FMFRamide

EVOLUTIONARY HOMOLOGS (part 3/3)

Functional specifity of FMRFamides

Numerous studies of plasticity inthe feeding behavior of Aplysia have shown that substantial plasticity is due toperipheral neuromodulation of the feeding musculature. Extensive previous workfocusing on the accessory radula closer (ARC) muscle has led to the realizationthat a major function of the modulation in that muscle may be to ensureefficient coordination between its contractions and those of its antagonistmuscles. For a more complete understanding, therefore, thesemuscles must also be studied. The radula opener muscles I7-I10 has now been studied. Using single isolated muscle fibers under voltage clamp, ioncurrents gated by voltage and by the physiological contraction-inducingneurotransmitter acetylcholine (ACh) have been studied and the effects of the physiologicalmodulators serotonin, myomodulins A and B, and FMRFamide. The results explainsignificant aspects of the electrophysiological behavior of the whole openermuscles, as well as why the opener and ARC muscles behave similarly in many waysyet differently in some key respects. Opener muscles express four types of Kcurrents: inward rectifier; A-type [IK(A)]; delayed rectifier [IK(V)], andCa2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and aleakage current. ACh activates a positive-reversing cationic current [IACh(cat)]and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ fromthe ARC in that, in the openers, activation of IK(A) occurs approximately 9 mVmore positive and there is much less IACh(Cl). In both muscles, IACh(cat) mostlikely serves to depolarize the muscle until ICa activates to supply Ca2+ forcontraction, but further depolarization and spiking is opposed by coactivationof IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) andIACh(Cl) may well be key factors that prevent spikes in the ARC but often allowthem in the opener muscles. As in the ARC, the modulators enhance ICa and sopotentiate contractions. They also activate a modulator-specific K current,which causes hyperpolarization and depression of contractions. Finally, in theopener muscles but not in the ARC, the modulators activate a depolarizingcationic current that may help phase-advance the contractions. Each modulatorexerts these effects to different degrees and thus has a distinct effect onvoltage and contraction size and shape. The overall effect will depend onthe specific combinations of modulators released in different behaviors. Byunderstanding the modulation in the opener muscles, as well as in the ARC, how the behavior of the two muscles iscoordinated under a variety of circumstances can now be understood (Scott, 1997).

The molluscan neuropeptide FMRFamide has a number of inhibitory actions on thesensory neurons and motoneurons mediating the defensive gill and siphonwithdrawal reflex pathway of Aplysia californica. Exogenous application ofFMRFamide has a biphasic, dual-polarity effect on the majority of LFS siphonmotoneurons, causing a transient depolarization followed by a prolongedhyperpolarization. FMRFamide induces this response in LFS neurons by causing anincrease in multiple ionic currents, including a transient Na+ current, a slowprolonged Na+ current, a 4-aminopyridine (4-AP)-sensitive K+ current and a4-AP-insensitive K+ current. A subset of LFS neurons exhibitsan exclusively excitatory, biphasic response to FMRFamide, consisting of atransient depolarization followed by a prolonged depolarization of reducedmagnitude. Over a period of 29 months, an increase inthe incidence of the exclusively excitatory response was consistently observed during the summer months (June to September). From October to May, an exclusively excitatoryresponse to FMRFamide in 19% of LFS neurons was observed; yet, in the summer months, 51% of LFS neurons exhibit this response pattern. The ionic basis of theexclusively excitatory response to FMRFamide was compared with the ionic mechanisms mediating the more frequently observed excitatory/inhibitory response. The exclusively excitatory response involves three of the same ionic components as the more typical excitatory/inhibitory response, including the activation of a transientNa+ current, a slow prolonged Na+ current and a 4-AP-insensitive K+ current. Theprincipal difference between the two response types is that FMRFamide fails toactivate a 4-AP-sensitive K+ current in those LFS neurons that exhibit anexclusively excitatory response to the peptide. In addition, LFS neurons with anexclusively excitatory response tend to show a coordinated increase in themagnitude of the inward current component of the FMRFamide response. Together,these changes during the summer months may enable this modulatory peptide tobring LFS neurons to suprathreshold levels of activity for eliciting a siphonwithdrawal and should substantially alter the neuromodulatory effects of thepeptide (Belkin. 1998).

This study examined differential modulation of motor neurons that innervate the same muscle but usedifferent excitatory transmitters in Aplysia. The medial portion of intrinsic buccal muscle 3 (I3m) is innervated by twoexcitatory motor neurons, B3 and B9. B3 uses glutamate as its fast transmitterand expresses the neuropeptide FMRFamide, whereas B9 uses acetylcholine as itsfast transmitter and expresses the neuropeptide SCP. This preparation was usedto study peptidergic modulation of muscles innervated by neurons that usedifferent fast excitatory transmitters. The effects of the application of the neuropeptides expressed in these neurons on excitatoryjunction potentials (EJPs) and contractions were determined. FMRFamide increases the amplitude of EJPs and contractions evoked by B3 while decreasing those evoked by B9. This is the first observation in buccal muscle of a substance that modulates twoexcitatory neurons innervating the same muscle in opposite directions. SCPincreases EJPs contraction amplitude, and the rate of muscle relaxation for bothmotor neurons. SCP potently increased cAMP levels in I3m asit does in other buccal muscles. Stimulation of B9 also causes increases cAMPlevels in I3m, providing independent evidence for SCP release. Stimulation of B9 increases both the contraction amplitude and relaxation rateof B3-evoked I3m contractions in a manner similar to that observed usingexogenous SCP. By inhibiting B9's cholinergic transmission with an antagonist, the modulatory effects of B9 could be observed in the absence of fastexcitatory effects. The magnitude of the modulation is dependenton the firing frequency and occurs at frequencies and patterns of firingrecorded for B9 during ingestive-like motor programs (Keating, 1999).

Fibers immunoreactive (IR) to serotonin (5-HT), the myomodulins(MMs), and FMRFamide were observed on the I7-I10 complex in the marine mollusk Aplysia californica. The I7-I10 muscle complex, which produces radula opening, isinnervated primarily by one motor neuron, B48. B48 is MM-IR and synthesizesauthentic MM(A). When B48 is stimulated in a physiological manner, cAMP levelsare increased in opener muscles. cAMP increases also are seen when the MMs areapplied to opener muscles but are not seen with application of the B48 primaryneurotransmitter acetylcholine (ACh). Possible physiological sources of 5-HT andFMRFamide are discussed. When modulators are applied to resting opener muscles,changes in membrane potential are observed. Specifically, 5-HT, MM(B), and lowconcentrations of MM(A) all depolarize muscle fibers. This depolarization isgenerally not sufficient to elicit myogenic activity in the absence of neuralactivity under 'rest' conditions. However, if opener muscles are stretchedbeyond rest length, stretch- and modulator-induced depolarizations can summateand elicit contractions. This only occurs, however, if 'depolarizing' modulatorsare applied alone. Thus other modulators [i.e., FMRFamide and highconcentrations of MM(A)] hyperpolarize opener muscle fibers and can preventdepolarizing modulators from eliciting myogenic activity. All modulators testedaffected parameters of motor neuron-elicited contractions of opener muscles.MM(B) and 5-HT increase contraction size over the range of concentrationstested, whereas MM(A) potentiates contractions when it is applied at lowerconcentrations but decreases contraction size at higher concentrations.FMRFamide decreases contraction size at all concentrations and does not affectrelaxation rate. Additionally, the MMs and 5-HT increase muscle relaxationrate, decrease contraction latency, and decrease the rate at which tension isdeveloped during motor neuron-elicited muscle contractions. Thus thesemodulators dramatically affect the ability of opener muscles to follow activityin the opener motor neuron B48. The possible physiological significance of thesefindings is discussed (Evans, 1999).

FMRFamide, synaptic modification and learning

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibitionfollowing noxious stimuli such as tail shock. This behavioral inhibition appears to bedue in part to transient presynaptic inhibition of the siphon sensory cells, which can bemimicked by application of the peptide FMRFamide. Although FMRFamide iswidespread in the Aplysia nervous system, an FMRFamide-containing inhibitoryneuron has not previously been identified. A search was carried out for such a neuron bycombining FMRFamide immunofluorescence with fluorescent dye backfilling from theabdominal ganglion, the location of the siphon sensory cells. These methods localize aneuron in the left pleural ganglion, termed LPL16. LPL16 isFMRFamide immunoreactive; it is excited by tail shock; stimulation of LPL16produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials andnarrowing of action potentials in the sensory cells in tetraethylammonium solution.These results indicate that LPL16 participates in the inhibitory effects of tail shock,and support the idea that FMRFamide plays a physiological role in the inhibition (Small, 1992).

At least two processes contribute to the modulation by 5-HT of the connectionsbetween sensory neurons and motor neurons in Aplysia. The first involves broadeningof the presynaptic spike through modulation of 5-HT-sensitive K+ channels that leadsto elevated levels of intracellular Ca2+ and increased release of transmitter. A secondprocess (or set of processes) apparently accounts for the amount of facilitation notproduced by presynaptic spike broadening. This spike duration-independent (SDI)process is particularly prominent in depressed synapses. A protocol was used in whichspikes are prebroadened into a range of durations in which further spike broadeningby itself has little or no effect on facilitation of the EPSP. 5-HT produces pronouncedfacilitation in depressed synapses under these conditions. Another modulatory agent,small cardioactive peptide (SCPb), also broadens spikes in sensory neurons but doesnot produce facilitation comparable to that produced by 5-HT. These results indicatethat 5-HT activates the SDI process whereas SCPb fails to do so. A 5 minpreexposure to the modulatory peptide FMRFamide inhibits 5-HT-induced activationof the SDI process, whereas a 1 min preexposure does not. Another process thatmay modulate synaptic efficacy in sensorimotor synapses involves a change in theproperties of the motor (follower) neuron, such as input resistance. FMRFamidedecreases the input resistance of postsynaptic neurons. This action could contribute tothe effects of FMRFamide when administered alone, but it does not appear to beresponsible for the inhibitory action of FMRFamide on 5-HT-induced facilitation.Neither 5-HT nor SCPb have a clear effect on input resistance. The actions of thesethree agents, therefore, seem to be differentially distributed among various pre- andpostsynaptic processes involved in the modulation of synaptic transmission (Pieroni, 1992).

Cell adhesion molecules play important roles in axon guidance and synapse formation.Recent studies suggest that the expression of some of these molecules can beregulated either by electrical activity or by specific neurotransmitters. The expressionof neural cell adhesion molecule (NCAM)-like molecules in Aplysia (see Drosophila Fasciclin II), designatedapCAM, is downregulated from the surface of sensory neurons by 5-HT, a transmitterknown to evoke long-term changes in the structure and function of these neurons. Whether the distribution of apCAM on the surface of other neurons can beregulated by treatments with other neurotransmitters known to evoke long-termfunctional and structural changes in Aplysia neurons was tested, as well as theconsequences of treatments with the neurotransmitters on the pattern of growthcone-neurite interactions. Applications of the neuropeptidePhe-Met-Arg-Phe-amide (FMRFamide) that evoke long-term synaptic depression alsoreduce apCAM expression on the surface of motor cell L7 via a mechanism thatappears to be similar to the mechanism mediating the 5-HT-induced change in thesensory cells. Specific treatments that affect apCAM distribution on the surface oftheir respective cells (5-HT on sensory cells and FMRFamide on motor cell L7) mimictreatment with monoclonal antibodies against apCAM by evoking a significantreduction in the fasciculation of growth cones with other neurites extending fromhomologous cells (Peter, 1994).

FMRFamide evokes long-term inhibition of the sensorimotor connection of Aplysia, including structural alterations in the presynaptic sensory cell. FMRFamide alsoevokes a down-regulation of the adhesion molecule apCAM from the surface of thepostsynaptic motor cell L7. The second messenger pathways mediatingthe long-term actions of FMRFamide on both the pre- and postsynaptic cells were examined to determine whether the activation of each pathway is required for the expression oflong-term functional and structural plasticity. Inhibition of the lipoxygenase pathway ofarachidonic acid metabolism, but not the cyclooxygenase pathway, blocks thelong-term changes in the presynaptic sensory cell evoked by FMRFamide. Thedown-regulation of apCAM in L7 appears to be mediated by cAMP-dependentactivation of protein kinase A. Blocking the cAMP-dependent changes also blocksFMRFamide-induced long-term functional and structural changes. These resultssuggest that the expression of long-term heterosynaptic inhibition in Aplysia mayrequire concomitant presynaptic and postsynaptic changes, each transduced byspecific second messenger systems (Wu, 1994).

Both 5-HT and FMRFamide evoke long-lasting changes in the efficacy of sensorimotor(SN-L7) synapses of Aplysia, structural alterations of the presynaptic sensory cell,and cell-specific downregulation in the distribution of the adhesion molecule apCAM.The cell-specific changes in apCAM, related to vertebrate NCAM and Drosophila Fas II, contribute to theformation of new presynaptic varicosities by 5-HT and the elimination of existingpresynaptic varicosities by FMRFamide. The formation of new sensoryvaricosities is directed by the presence of preexisting zones on the motor axon that areenriched for apCAM. Moreover, there was a further enrichment of apCAM levels atexisting sensory varicosities contacting the motor axon beginning at 1 hr and lasting 24hr after treatment with 5-HT. As was found for synapse formation during the earlystages of cell-cell interaction, incubation with anti-apCAM mAb blocks the5-HT-induced long-term changes in synaptic efficacy and the accompanying changesin sensory neuron structure. Long-term synaptic depression with FMRFamide isaccompanied by an overall decline of apCAM levels. Treatment with FMRFamideevokes an even greater decline in apCAM levels at sites of sensory varicosities thatprecede the structural changes and persist especially at sites where sensoryvaricosities are eliminated. These results suggest that neurotransmitters evoke bothcell- and site-specific changes in the levels of adhesion molecules that can influenceeither the formation or the elimination of presynaptic varicosities that accompanylong-term heterosynaptic modulation of a behaviorally relevant synaptic connection (Zhu, 1995).

Synaptic transmission and excitability in Aplysia sensory neurons (SNs) arebidirectionally modulated by 5-HT and FMRFamide. To explore the regionaldistribution of different functional receptors that modulate SN properties, changes in synaptic efficacy and excitability were examined upon brief focalapplications of the neuromodulators to different regions of SNs that haveestablished connections with motor cell L7 in culture. Short-term changes insynaptic efficacy are evoked only when 5-HT or FMRFamide is applied to regionswith SN varicosities along the surface of L7 axons. Applications to adjacent SNneurites with few varicosities in contact with L7 axons fail to evoke asignificant change in synaptic efficacy. The distribution of functionalreceptors mediating changes in excitability differ for 5-HT and FMRFamide.Whereas excitability increases are evoked only when 5-HT is applied to SN cellbodies, excitability decreases in SNs are evoked only when FMRFamide isapplied to regions along the L7 axon with SN varicosities. Without the targetcell, cell bodies of SNs express both 5-HT and FMRFamide receptors thatmodulate excitability. These results indicate that functional G-protein-coupledreceptors for two neuromodulators are distributed differentially along thesurface of a presynaptic neuron that forms chemical connections in vitro. Thisdifferential distribution of receptors on the presynaptic neuron is regulated bya target and does not require the physical presence of neurons that release theneuromodulators (Sun, 1996).

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergolong-term functional and structural modulation with appropriate behavioraltraining or with applications of specific neuromodulators. Expression ofmolecules within the presynaptic terminals may be regulated in parallel with thechanges evoked by the neuromodulators. Immunocytochemicalmethods were used to examine whether the level of sensorin, the SN-specific neuropeptide, ismodulated in SN varicosities by the location of interaction with the targetmotor cell L7 and by applications of either 5-HT, which evokes long-termfacilitation or FMRFamide, which evokes long-term depression of Aplysiasensorimotor connections in vitro. A significantly higher proportion of SNvaricosities are sensorin positive when they are in contact with the proximalaxons of L7, as compared to varicosities of the same SNs in contact with distal L7neurites. Both 5-HT and FMRFamide evoke changes in the efficacy and structureof sensorimotor connections that are accompanied by changes in the frequency ofsensorin-positive varicosities contacting the axons of L7. More preexisting SNvaricosities are stained after 5-HT, and fewer preexisting SN varicosities arestained after FMRFamide. These results suggest that the postsynaptic target andthe neuromodulators not only regulate overall structure but also regulate thelevel of SN neuropeptide at synaptic sites (Santarelli, 1996).

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