FMFRamide

EVOLUTIONARY HOMOLOGS (part 3/3)

Functional specifity of FMRFamides

Numerous studies of plasticity in the feeding behavior of Aplysia have shown that substantial plasticity is due to peripheral neuromodulation of the feeding musculature. Extensive previous work focusing on the accessory radula closer (ARC) muscle has led to the realization that a major function of the modulation in that muscle may be to ensure efficient coordination between its contractions and those of its antagonist muscles. For a more complete understanding, therefore, these muscles must also be studied. The radula opener muscles I7-I10 has now been studied. Using single isolated muscle fibers under voltage clamp, ion currents gated by voltage and by the physiological contraction-inducing neurotransmitter acetylcholine (ACh) have been studied and the effects of the physiological modulators serotonin, myomodulins A and B, and FMRFamide. The results explain significant aspects of the electrophysiological behavior of the whole opener muscles, as well as why the opener and ARC muscles behave similarly in many ways yet differently in some key respects. Opener muscles express four types of K currents: inward rectifier; A-type [IK(A)]; delayed rectifier [IK(V)], and Ca2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and a leakage current. ACh activates a positive-reversing cationic current [IACh(cat)] and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ from the ARC in that, in the openers, activation of IK(A) occurs approximately 9 mV more positive and there is much less IACh(Cl). In both muscles, IACh(cat) most likely serves to depolarize the muscle until ICa activates to supply Ca2+ for contraction, but further depolarization and spiking is opposed by coactivation of IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) and IACh(Cl) may well be key factors that prevent spikes in the ARC but often allow them in the opener muscles. As in the ARC, the modulators enhance ICa and so potentiate contractions. They also activate a modulator-specific K current, which causes hyperpolarization and depression of contractions. Finally, in the opener muscles but not in the ARC, the modulators activate a depolarizing cationic current that may help phase-advance the contractions. Each modulator exerts these effects to different degrees and thus has a distinct effect on voltage and contraction size and shape. The overall effect will depend on the specific combinations of modulators released in different behaviors. By understanding the modulation in the opener muscles, as well as in the ARC, how the behavior of the two muscles is coordinated under a variety of circumstances can now be understood (Scott, 1997).

The molluscan neuropeptide FMRFamide has a number of inhibitory actions on the sensory neurons and motoneurons mediating the defensive gill and siphon withdrawal reflex pathway of Aplysia californica. Exogenous application of FMRFamide has a biphasic, dual-polarity effect on the majority of LFS siphon motoneurons, causing a transient depolarization followed by a prolonged hyperpolarization. FMRFamide induces this response in LFS neurons by causing an increase in multiple ionic currents, including a transient Na+ current, a slow prolonged Na+ current, a 4-aminopyridine (4-AP)-sensitive K+ current and a 4-AP-insensitive K+ current. A subset of LFS neurons exhibits an exclusively excitatory, biphasic response to FMRFamide, consisting of a transient depolarization followed by a prolonged depolarization of reduced magnitude. Over a period of 29 months, an increase in the incidence of the exclusively excitatory response was consistently observed during the summer months (June to September). From October to May, an exclusively excitatory response 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 the exclusively 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 transient Na+ current, a slow prolonged Na+ current and a 4-AP-insensitive K+ current. The principal difference between the two response types is that FMRFamide fails to activate a 4-AP-sensitive K+ current in those LFS neurons that exhibit an exclusively excitatory response to the peptide. In addition, LFS neurons with an exclusively excitatory response tend to show a coordinated increase in the magnitude of the inward current component of the FMRFamide response. Together, these changes during the summer months may enable this modulatory peptide to bring LFS neurons to suprathreshold levels of activity for eliciting a siphon withdrawal and should substantially alter the neuromodulatory effects of the peptide (Belkin. 1998).

This study examined differential modulation of motor neurons that innervate the same muscle but use different excitatory transmitters in Aplysia. The medial portion of intrinsic buccal muscle 3 (I3m) is innervated by two excitatory motor neurons, B3 and B9. B3 uses glutamate as its fast transmitter and expresses the neuropeptide FMRFamide, whereas B9 uses acetylcholine as its fast transmitter and expresses the neuropeptide SCP. This preparation was used to study peptidergic modulation of muscles innervated by neurons that use different fast excitatory transmitters. The effects of the application of the neuropeptides expressed in these neurons on excitatory junction 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 two excitatory neurons innervating the same muscle in opposite directions. SCP increases EJPs contraction amplitude, and the rate of muscle relaxation for both motor neurons. SCP potently increased cAMP levels in I3m as it does in other buccal muscles. Stimulation of B9 also causes increases cAMP levels in I3m, providing independent evidence for SCP release. Stimulation of B9 increases both the contraction amplitude and relaxation rate of B3-evoked I3m contractions in a manner similar to that observed using exogenous SCP. By inhibiting B9's cholinergic transmission with an antagonist, the modulatory effects of B9 could be observed in the absence of fast excitatory effects. The magnitude of the modulation is dependent on the firing frequency and occurs at frequencies and patterns of firing recorded 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, is innervated primarily by one motor neuron, B48. B48 is MM-IR and synthesizes authentic MM(A). When B48 is stimulated in a physiological manner, cAMP levels are increased in opener muscles. cAMP increases also are seen when the MMs are applied to opener muscles but are not seen with application of the B48 primary neurotransmitter acetylcholine (ACh). Possible physiological sources of 5-HT and FMRFamide are discussed. When modulators are applied to resting opener muscles, changes in membrane potential are observed. Specifically, 5-HT, MM(B), and low concentrations of MM(A) all depolarize muscle fibers. This depolarization is generally not sufficient to elicit myogenic activity in the absence of neural activity under 'rest' conditions. However, if opener muscles are stretched beyond rest length, stretch- and modulator-induced depolarizations can summate and elicit contractions. This only occurs, however, if 'depolarizing' modulators are applied alone. Thus other modulators [i.e., FMRFamide and high concentrations of MM(A)] hyperpolarize opener muscle fibers and can prevent depolarizing modulators from eliciting myogenic activity. All modulators tested affected parameters of motor neuron-elicited contractions of opener muscles. MM(B) and 5-HT increase contraction size over the range of concentrations tested, whereas MM(A) potentiates contractions when it is applied at lower concentrations but decreases contraction size at higher concentrations. FMRFamide decreases contraction size at all concentrations and does not affect relaxation rate. Additionally, the MMs and 5-HT increase muscle relaxation rate, decrease contraction latency, and decrease the rate at which tension is developed during motor neuron-elicited muscle contractions. Thus these modulators dramatically affect the ability of opener muscles to follow activity in the opener motor neuron B48. The possible physiological significance of these findings is discussed (Evans, 1999).

FMRFamide, synaptic modification and learning

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. A search was carried out for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localize a neuron in the left pleural ganglion, termed LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing 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 connections between sensory neurons and motor neurons in Aplysia. The first involves broadening of the presynaptic spike through modulation of 5-HT-sensitive K+ channels that leads to elevated levels of intracellular Ca2+ and increased release of transmitter. A second process (or set of processes) apparently accounts for the amount of facilitation not produced by presynaptic spike broadening. This spike duration-independent (SDI) process is particularly prominent in depressed synapses. A protocol was used in which spikes are prebroadened into a range of durations in which further spike broadening by itself has little or no effect on facilitation of the EPSP. 5-HT produces pronounced facilitation in depressed synapses under these conditions. Another modulatory agent, small cardioactive peptide (SCPb), also broadens spikes in sensory neurons but does not produce facilitation comparable to that produced by 5-HT. These results indicate that 5-HT activates the SDI process whereas SCPb fails to do so. A 5 min preexposure to the modulatory peptide FMRFamide inhibits 5-HT-induced activation of the SDI process, whereas a 1 min preexposure does not. Another process that may modulate synaptic efficacy in sensorimotor synapses involves a change in the properties of the motor (follower) neuron, such as input resistance. FMRFamide decreases the input resistance of postsynaptic neurons. This action could contribute to the effects of FMRFamide when administered alone, but it does not appear to be responsible 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 these three agents, therefore, seem to be differentially distributed among various pre- and postsynaptic 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 be regulated either by electrical activity or by specific neurotransmitters. The expression of neural cell adhesion molecule (NCAM)-like molecules in Aplysia (see Drosophila Fasciclin II), designated apCAM, is downregulated from the surface of sensory neurons by 5-HT, a transmitter known 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 be regulated by treatments with other neurotransmitters known to evoke long-term functional and structural changes in Aplysia neurons was tested, as well as the consequences of treatments with the neurotransmitters on the pattern of growth cone-neurite interactions. Applications of the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) that evoke long-term synaptic depression also reduce apCAM expression on the surface of motor cell L7 via a mechanism that appears to be similar to the mechanism mediating the 5-HT-induced change in the sensory cells. Specific treatments that affect apCAM distribution on the surface of their respective cells (5-HT on sensory cells and FMRFamide on motor cell L7) mimic treatment with monoclonal antibodies against apCAM by evoking a significant reduction in the fasciculation of growth cones with other neurites extending from homologous cells (Peter, 1994).

FMRFamide evokes long-term inhibition of the sensorimotor connection of Aplysia, including structural alterations in the presynaptic sensory cell. FMRFamide also evokes a down-regulation of the adhesion molecule apCAM from the surface of the postsynaptic motor cell L7. The second messenger pathways mediating the 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 of long-term functional and structural plasticity. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism, but not the cyclooxygenase pathway, blocks the long-term changes in the presynaptic sensory cell evoked by FMRFamide. The down-regulation of apCAM in L7 appears to be mediated by cAMP-dependent activation of protein kinase A. Blocking the cAMP-dependent changes also blocks FMRFamide-induced long-term functional and structural changes. These results suggest that the expression of long-term heterosynaptic inhibition in Aplysia may require concomitant presynaptic and postsynaptic changes, each transduced by specific 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 the formation of new presynaptic varicosities by 5-HT and the elimination of existing presynaptic varicosities by FMRFamide. The formation of new sensory varicosities is directed by the presence of preexisting zones on the motor axon that are enriched for apCAM. Moreover, there was a further enrichment of apCAM levels at existing sensory varicosities contacting the motor axon beginning at 1 hr and lasting 24 hr after treatment with 5-HT. As was found for synapse formation during the early stages of cell-cell interaction, incubation with anti-apCAM mAb blocks the 5-HT-induced long-term changes in synaptic efficacy and the accompanying changes in sensory neuron structure. Long-term synaptic depression with FMRFamide is accompanied by an overall decline of apCAM levels. Treatment with FMRFamide evokes an even greater decline in apCAM levels at sites of sensory varicosities that precede the structural changes and persist especially at sites where sensory varicosities are eliminated. These results suggest that neurotransmitters evoke both cell- and site-specific changes in the levels of adhesion molecules that can influence either the formation or the elimination of presynaptic varicosities that accompany long-term heterosynaptic modulation of a behaviorally relevant synaptic connection (Zhu, 1995).

Synaptic transmission and excitability in Aplysia sensory neurons (SNs) are bidirectionally modulated by 5-HT and FMRFamide. To explore the regional distribution of different functional receptors that modulate SN properties, changes in synaptic efficacy and excitability were examined upon brief focal applications of the neuromodulators to different regions of SNs that have established connections with motor cell L7 in culture. Short-term changes in synaptic efficacy are evoked only when 5-HT or FMRFamide is applied to regions with SN varicosities along the surface of L7 axons. Applications to adjacent SN neurites with few varicosities in contact with L7 axons fail to evoke a significant change in synaptic efficacy. The distribution of functional receptors mediating changes in excitability differ for 5-HT and FMRFamide. Whereas excitability increases are evoked only when 5-HT is applied to SN cell bodies, excitability decreases in SNs are evoked only when FMRFamide is applied to regions along the L7 axon with SN varicosities. Without the target cell, cell bodies of SNs express both 5-HT and FMRFamide receptors that modulate excitability. These results indicate that functional G-protein-coupled receptors for two neuromodulators are distributed differentially along the surface of a presynaptic neuron that forms chemical connections in vitro. This differential distribution of receptors on the presynaptic neuron is regulated by a target and does not require the physical presence of neurons that release the neuromodulators (Sun, 1996).

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. Immunocytochemical methods were used to examine whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT, which evokes long-term facilitation or FMRFamide, which evokes long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7, as compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoke changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites (Santarelli, 1996).

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