly, improving the motion detection of differently colored objects with the same chromatic contrast (Longden, 2023).

Like primates, color and motion are initially largely processed along separate pathways in Drosophila, and their motion vision was not thought to be influenced by color vision based on studies using blue and green wavelengths. However, connectomic studies found that the photoreceptors used for color vision are also connected to cells in the motion pathway, and a study using sophisticated genetic manipulations indicated that color inputs expand the spectral range of motion vision through unknown cellular mechanisms. Drosophila forages on fruits, flowers, and water droplets that can all have a UV reflectance distinct from background foliage, and UV illumination is used commercially to identify ripe or damaged citrus fruit17. Therefore how color might contribute to object motion vision was explored using the behavioral and cellular responses of Drosophila to objects defined by UV-green color edges (Longden, 2023).

Drosophila has a visual system well suited to parsing UV and green components of the visual scene. Under every eye facet (ommatidium) are eight photoreceptor). The outer six, R1-6, are sensitive to a broad range of wavelengths, especially UV and green. They provide a luminance signal for pathways processing motion vision and also contribute to color vision. The inner two, R7 and R8, provide additional wavelength sensitivity for color vision, and also contribute to luminance processing. The R7 and R8 neurons are paired and come in two flavors, determined by the rhodopsins expressed, across most of the eye: one sensitive to short wavelength UV and blue respectively (in so-called pale ommatidia), and the other to long wavelength UV and green (in so-called yellow ommatidia). R1-6 and R7-8 project to different visual neuropils, the lamina and medulla, respectively, and like vertebrates, flies have visual processing pathways for the movement of bright edges relative to the background (ON-motion), and dark edges relative to the background (OFF-motion). The T4 and T5 cell types are directionally selective for ON- and OFF-motion, respectively2, and the circuitry connecting the photoreceptors to T4 and T5 neurons has been described in considerable detail (Longden, 2023).

To test if color contributes to motion vision in flies, this study developed a custom projector to display spatially precise UV and green patterns that corrected for impedimentary short-wave scattering; then behavioral responses were measured to expanding discs that varied between dark and bright UV with a green background. Flies responded to all intensities of UV discs, indicating that motion vision is not driven by a single luminance channel alone and that color therefore contributes. Notably, responses to ON-motion were much more sensitive to UV than those to OFF-motion in a range of Drosophila species. To identify the cells responsible, genetic reagents were developed to manipulate the function of classes of photoreceptors, and to identify cells downstream of R7, two-photon calcium imaging of neuronal activity and genetic silencing in behavioral experiments were used. Analysis of these results generated the counterintuitive prediction that the contribution of color to motion vision would not support the detection of green discs seen against UV, even though both UV and green discs have the same chromatic contrast, and remarkably, this was the case. Finally, this study determined that the mechanism can be employed in other visual systems to detect objects of other colors. These findings identify neurons linking photoreceptors required for color vision to the different spectral sensitivities of the motion vision pathways. They show how the mechanism can be selective for UV objects in Drosophila, and can be employed for color-selectivity in other visual systems (Longden, 2023).

This study has shown that color contributes to motion vision for UV-green edges in Drosophila. Behavioral responses to ON-motion were much more sensitive to UV than responses to OFF-motion , a difference requiring the R7-8 photoreceptors. The T4 and T5 cells that process ON- and OFF-motion, showed a corresponding difference in their sensitivity to UVe, and in the cells linking the R7-8 photoreceptors and T4 cells, there were consistent spectral differences between lamina monopolar cells and the medulla T4 input cells: L5 and Mi4 were less sensitive to UV, and L3 and Mi9 were more sensitive to UV, compared to the L1 driven Mi1 and Tm3. Silencing cells with divergent isoluminance levels (L3, Mi4 and Mi9) also reduced the difference between the ON- and OFF-motion isoluminance levels, indicating a causal role in behavioral tests. This study correctly predicted that if the augmented UV-sensitivity of ON-motion processing explained the contribution of color to motion vision, then green discs should not be visible against a UV background that was neither bright nor dark. Finally, the contribution of color to motion vision was shown to be not just a mechanism for resolving low-contrast UV edges, but can also be organized to preferentially support the motion detection of objects of specific colors (Longden, 2023).

Previous studies have shown that motion vision is colorblind for blue-green gratings in flies. It is proposed that these studies did not observe a contribution of color to motion vision because gratings induce both ON- and OFF-motion, so differences between these pathways cannot be isolated, and because blue stimuli only weakly drive the UV-sensitive R7 photoreceptors. That work was extended by not using gratings, and by developing methods to accurately display wide-field UV-green stimuli. Prior work also indicated that color might contribute to motion vision by broadening the spectral sensitivity of the luminance channel through unidentified cellular mechanisms, and subsequent EM reconstructions indicated that the R7 and R8 photoreceptors form synapses in the medulla with cells specifically presynaptic to T4. These results indicate that UV-sensitivity is maintained along the R7-L3-Mi9-T4 pathway and predict that R7 cells innervate L3. Indeed, it was recently demonstrated in an EM reconstruction study that R7 cells form substantial numbers of previously unreported synapses with L3 and other cells in the optic chiasm between the lamina and medulla. L3 has been recently identified as critical for encoding luminance information, particularly in dim light conditions. These experiments used only a restricted range of luminance levels, and was focused on providing a tight linkage between visual stimuli used for behavior and imaging. It was demonstrated that the UV-sensitivity of behavioral ON-motion responses scaled in proportion to a doubling of the green channel luminance, but it will be important to establish if the bright luminance of full daylight alters the effects: it is possible that UV augments the detection of approaching color objects only during the dawn and dusk periods favored by Drosophila melanogaster (Longden, 2023).

In future work it will be possible to causally test the contribution of cell types to UV-sensitivity along the ON-motion pathway by using multiple expression control systems to silence a cell type and independently image from downstream cells. However, understanding how sensitivity to UV propagates from R7 through the lamina and medulla circuitry to T4 cells is complex due to sign changes, asymmetric spectral tuning, and recurrent connections along the pathway. To focus on just one example, the Mi4 and Mi9 cell types, which are inhibitory to T4 cells, heavily synapse onto each other and reciprocal inhibition between these cell types may amplify their chromatic differences (Longden, 2023).

Although this study used expanding discs that triggered aversive turns, it is not thought that color motion vision is specifically adapted for predator evasion, particularly because it was possible to carefully map the UV-sensitivity of ON- and OFF-motion using moving edges. Flies are attracted to UV using motion-independent visual pathways and using a stimulus that generated aversive turns allowed ensuring that UV phototaxis was not masking deficits in motion vision. In natural situations, the chromatic motion vision mechanisms identified in this study may combine with phototaxis and other visual processing to identify salient edges as the fly navigates its path. How motion and chromatic signals are integrated in output cells of the optic lobes, such as the lobula columnar cells, many of which respond to looming including LC4, LC6, LC10, LC16 and LPLC2, is currently being explored. Across these cell types, it is possible that different pathways mediate the response to dark and bright discs (Longden, 2023).

This study established that augmented ON-motion UV-sensitivity is not limited to D. melanogaster but is also displayed by other drosophilids. Among invertebrates, color has been reported to contribute to motion vision in other insects including the honeybee and the butterfly Papilio xuthus, whose behavioral responses to moving colored ON- and OFF-edges indicated that responses to ON-motion were more sensitive to red, compared to responses to OFF-motion that were more sensitive to blue and green. If Papilio implements the chromatic mechanism that this study has proposed for Drosophila, then this would predict that its ON- and OFF-motion pathways support seeing red objects against green backgrounds, for example red flowers set against foliage. However, in Papilio, spectral information is preprocessed in the lamina through lateral connections that are not found in Drosophila, indicating earlier interactions between the color and other visual pathways. Tantalizingly, central brain neurons responding to gratings with chromatic contrast have been discovered that project to the medulla, but the supporting cellular mechanisms remain unknown (Longden, 2023).

In vertebrates, differences in the spectral sensitivity to ON- and OFF-motion have not been thoroughly investigated, and if present they could support a contribution of color to motion vision as was found for Drosophila. Larval zebrafish use UV-ON processing to detect paramecia while foraging, and the mechanism described in this study has the potential to operate in zebrafish to enhance the detection of approaching paramecia. Mice are also sensitive to UV and green wavelengths, and since they have the greatest chromatic sensitivity in the visual circuits viewing the sky, they are thought to use color vision to detect approaching predators. Again, the mechanism described in this study is in theory directly applicable to the mouse visual system: it predicts that if OFF-motion responses were more sensitive to UV than for ON-motion, this would favor the detection of an approaching object seen against a UV-rich sky. In mice, ON and OFF retinal ganglion cells have been recently reported that nonlinearly integrate UV and green to allow an OFF cell, for example, to have different isoluminance levels for UV-OFF and green-ON and for green-OFF and UV-ON. In this cell the spectral divergence of ON and OFF processing supports the detection of a light decrement, whether the decrement is in green or UV wavelengths (UV-OFF and green-OFF), and in other cells the nonlinearities were UV-selective (UV-ON and UV-OFF). These nonlinearities are algorithmically very similar to the mechanism proposed in this stduy and indicate a potential platform for motion-sensitive cells downstream to be sensitive to isoluminant motion, and to preferentially detect objects rich in UV or green, relative to the background. In primates, motion-sensitive cells in area MT contribute to the smooth pursuit tracking of objects and frequently retain some degree of chromatic sensitivity, such that around the isoluminance level the response to motion is decreased, but not to zero. Any implementation in another animal would have to be integrated with many aspects of visual processing, such as luminance, and may not involve directional selectivity, for example. Nevertheless, the current results reveal that, in addition to allowing the animal to view isoluminant edges, an individual cell’s chromatic sensitivity may be organized to enhance the detection of motion of targets of a particular color (Longden, 2023).

In summary, this study has shown how UV contributes to the processing of ON-motion in Drosophila in a way that enhances responses to expanding UV discs. Key cellular components are reported of how color contributes to motion vision in flies, the R7-8 and T4 cells, and how cells linking them show consistent differences in spectral tuning and alter the behavior when silenced. How a spectral divergence in ON- and OFF-motion processing can be used to favor objects of a specific color was shown, an insight that is directly applicable to many vertebrate and invertebrate sensory systems (Longden, 2023).

Synaptic targets of photoreceptors specialized to detect color and skylight polarization in Drosophila

Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, this study has systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and light microscopy was used to confirm many of the findings. Known and novel downstream targets were identified that are selective for different wavelengths or polarized light, and their projections were followed to other areas in the optic lobes and the central brain. The results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. This reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs (Kind, 2021).

Both the wavelength and the polarization angle of light contain valuable information that can be exploited by many visual animals. For instance, color gradients across the sky can serve as navigational cues, and skylight's characteristic pattern of linear polarization can also inform navigation by indicating the orientation relative to the sun. The spectral content of light is detected by groups of photoreceptor cells containing rhodopsin molecules with different sensitivities, often organized in stochastic retinal mosaics, and specialized, polarization-sensitive photoreceptors have been characterized in many species, both vertebrates and invertebrates. These two visual modalities, color and polarization vision, require the processing of signals over a wide range of spatial and temporal scales, and many questions remain about how the signals from functionally specialized photoreceptors are integrated in downstream neurons. Are color and polarization signals mixed at an early stage, or are they processed by different, modality-specific cell types? Do separate pathways exist that selectively process and convey information from photoreceptor types in the retinal mosaic to targets in the central brain? The full scope of the early synaptic stages of color and polarization circuitry is unknown in any animal, and the analysis of electron microscopy (EM) connectomes is ideally suited to exhaustively answer these questions, especially when corroborated with genetic labeling of cell types and circuit elements imaged with light microscopy. This study makes significant progress on these questions by mapping the neuronal connections of specialized, identified photoreceptors within the Drosophila full adult fly brain data set and validates many of these results by using the powerful genetic tools available in Drosophila (Kind, 2021).

While studies in many insects have contributed to the understanding of polarized light and color vision, the visual system of Drosophila offers many advantages for the exploration of neural circuits. Anatomical studies are facilitated by the stereotyped, repetitive structure of the optic lobes, with many cell types, the so-called columnar neurons, found in repeated circuit units, called visual columns, that are retinotopically arranged and each correspond to one of the ~800 unit eyes (ommatidia) of the compound eye. Over 100 optic lobe cell types have been described in classical Golgi work, by more recent studies combining genetic labeling with light microscopy and, for some cell types, through EM reconstructions that have revealed not only cell morphologies but also detailed synaptic connectivity. Furthermore, genetic tools, and, most recently, gene expression data are available for many optic lobe cell types. An enabling feature of this emerging body of work is that in nearly all cases, distinct cell types can be reliably identified across data sets, such that new studies often directly enrich prior ones (Kind, 2021).

Each Drosophila ommatidium contains eight photoreceptors whose output is processed in a series of neuropils called the lamina, medulla, lobula, and lobula plate that together form the optic lobes of the fly. Outer photoreceptors R1-6 project to the lamina neuropil, and serve as the main input to the motion vision circuitry; inner photoreceptors R7 and R8 pass through the lamina without connections and project directly to the deeper medulla neuropil, which also receives lamina projections. The organization of the inner photoreceptors along the dorsal rim area (DRA) of the eye characteristically differs from that of the rest of the retina. In the non-DRA part of the retina, R7 and R8 differ in their axonal target layers, with R7 projecting to layer M6, and R8 to layer M3. R7 and R8 also differ in their rhodopsin expression, being sensitive to short wavelength ultraviolet (UV, R7) and blue (R8), respectively, in so-called 'pale' ommatidia, and to long wavelength UV (R7) and green (R8) in 'yellow' ommatidia. Pale and yellow ommatidia are distributed randomly, at an uneven ratio that is conserved across insects. Meanwhile, DRA ommatidia are morphologically and molecularly specialized for detecting skylight polarization, that Drosophila can use to set a heading. In the DRA, the inner photoreceptors express the same UV rhodopsin (Rh3) and detect perpendicular angles of polarized UV light. In contrast to the rest of the medulla, R7-DRA and R8-DRA project to the same medulla layer (M6), where their targets include polarization-specific cell types. Across insects, a 'compass pathway' connects the DRA to the central brain via the anterior optic tubercle (AOTU). Anatomical and functional data from Drosophila suggests that the non-DRA medulla is also connected to the compass pathway, potentially forming parallel pathways for processing different celestial cues (Kind, 2021).

EM studies have already revealed some of the circuitry downstream of R7 and R8. For example, axons of R7 and R8 from the same ommatidium are reciprocally connected with inhibitory synapses, leading to color-opponent signals in their presynaptic terminals. Interestingly, R7-DRA and R8-DRA also inhibit each other. Other known R7 and R8 targets in the main medulla include local interneurons (e.g. Dm8) and projection neurons that provide connections to deeper optic lobe regions (e.g. Tm5 and Tm20 neurons). A previous light microscopy study identified a single cell type, Tm5a, that is specific for yellow medulla columns; this neuron has been used to identify pale and yellow columns in an EM volume. Using genetic labeling techniques, four classes of TmY cells have also been reported as specific targets of pale versus yellow photoreceptors, yet previous connectomic studies did not reveal similar cells. The currently most comprehensive EM study of the medulla reconstructed the connections between neurons in seven neighboring medulla columns, revealing a detailed, yet incomplete, inventory of cell types connected to R7-8. This data set, now publicly available, is remarkable for its dense reconstruction of columnar circuits, but could not be used to identify many multicolumnar neurons, that were cut-off at the edge of the data volume, leaving ~40% of R7-8 synapses onto unidentified cell types. In addition, no EM-based reconstructions of DRA neurons and their connections are currently available (Kind, 2021).

This study presents a comprehensive reconstruction of all inner photoreceptor synaptic outputs and inputs, from pairs of pale and yellow columns and from three DRA columns in the data set. These reconstructions were carried out within a full-brain volume, such that, for the first time, nearly all neurons connected to these photoreceptors were identified. Light microscopy revealed a large visual projection neuron (VPN) with distinctive morphology, named accessory medulla cell type 12 (aMe12), that selectively innervates pale ommatidia across the medulla. This cell was reconstructed and subsequently used to identify pale and yellow columns, from which the connectivity of R7 and R8 was enumerated with known and novel cell types within the optic lobes and projecting to the central brain, including more cells with pale-yellow specificity, and synapses on axons between neuropils. In the DRA, it was shown that cellular diversity is reduced, with local interneurons and projection neurons to the AOTU dominating, and connections to the lobula virtually missing. Circuit motifs shared between DRA and central columns were identified, and modality-specific cell types were identified, including cells with interhemispheric connections and projections to the central brain. Together, this study identified the connected neurons that account for 96% of these inner photoreceptor synapses, a comprehensive set of the neurons that comprise the first step of the pathways through which color and polarization signals are transduced to the rest of the brain (Kind, 2021).

This systematic reconstruction of all synaptic inputs and outputs of identified, functionally specialized Drosophila photoreceptors (pale and yellow R7-8, R7-DRA, and R8-DRA) provides a comprehensive inventory of the first steps of the color and polarization pathways, from which all the computations of the dependent behaviors stem. These data revealed core connectomic motifs shared across column types, multiple new photoreceptor targets, and uncovered additional cell types as being connected to specific photoreceptor subtypes conveying specific color and polarization information to the central brain (Kind, 2021).

Previously reported synaptic partners of the inner photoreceptors in the non-DRA medulla were confirmed, and new photoreceptor targets were identified. As prior reconstructions were incomplete, it was unclear whether the unidentified connections were mainly onto new target neurons or represented more connections onto known cell types; the current reconstructions revealed both types of omissions. One functionally important set of missed connections are the synapses between inner photoreceptors from the same central and DRA ommatidia, which this study found to be stronger than previously reported, due to significant numbers of synapses outside the medulla. These synapses likely contribute to color-opponent responses seen in central R7 and R8 terminals and the polarization-opponent signals measured from DRA photoreceptors. The reconstructions also support a larger-scale opponent process mediated by multicolumnar Dm9 cells, which also formed some synapses outside the medulla neuropil (Kind, 2021).

Other cell types also received inner photoreceptor input outside the medulla, notably the lamina monopolar cells L1 and L3. These lamina connections indicate that chromatic comparisons arising from R7 and R8 may feed into the motion vision pathway, and identifies a new site for interplay between the 'color' and 'motion' pathways. Together, these observations suggest that synapses in an unexpected location, outside the main synaptic layers of the medulla, could play a significant role in early visual processing (Kind, 2021).

In the non-DRA medulla, this study found, for the first time in an EM study, strong synaptic connections between R7s and MeTu cells that project to the AOTU, confirming previous claims based on light microscopy. This finding may reconcile disparate observations, such as a role in wavelength-specific phototaxis for cells matching MeTu morphology, as well as measurements of color-sensitive signals in the AOTU of bees. Previous anatomical studies partitioned MeTu cells into distinct subclasses that terminate in discrete subdomains of the AOTU. T identified modality-specific MeTu-DRA cells that only integrate from the polarization-sensitive R7-DRA photoreceptors, while avoiding synaptic contacts with color-sensitive pale or yellow R7s. Our MeTu and MeTu-DRA cells target adjacent subdomains within the small unit of the AOTU (Figure 10E), in agreement with proposals that parallel channels convey different forms of visual information from the eye to the central complex via the AOTU (Kind, 2021).

This study also identified connections from inner photoreceptors to several cell types either not previously described or not known to be photoreceptor targets, thus setting up a clear expectation that these cells should contribute to color or polarization vision. ML1 is a new, major target of R8, a cell type that connects the medulla to the lobula via a previously unknown, non-columnar pathway and the central brain. Previous studies have identified an important role for Tm5a/b/c and Tm20 cell types for chromatic processing in the lobula, and this study has confirmed that these cell types are targets of R7-8. Whether these Tm neurons and ML1 cells have common targets in the lobula, feed into shared central pathways or contribute to separate channels remain open questions; the lobula arbors of the Tm and ML1 cells are mostly in different layers arguing against direct synaptic interactions between the cells (Kind, 2021).

The reconstruction of DRA photoreceptor targets confirmed the modality-specific connectivity of Dm-DRA cell types within layer M6: Dm-DRA1 to R7-DRA, and Dm-DRA2 to R8-DRA (see Comparison of central versus dorsal rim area (DRA), and pale versus yellow pathways). The R7-DRA and R8-DRA cells respond to orthogonal orientations of polarized light at each location of the DRA, so Dm-DRA1 and Dm-DRA2 likely process orthogonal e-vector orientations spatially averaged by pooling over ~10 ommatidia. The data also revealed additional DRA pathways into the PLP in the central brain via VPN-DRA cells, as well as to the contralateral DRA, via MeMe-DRA cells. Such interhemispheric connections have been demonstrated in larger insects, but not in Drosophila, and their synaptic input was not known. Interocular transfer contributes to navigation by desert ants that can see the celestial polarization but not visual landmarks, but the interactions between the DRA regions remain poorly understood. The identification of MeMe-DRA neurons may enable the mechanisms of such phenomena to be explored (Kind, 2021).

For color vision, this study has confirmed the pale-yellow specificity of Tm5a and Tm5b cells. The aMe12 and ML-VPN1 cell types were identified as pale-specific, and the yellow bias of Tm5c and the pale bias of Mi9 were identified as well. Detailed analysis of Dm8 inputs confirmed that these neurons receive most of their photoreceptor input in a central home column, consistent with pale and yellow subtypes of Dm8 cells having distinct chromatic properties. The selective photoreceptor input to these cell types, combined with input to columnar cells from a single photoreceptor subtype, indicates that wavelength-specific information is maintained in the medulla and lobula. The projections of aMe12 and ML-VPN1 to the central brain indicate the possibility that wavelength-specific photoreceptor responses are directly conveyed into the central brain by these cells, although they likely integrate input from other chromatically sensitive cell types (Kind, 2021).

By focusing on a small number of columns, this study has delivered a near-complete picture of local connectivity, but the possibility cannot be ruled out that certain cell types may have been overlooked. For instance, an arbitrary threshold was chosen of two to three synapses below which attempts were not made to reconstruct synaptic targets to the extent required to uniquely identify them. As a result, in principle, this study may have missed large cells that receive small, but significant inputs across many columns. Furthermore, regional specializations in the optic lobes, such that specific cell types might be found outside of the seed columns, cannot be ruled out. For example, some MeTu cells are only found in the dorsal third of the medulla, where incoming R7 cells are known to co-express Rh3 and Rh4 rhodopsins (Kind, 2021).

Taken together, the data presented in this study provides access to the full complement of R7 and R8 photoreceptor targets from functionally specialized optical units. By reconstructing these local circuits within a full-brain EM volume, it was possible to establish the complete morphology of large, multicolumnar cell types, that are strongly connected to photoreceptors, but had eluded previous connectomic reconstruction efforts. The sparse tracing approach implemented and described in this study enabled efficient identification of the complete set of upstream and downstream partners of the inner photoreceptors, in a manner that is complementary to the dense connectomes generated from smaller-scale medulla volumes. As an example of this synergistic usage of complementary data sets, this study has returned to the 7-column data and used whole-cell morphologies to match the bodies of previously unidentified photoreceptor targets. In that process, strong candidates have been established for MeTu, ML1, and perhaps for aMe12 and several aspects of their connectivity were confirmed in a previous data set. In so doing, this study now has access to the additional connectivity data provided by the 7-column dense reconstruction. While full exploration and follow-up analyses of these combined data is beyond the scope of this work, the combined analysis a previous study and the medulla-7-column connectome revealed several intriguing connectivity patterns, including new candidate paths for the integration of output from different photoreceptor types. For example, the MeTu cells that are postsynaptic to R7 also receive indirect R8 input via the R8 target Mi15, and ML1 combines direct R8 input with indirect input from outer photoreceptors via lamina neurons. As further connectome data sets are completed, this comparative interplay between data sets with unique advantages and limitations will be an important step in both cross-validating and extending the applicability of all related data sets (Kind, 2021).

Reconstruction of the DRA photoreceptor targets provides the first EM-based connectomic data set for modality-specific cell types likely to process skylight information in any insect and will be important for developing refined models of skylight navigation. Core motifs shared between DRA and central columns are prime candidates for circuit elements that perform computations, such as establishing opponency, that are key for both polarization and color processing, whereas cell types with preferential connections to either pale or yellow columns are promising candidates for the study of specific aspects of color processing in the insect brain. This comprehensive catalog of the neurons carrying signals from R7 and R8 photoreceptors deeper into the brain establishes a broad foundation for further studies into the mechanistic basis of color vision and its contributions to perception and behavior (Kind, 2021).

Color Processing in the Early Visual System of Drosophila

Color vision is encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. This study examined the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. Color-opponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors (see the Graphical Abstract) and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently (Schnaitmann. 2018).

Color vision enables animals to distinguish spectral stimuli independent of their relative intensities and provides an extra dimension to vision that facilitates discrimination tasks and intra-specific communication. In pollinators such as honeybees, color vision plays a crucial role in flower recognition and, thus, has both ecological and economic importance. Color vision requires possession of at least two photoreceptor types with different spectral sensitivities and the ability to compare their outputs. Antagonistic interactions between different channels are a hallmark of sensory processing that enhances stimulus contrast and maximizes information transfer. In color vision, this opponency between spectral channels solves the critical shortcoming that any single photoreceptor cannot distinguish between changes in brightness and spectral information (Schnaitmann. 2018).

Much knowledge of color-opponent processing is based on vertebrates. In humans and other trichromatic primates, the signals of short (S), middle (M), and long (L) wavelength-sensitive cone photoreceptors are combined antagonistically to create two spectrally opponent pathways. In the first pathway, L and M cone signals mutually inhibit each other. In the second pathway S cone signals and the summed signals of L and M cones mutually inhibit each other. These color opponencies correspond with the red-green and blue-yellow opponent axes of human and macaque color perception and are implemented at the first visual synapse. Different types of cones converge onto horizontal cells, and the latter establish reciprocal sign-inverting synapses with cone terminals. This circuit adapts photoreceptor output to the intensity of ambient light, enhances achromatic and chromatic image contrast, and renders the terminals of vertebrate cones color-opponent. Furthermore, it provides the basis for the center-surround organization of cone terminals and bipolar cells. However, the exact biophysical mechanisms that underlie this critical processing stage in the retina are still unresolved (Schnaitmann. 2018).

Compared with the vertebrate retina, less is known about color-opponent processing in insects. Color-opponent neurons have been recorded in a few species, but the lack of genetic amenability prohibited identification of the underlying circuits and synaptic mechanisms. Recent studies of sensory processing in Drosophila suggest that such insights may be revealed in this model organism; however, color-opponent neurons have not been previously identified. Such neurons are expected because fruit flies exhibit a multitude of wavelength-dependent behaviors, including phototaxis, spectral preference, and color memory. Moreover, Drosophila is a classic model for studies of visual system development and connectivity and offers almost unlimited genetic amenability. In the Drosophila eye, each ommatidium houses six outer photoreceptors, R1–R6, and a pair of superimposed inner photoreceptors, R7/R8 that release histamine as neurotransmitter. The broadband-sensitive R1–R6 photoreceptors express rhodopsin1 (rh1), project to the lamina, and provide the major input to the motion vision system. Although not required for color vision, R1–R6 photoreceptors were recently shown to also contribute to it. In R7/R8 photoreceptor pairs, precise genetic control of rhodopsin expression determines the two major types of ommatidia, 'pale' (p) and 'yellow' (y), that are stochastically distributed over the main part of the eye. R7p and R8p photoreceptors express rh3 with maximum sensitivity in the short-UV and rh5 with maximum sensitivity in the blue spectral range, respectively. R7y and R8y photoreceptors express rh4 with maximum sensitivity in the long-UV and rh6 with maximum sensitivity in the green spectral range, respectively. These four types of inner photoreceptors provide the major input to the color vision system in the medulla, to which they project without making chemical synapses in the lamina (Schnaitmann. 2018).

Direct investigation of color-opponent processing using electrophysiological recordings has so far proved elusive by the technical difficulties associated with the stacked arrangement of the R7 and R8 cells and their postsynaptic partners in Drosophila. However, behavioral studies on Lucilia and Drosophila suggest that flies compare the signals of R7 and R8 photoreceptors of the same type of ommatidia (p and y) to distinguish color. Intracellular recordings from distal segments of photoreceptors in large dipteran flies and most other insect species revealed no sign of spectral inhibition; therefore, this comparison is thought to be implemented downstream of Drosophila inner photoreceptors. Candidate color-opponent neurons postsynaptic to inner photoreceptors have been revealed in behavioral, genetic, and anatomical studies in Drosophila and include Tm9, Tm20, Tm5a/b/c, and certain TmY cells. However, these studies do not exclude the possibility of color-opponent processing at the level of photoreceptors. For example, intracellular recordings in a few butterfly species revealed excitatory and inhibitory response components in particular photoreceptors. Finally, the responses of photoreceptor terminals have not been recorded in any insect species so far. This leaves open the question of whether local inhibitory circuit mechanisms render photoreceptor output color-opponent and, if so, how these mechanisms compare with processing in the vertebrate retina (Schnaitmann. 2018).

This study reports physiological recordings from inner photoreceptor terminals of Drosophila, combining two-photon calcium imaging, spectral stimulation, and use of the fluorescent genetically encoded calcium reporter Twitch-2C. Presynaptic UVshort/blue and UVlong/green color opponencies are evident in the terminals of the inner photoreceptors R7/R8 of p and y ommatidia. Genetic dissection of the peripheral visual circuits enabled identification of the photoreceptor interactions underlying color-opponent processing: R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other at the level of their presynaptic terminals, whereas R1–R6 do not contribute to spectrally opponent processing in R7/R8 photoreceptor terminals. Two concurrent circuit mechanisms that involve the distinct histamine receptors HisCl1 and Ort mediate this processing. Direct inhibitory synaptic interactions between the terminals of R7/R8 pairs are mediated by HisCl1, and feedback inhibition with similar spectral tuning requires expression of Ort. These results illustrate that the Drosophila visual system subtracts different spectral channels at the first synapse, reminiscent of processing in the vertebrate retina, albeit by entirely different synaptic and cellular mechanisms (Schnaitmann. 2018).

Based on the spectral sensitivities of the Rhodopsins expressed in the four types of inner photoreceptors in Drosophila, R7 and R8 cells have been implicated in color vision for decades. Recent studies corroborated this view and provided further insights into the neural underpinnings of Drosophila color vision. However, because of the lack of physiological recordings from neurons in the color pathway, it is still unknown how color information is processed in Drosophila at the cellular and circuit level. This study reports an optophysiological approach that enables the analysis of neural responses to spectral stimuli in Drosophila. Based on this approach, spectral processing was investigated in R7/R8 inner photoreceptor terminals in the medulla. This study demonstrates that color opponency, a hallmark of spectral processing, is observable at the level of the first visual synapse in presynaptic terminals of the inner photoreceptors R7/R8. Two concurrent neuronal circuit mechanisms that involve distinct histamine receptors implement the comparison of R7 and R8 photoreceptor signals in the p and y pathways (Schnaitmann. 2018).

Recordings in norpA mutant flies combined with norpA rescue in pairwise combinations of photoreceptor types revealed mutual inhibitory interactions between R7 and R8 of the same type of ommatidia. These interactions provide the cellular basis for UVshort/blue and UVlong/green color-opponent responses in p and y photoreceptor terminals, respectively. Similar opponencies were previously posited to underlie color discrimination in dipteran flies). Thus, it is proposed that the photoreceptor terminals of the four types of inner photoreceptors mark the onset of two parallel color-opponent pathways that mediate color vision in Drosophila. The recently identified Tm5a/b/c, Tm9, Tm20, and TmY cells that are postsynaptic to R7/R8 likely represent further elements of these pathways). In particular, it has been suggested that Tm5a/b/c and Tm20 cells are elements of redundant color vision pathways. Blocking of all of these cell types, but not of single types or combinations, is required for complete loss of color discrimination. The finding of presynaptic color opponency in R7/R8 photoreceptors suggests that all of these neurons receive color-opponent input and do not generate color opponency de novo. These neurons likely participate in higher color processing, such as the spatial integration of spectral inputs, which is suggested for Tm5b/c cells based on their multi-columnar arborizations. Furthermore, synaptic connections from the outer photoreceptors R1–R6 to L3 lamina neurons and from L3 to some of the candidate neurons might explain the contribution of R1–R6 to color vision. At the level of R7/R8 terminals, no evidence was found for a role of R1–R6 photoreceptors in color-opponent processing (Schnaitmann. 2018).

The genetic, anatomical, and physiological experiments identify the two Drosophila histamine receptors HisCl1 and Ort) as key elements of direct and indirect inhibitory circuit mechanisms, respectively. In prior studies, chemical synaptic contacts of unknown function have been observed between R7/R8 photoreceptors by serial EM. The results on HisCl1 expression, and physiological recording suggest that these synapses are inhibitory and mediate color-opponent processing. Therefore, HisCl1 is of varying importance in R7 and R8 photoreceptors: in R7 terminals, hisCl1 expression is not required but is sufficient to generate color opponency; in R8 terminals, hisCl1 expression is required for color opponency, and it is only sufficient when ort expression in the visual circuits is intact. Interestingly, the varying importance of hisCl1-mediated direct inhibition between R7/R8 correlates well with the reported number of synapses between R7 and R8 photoreceptors. Serial EM reconstruction of one medulla column revealed 5 and 11 synapses from R7 onto R8 and vice versa, respectively (Schnaitmann. 2018).

Although ort is not expressed in photoreceptors, it nevertheless mediates color-opponent processing in concert with hisCl1. Physiological recordings reveal that ort is of varying importance for color opponency in R7 and R8, comparable with the results for hisCl1. Intact ort expression in the visual circuits is not required but sufficient for color-opponent processing in R7 terminals. In contrast, ort expression in the visual circuits is required for color opponency in R8 terminals, and it is only sufficient together with hisCl1 expression in R8 photoreceptor. The medulla neurons mediating ort-dependent feedback inhibition to R7/R8 terminals remain unknown. Among the many ort-expressing neurons in the medulla, several neuron types have been identified; however, none of these cell types establish feedback synapses onto R7/R8 photoreceptors. Work on polarization vision in flies has demonstrated that inner photoreceptors in the dorsal rim area similarly display antagonistic responses when the orientation of the e-vector is altered. If these inhibitory interactions are mediated by circuit mechanisms similar to the ones described in this study remains to be investigated. Along with the current results, the prior findings suggest that presynaptic calcium in Drosophila photoreceptor terminals is altered by local circuit interactions that enhance contrast (Schnaitmann. 2018).

Because the distal segments of the inner photoreceptors of large flies and of most other insects exhibit exclusively depolarizing non-opponent voltage responses, the observed color opponency in Drosophila inner photoreceptor terminals is unexpected, and it is proposed that spectrally antagonistic processing emerges only locally in photoreceptor terminals. Assuming that this local inhibitory signal does not propagate backward to the distal photoreceptor segments that have been recorded intracellularly, local processing would explain and reconcile the reported differences between insect species (Schnaitmann. 2018).

Recordings from R7/R8 terminals are consistent with mutually antagonistic processing of photoreceptors in the vertebrate retina. However, substantial differences in the molecular, synaptic, and network implementation demonstrate surprising variability in the underlying circuits (Schnaitmann. 2018).

Vertebrate photoreceptors employ cyclic guanosine monophosphate (cGMP) signaling, hyperpolarize, and reduce the release of glutamate in response to light. As a consequence, horizontal cells and OFF-bipolar cells expressing sign-conserving α-amino-3-hydroxy-5-methyl-4-isoxazol-propionacid (AMPA)/kainate-receptors receive less excitation, whereas ON-bipolar cells expressing sign-inverting metabotropic glutamate receptor 6 (mGluR6) receive less inhibition. In contrast, Drosophila photoreceptors employ phosphoinositide signaling and depolarize in response to light. This increases presynaptic calcium influx and the release of histamine, which binds to inhibitory Ort and HisCl1 receptors on postsynaptic cells. The results show that, in addition to interactions with Ort-expressing second-order neurons of the medulla, direct HisCl1-mediated interactions between inner photoreceptors play an important role in Drosophila color vision. Direct chemical synaptic interactions between different types of cones do not exist in the vertebrate retina. Vertebrate horizontal cells are the key players in early color-opponent processing: they receive input from different types of cones, synapse onto bipolar cells, and feed back onto cones with a sign-inverting synapse. Light-induced disinhibition of cone terminals is the fundamental mechanism underlying opponent L/M, and S/(L+M) interactions in cone terminals. If neurons analogous to horizontal cells exist in the Drosophila visual system has to be revealed. If so, they should participate in the Ort-dependent opponency mechanism revealed in this study. Based on the data and similar findings regarding the vertebrate retina, it is proposed that presynaptic color opponency in photoreceptor terminals is an important processing principle of color vision that is shared across taxa and that evolved in different taxa independently (Schnaitmann. 2018).

Septins tune lipid kinase activity and PI(4,5)P(2) turnover during G-protein-coupled PLC signalling in vivo

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P(2), its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P(2) synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P(2) is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P(2) hydrolysis is unknown. This study, identified a unique isoform dPIP5K(L), that is both necessary and sufficient to mediate PI(4,5)P(2) synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5K(L) activity in vitro and PI(4,5)P(2) resynthesis in vivo; co-depletion of dPIP5K(L) reverses the enhanced rate of PI(4,5)P(2) resynthesis in vivo. Thus, this work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P(2) hydrolysis (Kumari, 2022).

Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors

The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. This study sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, flies were aged or exposed to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, genes were identified that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of phototransduction. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. It is proposed that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures (Escobedo, 2022).

Insect vision starts with light absorption by visual pigments based on opsins that drive Gq-type G protein-mediated phototransduction. Since Drosophila, the most studied insect in vision research, has only Gq-coupled opsins, the Gq-mediated phototransduction has been solely focused on insect vision for decades. However, genome projects on mosquitos uncovered non-canonical insect opsin genes, members of the Opn3 or c-opsin group composed of vertebrate and invertebrate non-visual opsins. This study reports that a homologue of Opn3, MosOpn3 (Asop12) is expressed in eyes of a mosquito Anopheles stephensi. In situ hybridization analysis revealed that MosOpn3 is expressed in dorsal and ventral ommatidia, in which only R7 photoreceptor cells express MosOpn3. It was also found that Asop9, a Gq-coupled visual opsin, exhibited co-localization with MosOpn3. Spectroscopic analysis revealed that Asop9 forms a blue-sensitive opsin-based pigment. Thus, the Gi/Go-coupled opsin MosOpn3, which forms a green-sensitive pigment, is co-localized with Asop9, a Gq-coupled opsin that forms a blue-sensitive visual pigment. Since these two opsin-based pigments trigger different phototransduction cascades, the R7 photoreceptors could generate complex photoresponses to blue to green light. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods (Koyanagi, 2022).

Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, this study took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. The impacts were analyzed of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. Chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, no apparent effects of vitamin A deficiency on the ocular lipidome were found. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated (Kumar, 2022).

Connectome of the lamina reveals the circuit for early color processing in the visual pathway of a butterfly

Connectomics has become a standard neuroscience methodology in a few model animals, with the visual system being a popular target of study. Combining connectomics with circuit and behavioral physiology, recent studies on the color vision of the fruit fly Drosophila melanogaster have focused on the mechanisms underlying early wavelength processing in the optic ganglia. However, the color vision capabilities of D. melanogaster are limited, compared with many flower-visiting insects. For example, a butterfly Papilio xuthus has six spectral classes of photoreceptors. Each ommatidium contains nine photoreceptors in one of three fixed combinations, making the eye an array of three spectrally distinct ommatidia types. Behaviorally, P. xuthus can detect 1 nm differences in light wavelength across the spectrum from ultraviolet to red, outperforming humans. What is the neuronal basis of such precise color vision? How does such a system evolve? Addressing these questions requires comparative studies at the circuit level. This study performed a connectome analysis in the first optic ganglion, the lamina, of P. xuthus. The lamina comprises cartridges, each typically containing nine photoreceptor axons from a single ommatidium and four second-order neurons. Abundant inter-photoreceptor connections were found that are absent in the lamina of D. melanogaster. Connections were identified between neighboring cartridges, particularly those receiving inputs from spectrally distinct ommatidia. The linear summation of synaptic connections well explains the spectral sensitivity of photoreceptors and second-order neurons in the lamina (Matsushita, 2022).

Effects of the IQ1 motif of Drosophila myosin-5 on the calcium interaction of calmodulin

In Drosophila compound eyes, myosin-5 (DmMyo5) plays a key role in organelle transportation, including transporting pigment granules from the distal end to the proximal end of the photoreceptor cells to regulate the amount of light reaching the photosensitive membrane organelle rhabdomere. It is generally accepted that, upon exposure to light, the dark-adapted compound eyes produce a rapid rise of free Ca(2+) concentration, which in turn activates DmMyo5 to transport pigment granules. Considering the dynamic and compartmentation of Ca(2+) signaling in photoreceptor cells during light exposure, it is necessary to understand the kinetics of Ca(2+) interaction with DmMyo5. This study investigated the interaction of Ca(2+) with Drosophila calmodulin (CaM) in complex with the IQ1 of DmMyo5 using steady-state and kinetic approaches. These results show that IQ1 binding substantially increases the Ca(2+) affinity of CaM and decreases the dissociation rate of Ca(2+) from CaM. In addition, it was found that Mlc-C, the light chain associated with the IQ2 of DmMyo5, has little effect on the Ca(2+) kinetics of CaM in IQ1. It is proposed that, by decreasing the Ca(2+) dissociation rate from CaM, IQ1 delays the deactivation of DmMyo5 after Ca(2+) transition, thereby prolonging the DmMyo5-driven transportation of pigment granules (Zhang, 2022).

Diacylglycerol Activates the Drosophila Light Sensitive Channel TRPL Expressed in HEK Cells

Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP(2)) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. This study re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, this study added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, a picosecond-fast optically activated DAG analogue, OptoDArG, was used. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPL(F557I) pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions (Rhodes-Mordov, 2023).

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gα(q) and phospholipase Cβ. Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. Attempts were made to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Using genetics, lipid analysis, and Ca(2+) imaging, this study found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca(2+) influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. It is proposed that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells (Sokabe, 2023).

Azimuthal invariance to looming stimuli in the Drosophila giant fiber escape circuit

Spatially invariant feature detection is a property of many visual systems that rely on visual information provided by two eyes. However, how information across both eyes is integrated for invariant feature detection is not fully understood. This study investigated spatial invariance of looming responses in descending neurons (DNs) of Drosophila melanogaster. It was found that multiple looming responsive DNs integrate looming information across both eyes, even though their dendrites are restricted to a single visual hemisphere. One DN, the giant fiber (GF), responds invariantly to looming stimuli across tested azimuthal locations. It was confirmed that visual information propagates to the GF from the contralateral eye, through an unidentified pathway, and it was demonstrated that the absence of this pathway alters GF responses to looming stimuli presented to the ipsilateral eye. The data highlight a role for bilateral visual integration in generating consistent, looming-evoked escape responses that are robust across different stimulus locations and parameters (Jang, 2023).

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list of proteins involved in visual signal transduction

date revised:  2) December 2024

Zygotically transcribed genes

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