Neuropeptide cotransmitters released from an identified cholinergic motor neuron modulate neuromuscular efficacy in Aplysia

Intrinsic buccal muscle 5 (I5) in Aplysia is innervated by 2 motor neurons (termed B15 and B16). In addition to the classical transmitter ACh, B15 also contains the 2 neuropeptides SCPA and SCPB. In a previous study, we demonstrated that the SCPs were released from the terminals of B15 in the I5 muscle and that this release was sufficient to raise cAMP levels in I5 muscle fibers. Significant peptide release occurred only when B15 was stimulated at high frequency or at lower frequencies with a relatively long burst duration (Whim and Lloyd, 1989). In the present article, we examine the possibility that the SCPs released from B15 modulate I5 muscle contractions produced by stimulation of the second motor neuron, B16. Application of exogenous SCPs to I5 muscles increased the amplitude and relaxation rate of B16-evoked contractions. Stimulation of B15 using paradigms that have been shown previously to cause release of the SCPs resulted in a long-lasting increase in the amplitude and relaxation rate of muscle contractions evoked by B16. This modulation is unlikely to be due to the B15-induced muscle contractions themselves, because modulation of B16-evoked contraction amplitude and relaxation rate was observed when the contractions were blocked transiently by a cholinergic antagonist during B15 stimulation. Conversely, stimulation of B15 at frequencies that produce no measurable release of the SCPs did not elicit significant modulation of B16-evoked contractions. The minimum B15 stimulation frequency required to elevate muscle cAMP levels or to modulate B16-evoked contractions was found to be within the physiological range at which B15 fires during feeding. Therefore, the mechanism underlying the modulation of B16-evoked contractions by B15 is likely to involve the release of the SCPs from B15 terminals in the I5 muscle. With respect to behavior, this modulation of muscle contractions would be most likely to occur during food-induced arousal when both motor neurons fire at high frequency with brief interburst intervals.     

Modulation of peptide release from single identified Aplysia neurons in culture.

Aplysia neurons B1 and B2 contain large amounts of the neuropeptides SCPA and SCPB. When grown in culture, individual B1 and B2 cells incorporate 35S-methionine into the SCPs, which can be released in a stimulus- and calcium-dependent fashion (Lloyd et al., 1986). We now show that single cells can be stimulated in a manner to evoke release of the SCPs that declines only slightly with repeated stimulation. This has allowed us to examine the ability of several physiologically relevant agonists to modulate the stimulus-evoked release of the SCPs. Bath application of either FMRFamide or 5-HT resulted in a significant decrease in the amount of SCPs released by intracellular stimulation of B1 or B2. The action of 5-HT was dose dependent with an inhibition of release of approximately 70% at a concentration of 100 microM. SCPA did not significantly affect release. The bath application of several compounds that are expected to elevate intracellular levels of cAMP were also found to depress release. To investigate the possibility that the agonists inhibited the release of the SCPs via a hyperpolarization of membrane potential (and perhaps a loss of spikes in the neurites), we examined the actions of 5-HT, FMRFamide, and SCPA on several electrophysiological parameters intended to monitor the level of cell excitability. Surprisingly, even though 5-HT depressed the release of the SCPs from both cells, it depolarized and increased the excitability of B1, and hyperpolarized and decreased the excitability of B2. Furthermore, in contrast to the effects seen in culture, 5-HT depolarized both B1 and B2 in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP only partially mediates the actions of serotonin at lobster neuromuscular junctions

Serotonin (5-HT) has multiple physiological actions at lobster neuromuscular junctions, including facilitation of transmitter release from nerve terminals and an increase in the tone and excitability of muscle fibers. These physiological effects of 5-HT are accompanied by a rise in intracellular levels of cAMP. We have used combined biochemical and physiological approaches to investigate whether cAMP directly mediates the physiological actions of the hormone. Based on the following lines of evidence, we conclude that the postsynaptic increase in muscle tone occurs independently of cAMP and that while the cyclic nucleotide does play a role in the facilitation of transmitter release by 5-HT, there is also a cAMP-independent component to this facilitation. (1) Agents that mimic the action of 5-HT on cAMP levels (forskolin, IBMX, SQ20009, 8-bromo cAMP) fail to mimic the postsynaptic actions of the amine. These agents do facilitate transmitter release, although none of them has as large an effect as does 5-HT. (2) When 5-HT is removed, presynaptic facilitation decays as the sum of 2 exponentials with very different time courses. The rate of the slower process is similar to the rate of breakdown of cAMP, while the faster process and the postsynaptic response decay significantly more rapidly. (3) IBMX retards the breakdown of cAMP and simultaneously retards the decay of the slower presynaptic process, with little or no effect on the other responses. (4) IBMX and forskolin potentiate the effect of 5-HT on cAMP levels and selectively enhance the slowly decaying presynaptic component with little or no effect on the other responses.     

Modulation of calcium current and diverse K+ currents in identified Hermissenda neurons by small cardioactive peptide B

The molluscan neuropeptides, small cardioactive peptides A and B (SCPA,B), are known to modulate the responses of many molluscan central and peripheral target cells (see review by Lloyd, 1986), but their full range of physiological actions remains unknown. External application of SCPB (1-10 microM) modified diverse ionic conductances in a set of giant identifiable neurons in the brain of the marine mollusk Hermissenda crassicornis. SCPB caused a transient depolarization and increased input resistance that enhanced or promoted cell firing. Under voltage-clamp, SCPB reduced a "background" residual current (IR), reduced early transient K+ current (IA), reduced a delayed K+ current (IK(V], and enhanced ICa, IBa, and a Ca2+-activated K+ current, IK(Ca). In tetraethylammonium chloride (TEA) saline, SCPB enhanced the amplitude and duration and reduced the threshold of evoked Ca and Ba spikes. Immunocytochemical staining techniques have localized an endogenous SCPB-like peptide in numerous somata and their neurites in the nervous system of Hermissenda (Longley and Longley, 1985; Watson and Willows, 1986). These data are consistent with a role for SCPB as a neurotransmitter/neurohormone modulator of neuronal excitability in Hermissenda. A neurotransmitter role for endogenous SCPs has been proposed for a synaptic pair of cultured neurons in the Aplysia buccal ganglion (Lloyd et al., 1986). SCPB has been implicated in the control of feeding motor output in Aplysia (Sossin et al., 1986) and Tritonia (Willows and Watson, 1986), and in the presynaptic facilitation of sensory neurons mediating the gill and siphon defensive withdrawal reflex in Aplysia (Abrams et al., 1984).     

Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.     

Localization of potential serotonergic facilitator neurons in Aplysia by glyoxylic acid histofluorescence combined with retrograde fluorescent labeling

A variety of evidence suggests that 5-HT participates in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) have shown that the 5-HT neurotoxin 5,7-DHT markedly reduces both the synaptic facilitation and behavioral dishabituation produced by tail shock. To provide more direct evidence for a role of 5-HT, I have used histological techniques to try to locate individual serotonergic facilitator neurons. I first used a modification of the glyoxylic acid histofluorescence technique to map serotonergic and dopaminergic neurons in the CNS of Aplysia. Intracellular fluorescent labeling combined with histofluorescence indicates that the previously identified L29 facilitator neurons are not serotonergic. Nerve transection experiments suggest that most of the perisomatic 5-HT histofluorescence in the abdominal ganglion (the location of the siphon sensory cells) comes from neurons whose cell bodies are located in the pedal or cerebral ganglia. As there are at least 500 serotonergic neurons in those ganglia, I combined retrograde fluorescent labeling with histofluorescence to identify a small subset of those neurons which send processes to the abdominal ganglion and are therefore potential serotonergic facilitators. In the following paper, Mackey et al. (1989) show that stimulation of 2 of those neurons in the cerebral ganglia (the CB1 cells) produces presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the withdrawal reflex.     

Biochemical and immunocytological localization of molluscan small cardioactive peptides in the nervous system of Aplysia californica

High pressure liquid chromatography (HPLC) followed by bioassay on isolated snail hearts were used to locate two related peptides, termed small cardioactive peptides A and B (SCPA and SCPB) in each of the central ganglia of Aplysia. The peptides are most concentrated in the buccal ganglia, the ganglia involved in the control of feeding movements. Immunocytology with antisera raised to conjugated SCPB stained three groups of neurons in the buccal ganglia. One group consisted of relatively small neurons that were tightly clustered. The second group was comprised of larger neurons that were more scattered. The third group was made up of several neurons including the two largest in the ganglia, identified cells B1 and B2. B1 and B2 and other neurons in this group innervate the gut by way of the esophageal nerve. HPLC-bioassay of single, individually dissected B1 or B2 neurons demonstrated that the two peptides are present in a single cell. For B2, but not B1, choline injected into the cell body was converted to the conventional transmitter, acetylcholine. This indicates that, in addition to the two peptides, B2 also contains choline acetyltransferase, and raises the possibility that acetylcholine and the SCPs may act as co-transmitters in B2. Strong immunocytological staining of fibers and varicosities was observed in the neuropilar region of the cerebral, pleural, pedal, and abdominal ganglia. In addition to the buccal ganglia, immunoreactive neurons were observed in all of the other central ganglia. The high concentration of the SCPs and the relatively large number of immunoreactive neurons in the buccal ganglion suggest a particularly important role of these peptides specifically in feeding behavior. However, the widespread occurrence of the SCPs in fibers and neuronal cell bodies throughout the nervous system suggests that these peptides also may have additional behavioral functions in Aplysia.     

Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.     

Selective modulation of spike duration by serotonin and the neuropeptides, FMRFamide, SCPB, buccalin and myomodulin in different classes of mechanoafferent neurons in the cerebral ganglion of Aplysia

An examination of the cellular properties and synaptic outputs of mechanoafferent neurons found on the ventrocaudal surface of the cerebral ganglion of Aplysia indicated that the cerebral mechanoafferent (CM) neurons are a heterogeneous population of cells. Based on changes in action potential duration in response to bath applications of 5-HT in the presence of TEA, CM neurons could be divided into 2 broad classes: mechanoafferents whose spikes broaden in response to 5-HT (CM-SB neurons) and mechanoafferents whose spikes narrow in response to 5-HT (CM-SN neurons). Morphological and electrophysiological studies of the CM-SN neurons indicated that they were comprised of previously identified interganglionic cerebral-buccal mechanoafferent (ICBM) neurons and a novel set of sensory neurons that send an axon into the LLAB cerebral nerve and have perioral zone receptive fields that are similar to those of ICBM neurons. Changes in spike width due to 5-HT were correlated with changes in synaptic output as indicated by the magnitudes of EPSPs evoked in postsynaptic neurons. Electrical stimulation of cerebral nerves and connectives also produced spike narrowing or broadening, and the sign of the effect was a function of the parameters of stimulation. Both heterosynaptic facilitation and heterosynaptic depression of EPSPs evoked in follower cells could be demonstrated. A variety of putative neuromodulators other than 5-HT were also found to affect the duration of action potentials in both classes of CM neurons. FMRFamide had effects opposite to that of 5-HT. SCPB and a recently characterized Aplysia neuropeptide, buccalin, broadened the spikes of both CM classes. Another neuropeptide, myomodulin, decreased the duration of CM-SB neuron spikes but had no effect on CM-SN spikes. Since the CM neurons appear to mediate a variety of competing behaviors, including feeding, locomotion, and defensive withdrawal, the various neuromodulator actions may contribute to the mechanisms whereby behaviors are selected and modified.     

Synaptic facilitation by ectopic octopamine and 5-HT receptors in Aplysia

The cAMP pathway plays a critical role in synaptic plasticity. We assessed using the ectopic expression of octopamine (OA) receptor, the contribution of the cAMP pathway to short-term facilitation of sensory-motor synapses in Aplysia. When synaptic connections were depressed to 20-30% of their initial EPSP amplitude, the application of OA to sensory cells expressing OA receptor showed significant synaptic facilitation, but this was less than the synaptic facilitation shown by 5-HT treatment. We also found that synaptic facilitation was further enhanced when OA was treated in the presence of 5-HT at non-depressed synapses, but not at depressed synapses. These results imply that the role of cAMP in synaptic facilitation is reduced as the synapse becomes depressed due to repeated activity.     

Biochemical and immunocytological localization of the neuropeptides FMRFamide, SCPA, SCPB, to neurons involved in the regulation of feeding in Aplysia

The localization of the neuropeptide FMRFamide in the buccal ganglia and buccal muscles of Aplysia was studied by immunocytology and high-pressure liquid chromatography (HPLC) combined with either a sensitive bioassay or 35S-methionine labeling. Immunocytology with an antiserum directed to FMRFamide stained a large number of fibers, varicosities, and neuronal somata. Two groups of stained neurons were of particular interest. One was the S cells, a group comprised of many small neurons, the majority of which were stained. HPLC of pooled labeled S cells confirmed that at least some of these neurons synthesize FMRFamide. The other group of stained neurons were in the ventral cluster, a group comprised of a small number of large neurons, many of which are motor neurons that innervate the buccal muscles involved in producing biting and swallowing movements. Several of the ventral neurons were previously shown to contain 2 other neuropeptides, the small cardioactive peptides SCPA and SCPB. These neurons are sufficiently large to permit HPLC analyses of the neuropeptides synthesized by individual neurons. This procedure confirmed that individual ventral neurons synthesized FMRFamide, or the SCPs, or all 3 peptides. The coexistence of FMRFamide and the SCPs in the same neuron was confirmed by simultaneous staining of sections from the buccal ganglia with a monoclonal antibody to the SCPs and an antiserum to FMRFamide. The coexistence of the 3 peptides in the same neuron was surprising in light of the observations that these peptides often have opposite biological activity. The ventral neurons are large and potentially identifiable as individuals. Thus, these neurons may be particularly useful for studying the physiological and behavioral roles of neuropeptides in generating complex behaviors.     

G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse

At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-gamma-S, prevented by pre-injection of guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered.     

Serotonin increases GABA release in rat entorhinal cortex by inhibiting interneuron TASK-3 K+ channels

Whereas the entorhinal cortex (EC) receives profuse serotonergic innervations from the raphe nuclei in the brain stem and is critically involved in the generation of temporal lobe epilepsy, the function of serotonin (5-hydroxytryptamine, 5-HT) in the EC and particularly its roles in temporal lobe epilepsy are still elusive. Here we explored the cellular and molecular mechanisms underlying 5-HT-mediated facilitation of GABAergic transmission and depression of epileptic activity in the superficial layers of the EC. Application of 5-HT increased sIPSC frequency and amplitude recorded from the principal neurons in the EC with no effects on mIPSCs recorded in the presence of TTX. However, 5-HT reduced the amplitude of IPSCs evoked by extracellular field stimulation and in synaptically connected interneuron and pyramidal neuron pairs. Application of 5-HT generated membrane depolarization and increased action potential firing frequency but reduced the amplitude of action potentials in presynaptic interneurons suggesting that 5-HT still increases GABA release whereas the depressant effects of 5-HT on evoked IPSCs could be explained by 5-HT-induced reduction in action potential amplitude. The depolarizing effect of 5-HT was mediated by inhibition of TASK-3 K(+) channels in interneurons and required the functions of 5-HT(2A) receptors and Galpha(q/11) but was independent of phospholipase C activity. Application of 5-HT inhibited low-Mg(2+)-induced seizure activity in slices via 5-HT(1A) and 5-HT(2A) receptors suggesting that 5-HT-mediated depression of neuronal excitability and increase in GABA release contribute to its anti-epileptic effects in the EC.     

Association of neuroactive peptides with the protein secretory pathway in identified neurons of Aplysia californica: immunolocalization of SCPA and SCPB to the contents of dense-core vesicles and the trans face of the Golgi apparatus

The subcellular distribution of two molluscan neuropeptides, the small cardioactive peptides A and B (SCPA and SCPB), has been determined in two identified Aplysia buccal ganglion neurons, B1 and B2. These neurons were previously shown to synthesize and release these neuropeptides. B1 and B2, identified by their size and location within the ganglion, were labeled by intrasomatic injection of an electron-dense particulate marker (ferritin or Imposil) permitting the unequivocal identification of their somata and proximal processes in thin sections. The somatic cytoplasm of both neurons had a conspicuous population of large dense-core vesicles along with a smaller number of compound vesicles and small lucent vesicles. All three vesicle types are found in the neurites within the neuropil and proximal axon in the esophageal nerve. Immunoreactivity was localized on the surface of thin sections by the indirect immunogold method. The primary antiserum was shown to recognize both SCPA and SCPB after the neuropeptides had been immobilized on protein-coated nitrocellulose membranes by means of glutaraldehyde, the primary fixative used to immobilize SCPA and SCPB in situ. SCP immunoreactivity was present in the lumens of the dense-core vesicles distributed throughout the cytoplasm of B1 and B2 and in dense-core regions of the Golgi apparatus in the somatic cytoplasm. Taken together with biochemical evidence that B1 and B2 synthesize and release SCPs, these data suggest that the neuropeptides are sequestered into the protein secretory pathway of B1 and B2, a distribution that supports the notion that the SCPs function physiologically as neurotransmitters or neuromodulators.     

Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia

Neuropeptide synthesis was determined for individual identified ventral-cluster neurons in the buccal ganglia of Aplysia. Each of these cells was shown to be a motor neuron that innervates buccal muscles that generate biting and swallowing movements during feeding. Individual neurons were identified by a battery of physiological criteria and stained with intracellular injection of a vital dye, and the ganglia were incubated in 35S-methionine. Peptide synthesis was determined by measuring labeled peptides in extracts from individually dissected neuronal cell bodies analyzed by HPLC. Previously characterized peptides found to be synthesized included buccalin, FMRFamide, myomodulin, and the 2 small cardioactive peptides (SCPs). Each of these neuropeptides has been shown to modulate buccal muscle responses to motor neuron stimulation. Two other peptides were found to be synthesized in individual motor neurons. One peptide, which was consistently observed in neurons that also synthesized myomodulin, is likely to be the recently sequenced myomodulin B. The other peptide was observed in a subset of the neurons that synthesize FMRFamide. While identified motor neurons consistently synthesized the same peptide(s), neurons that innervate the same muscle often express different peptides. Neurons that synthesized the SCPs also contained SCP-like activity, as determined by snail heart bioassay. Our results indicate that every identified motor neuron synthesizes a subset of these methionine-containing peptides, and that several neurons consistently synthesize peptides that are likely to be processed from multiple precursors.     

Differential effects of serotonin, FMRFamide, and small cardioactive peptide on multiple, distributed processes modulating sensorimotor synaptic transmission in Aplysia.

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. We used a protocol in which spikes were 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 produced pronounced facilitation in depressed synapses under these conditions. Another modulatory agent, small cardioactive peptide (SCPb), also broadened spikes in sensory neurons but did 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 the same. A 5 min preexposure to the modulatory peptide FMRFamide inhibited 5-HT-induced activation of the SDI process, whereas a 1 min preexposure did not. Another process(es) that may modulate synaptic efficacy in sensorimotor synapses involves changes in the properties of the motor (follower) neuron, such as input resistance. FMRFamide decreased the input resistance of postsynaptic neurons. This action could contribute to the effects of FMRFamide when administered alone, but it did not appear to be responsible for the inhibitory action of FMRFamide on 5-HT-induced facilitation. Neither 5-HT nor SCPb had 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.     

Enhanced synaptic transmission at identified synaptic connections in the cerebral ganglion of Aplysia.

The identified A-B neuron synaptic connections in the cerebral ganglion of Aplysia exhibited a novel form of enhanced synaptic transmission. A brief high-frequency train of action potentials (2 s, 10-30 Hz) in the presynaptic A neurons produced a long-lasting increase in the amplitude of excitatory postsynaptic potentials (EPSPs) in B neurons. The increase in synaptic efficacy was termed slow developing potentiation (SDP) since the EPSP amplitude increased slowly with the peak occurring 5 min after the tetanizing train. Peak EPSP amplitudes increased relative to the initial EPSP by an average of greater than 250%. SDP decayed as a single exponential with a time constant of tau = 24 min. The enhanced transmission was neuron specific. Only the connections made by the tetanized A neuron were potentiated. However, potentiation apparently occurred at all the synapses made by the tetanized A neuron. Tetanizing the postsynaptic B neurons neither induced, nor when paired with A neuron tetanization, increased SDP. SDP appears to be primarily due to increased transmitter release by the presynaptic neuron.     

Long-lasting Potentiation of a Direct Central Connection between Identified Motor Neurons in the Locust

The plasticity of the direct central connection between the fast extensor and the posterior fast flexor tibiae motor neurons in the locust ( Schistocerca gregaria ) metathoracic ganglion was studied. An action potential in the fast extensor results in a monosynaptic excitatory postsynaptic potential (EPSP) in the flexor motor neuron. Antidromic stimulation of the fast extensor at 100 Hz for 3.5 s resulted in a long-lasting potentiation of the EPSP amplitude. The potentiation was not dependent on feedback caused by movement of the tibia, and was associated with an increase in the input resistance of the flexor motor neuron. The potentiation was heterosynaptic, and was not affected by bath application of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid. The potentiation was voltage dependent, as hyperpolarizing the flexor motor neuron during the stimulation blocked the development of the potentiation whereas depolarizing the flexor in the absence of presynaptic activity caused potentiation of subsequent fast extensor-evoked EPSPs. The depolarization-induced potentiation was calcium dependent. Antidromic stimulation of the fast extensor at 100 Hz for 3.5 s also caused modulation of the presynaptic action potential. The spike duration was increased and the amplitude of the afterhyperpolarization reduced. These effects were dependent on movement of the tibia. Bath application of the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin blocked the changes in the presynaptic spike. The modulation was probably due to the release of 5-HT from proprioceptive afferents that monitor movement of the tibia about the femur. The modulation of the presynaptic action potential increases transmitter release onto the flexor motor neurons, and this acts in synergy with the postsynaptic modulation to potentiate the connection.