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Kinases play critical roles in synaptic and neuronal changes involved in the formation of memory. However, significant gaps exist in the understanding of how interactions among kinase pathways contribute to the mechanistically distinct temporal domains of memory ranging from short-term memory to long-term memory (LTM). Activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathways are critical for long-term enhancement of neuronal excitability (LTEE) and long-term synaptic facilitation (LTF), essential processes in memory formation. This study provides new insights into how these pathways contribute to the temporal domains of memory, using empirical and computational approaches. Empirical studies of Aplysia sensory neurons identified a positive feedforward loop in which the PKA and ERK pathways converge to regulate RSK, and a negative feedback loop in which p38 MAPK inhibits the activation of ERK and RSK. A computational model incorporated these findings to simulate the dynamics of kinase activity produced by different stimulus protocols and predict the critical roles of kinase interactions in the dynamics of these pathways. These findings may provide insights into the mechanisms underlying aberrant synaptic plasticity observed in genetic disorders such as RASopathies and Coffin-Lowry syndrome., (© 2021. The Author(s).)

The MAPK isoforms ERK and p38 MAPK are believed to play opposing roles in long-term synaptic facilitation (LTF) induced by serotonin (5-HT) in Aplysia . To fully understand their roles, however, it is necessary to consider the dynamics of ERK and p38 MAPK activation. Previous studies determined that activation of ERK occurred ∼45 min after a 5-min pulse of 5-HT treatment. The dynamics of p38 MAPK activation following 5-HT are yet to be elucidated. Here, the activity of p38 MAPK was examined at different times after 5-HT, and the interaction between the ERK and p38 MAPK pathways was investigated. A 5-min pulse of 5-HT induced a transient inhibition of p38 MAPK, followed by a delayed activation between 25 and 45 min. This activation was blocked by a MAPK kinase inhibitor, suggesting that similar pathways are involved in activation of ERK and p38 MAPK. ERK activity decreased shortly after the activation of p38 MAPK. A p38 MAPK inhibitor blocked this decrease in ERK activity, suggesting a causal relationship. The p38 MAPK activity ∼45 min after different stimulus protocols was also characterized. These data were incorporated into a computational model for the induction of LTF. Simulations and empirical data suggest that p38 MAPK, together with ERK, contributes to the efficacy of spaced stimulus protocols to induce LTF, a correlate of long-term memory (LTM). For example, decreased p38 MAPK activity ∼45 min after the first of two sensitizing stimuli might be an important determinant of an optimal interstimulus interval (ISI) for LTF induction.

Aplysia sensorimotor synapses provide a useful model system for analyzing molecular processes that contribute to heterosynaptic plasticity. For example, previous studies demonstrated that multiple kinase cascades contribute to serotonin (5-HT)-induced short-term synaptic facilitation (STF), including protein kinase A (PKA) and protein kinase C (PKC). Moreover, the contribution of each kinase is believed to depend on the state of the synapse (e.g., depressed or nondepressed) and the time after application of 5-HT. Here, a previously unappreciated role for PKC-dependent processes was revealed to underlie the maintenance of STF at relatively nondepressed synapses. This PKC dependence was revealed when the synapse was stimulated repeatedly after application of 5-HT. The contributions of the PKA and PKC pathways were examined by blocking adenylyl cyclase-coupled 5-HT receptors with methiothepin and by blocking PKC with chelerythrine. STF was assessed 20 s after 5-HT application. The effects of PKC were consistent with enhanced mobilization of transmitter, as assessed by application of hypertonic sucrose solutions to measure the readily releasable pool of vesicles and recovery of the readily releasable pool after depletion. A computational model of transmitter release demonstrated that a PKC-dependent mobilization process was sufficient to explain the maintenance of STF at nondepressed synapses and the facilitation of depressed synapses., (Copyright © 2014 the American Physiological Society.)

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be topoisomerase II inhibition, DNA cleavage, and free radical generation. However, in non-neuronal cells, DOX also inhibits the expression of dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK), two MAPK isoforms important for long-term memory (LTM) formation. Activation of these kinases by DOX in neurons, if present, could have secondary effects on cognitive functions, such as learning and memory. The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia to examine the effects of DOX on levels of phosphorylated ERK (pERK) and phosphorylated p38 (p-p38) MAPK. In addition, Aplysia neurons were used to examine the effects of DOX on long-term enhanced excitability, long-term synaptic facilitation (LTF), and long-term synaptic depression (LTD). DOX treatment led to elevated levels of pERK and p-p38 MAPK in SNs and cortical neurons. In addition, it increased phosphorylation of the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs. DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to overriding inhibitory effects of p-p38 MAPK, because LTF was rescued in the presence of an inhibitor (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) of p38 MAPK. These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway., (Copyright © 2014 the authors 0270-6474/14/3413289-12$15.00/0.)

Serotonin (5-HT)-induced long-term facilitation (LTF) of the Aplysia sensorimotor synapse depends on enhanced gene expression and protein synthesis, but identification of the genes whose expression and regulation are necessary for LTF remains incomplete. In this study, we found that one such gene is synapsin, which encodes a synaptic vesicle-associated protein known to regulate short-term synaptic plasticity. Both synapsin mRNA and protein levels were increased by 5-HT. Upregulation of synapsin protein occurred in presynaptic sensory neurons at neurotransmitter release sites. To investigate the molecular mechanisms underlying synapsin regulation, we cloned the promoter region of Aplysia synapsin, and found that the synapsin promoter contained a cAMP response element (CRE), raising the possibility that the transcriptional activator CRE-binding protein 1 (CREB1) mediates 5-HT-induced regulation of synapsin. Indeed, binding of CREB1 to the synapsin promoter was increased following treatment with 5-HT. Furthermore, increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 near the CRE site are consistent with transcriptional activation by CREB1. RNA interference (RNAi) targeting synapsin mRNA blocked the 5-HT-induced increase in synapsin protein levels and LTF; in the absence of 5-HT treatment, basal synapsin levels were unaffected. These results indicate that the 5-HT-induced regulation of synapsin levels is necessary for LTF and that this regulation is part of the cascade of synaptic events involved in the consolidation of memory.

Long-term memory and plasticity, including long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse, depend on the activation of transcription factors that regulate genes necessary for synaptic plasticity. In the present study we found that treatment with 5-HT and behavioral training produce biphasic changes in the expression of CREB2, a transcriptional repressor. An immediate increase in CREB2 protein was followed by a subsequent decrease. The effects of these treatments persist for at least 24 h and are observed in isolated sensory neurons. This study suggests that the dynamics of CREB2 expression could contribute to the consolidation of memory.

Synapsin is a synaptic vesicle-associated protein implicated in the regulation of vesicle trafficking and transmitter release, but its role in heterosynaptic plasticity remains elusive. Moreover, contradictory results have obscured the contribution of synapsin to homosynaptic plasticity. We previously reported that the neuromodulator serotonin (5-HT) led to the phosphorylation and redistribution of Aplysia synapsin, suggesting that synapsin may be a good candidate for the regulation of vesicle mobilization underlying the short-term synaptic plasticity induced by 5-HT. This study examined the role of synapsin in homosynaptic and heterosynaptic plasticity. Overexpression of synapsin reduced basal transmission and enhanced homosynaptic depression. Although synapsin did not affect spontaneous recovery from depression, it potentiated 5-HT-induced dedepression. Computational analysis showed that the effects of synapsin on plasticity could be adequately simulated by altering the rate of Ca(2+)-dependent vesicle mobilization, supporting the involvement of synapsin not only in homosynaptic but also in heterosynaptic forms of plasticity by regulating vesicle mobilization.

The sensorimotor synapse of Aplysia exhibits long-term facilitation (LTF) and long-term depression (LTD) elicited by the neuromodulator serotonin (5-HT) and the peptide Phe-Met-Arg-Phe-NH(2), respectively. 5-HT-induced LTF engages extracellular-regulated kinase (Erk) and CREB1, whereas FMRFa-induced LTD engages p38 MAPK (mitogen-activated protein kinase) and CREB2. The interaction of the 5-HT and FMRFa pathways was recently investigated in Aplysia at the level of gene expression. However, little is known about crosstalk of these pathways at the level of the second messenger cascades. We investigated the potential interaction of the 5-HT and FMRFa pathways at the level of the Erk cascade. We found that FMRFa inhibited basal Erk activity through p38 MAPK. FMRFa also inhibited 5-HT-induced phosphorylation of Erk and nuclear accumulation of phospho-ERK, suggesting that FMRFa may place inhibitory constraints on memory formation through regulation of the Erk MAPK cascade.

The 5-HT-induced synaptic plasticity of Aplysia sensorimotor synapses has typically been probed by firing a single presynaptic spike. In this study, 5-HT-induced synaptic plasticity was probed with brief bursts of spikes (10 Hz, 1 s), which are more behaviorally relevant stimuli. Because such bursts provide a greater challenge to the release machinery than single spikes, their use may reveal additional aspects of synaptic modulation, and, in particular, the role of extracellular signal-regulated protein kinase (ERK), which has recently been implicated in several examples of short- and long-term synaptic plasticity. Excitatory postsynaptic currents (EPSCs) were characterized by their amplitudes. In addition, two kinetic measurements, time to peak and decay time constant, were determined for the initial and last EPSCs of each burst. Application of 5-HT produced a uniform increase in gain by facilitating each EPSC elicited during a burst of spikes without affecting the kinetics of the initial or last EPSC. These data suggest that short-term facilitation during a burst is mediated largely by processes such as those that affect the size of the releasable pool or rate of vesicle mobilization rather than by an increase in the duration of the presynaptic action potential. An ERK cascade inhibitor (U0126) had no effect on the 5-HT-mediated facilitation of either the initial EPSC or EPSCs elicited late in the burst.

Present models of long-term sensitization in Aplysia californica indicate that the enhanced behavioral response is due, at least in part, to outgrowth of sensory neurons mediating defensive withdrawal reflexes. Presumably, this outgrowth strengthens pre-existing connections by formation of new synapses with follower neurons. However, the relationship between the number of sensorimotor contacts and the physiological strength of the connection has never been examined in intact ganglia. As a first step in addressing this issue, we used confocal microscopy to examine sites of contact between sensory and motor neurons in naive animals. Our results revealed relatively few contacts between physiologically connected cells. In addition, the number of contact sites was proportional to the amplitude of the EPSP elicited in the follower motor neuron by direct stimulation of the sensory neuron. This is the first time such a correlation has been observed in the central nervous system. Serotonin is the neurotransmitter most closely examined for its role in modulating synaptic strength at the sensorimotor synapse. However, the structural relationship of serotonergic processes and sensorimotor synapses has never been examined. Surprisingly, serotonergic processes usually made contact with sensory and motor neurons at sites located relatively distant from the sensorimotor synapse. This result implies that heterosynaptic regulation is due to nondirected release of serotonin into the neuropil.

Only a small fraction of neurotransmitter-containing synaptic vesicles (SVs), the readily releasable pool, is available for fast Ca(2+)-induced release at any synapse. Most SVs are sequestered at sites away from the plasma membrane and cannot be exocytosed directly. Recruitment of SVs to the releasable pool is thought to be an important component of short-term synaptic facilitation by serotonin (5-HT) at Aplysia sensorimotor synapses. Synapsins are associated with SVs and hypothesized to play a central role in the regulation of SV mobilization in nerve terminals. Aplysia synapsin was cloned to examine its role in synaptic plasticity at the well characterized sensorimotor neuron synapse of this animal. Acute 5-HT treatment of ganglia induced synapsin phosphorylation. Immunohistochemical analyses of cultured Aplysia neurons revealed that synapsin is distributed in distinct puncta in the neurites. These puncta are rapidly dispersed after treatment of the neurons with 5-HT. The dispersion of synapsin puncta by 5-HT was fully reversible after washout of the modulator. Both 5-HT-induced phosphorylation and dispersion of synapsin were mediated, at least in part, by cAMP-dependent protein kinase and mitogen-activated protein kinase. These experiments indicate that synapsin and its regulation by 5-HT may play an important role in the modulation of SV trafficking in short-term synaptic plasticity.

The buccal ganglia of Aplysia contain a central pattern generator (CPG) that mediates rhythmic movements of the buccal apparatus during feeding. Activity in this CPG is believed to be regulated, in part, by extrinsic serotonergic inputs and by an intrinsic and extrinsic system of putative dopaminergic cells. The present study investigated the roles of dopamine (DA) and serotonin (5-HT) in regulating feeding movements of the buccal apparatus and properties of the underlying neural circuitry. Perfusing a semi-intact head preparation with DA (50 microM) or the metabolic precursor of catecholamines (L-3-4-dihydroxyphenylalanine, DOPA, 250 microM) induced feeding-like movements of the jaws and radula/odontophore. These DA-induced movements were similar to bites in intact animals. Perfusing with 5-HT (5 microM) also induced feeding-like movements, but the 5-HT-induced movements were similar to swallows. In preparations of isolated buccal ganglia, buccal motor programs (BMPs) that represented at least two different aspects of fictive feeding (i.e., ingestion and rejection) could be recorded. Bath application of DA (50 microM) increased the frequency of BMPs, in part, by increasing the number of ingestion-like BMPs. Bath application of 5-HT (5 microM) did not significantly increase the frequency of BMPs nor did it significantly increase the proportion of ingestion-like BMPs being expressed. Many of the cells and synaptic connections within the CPG appeared to be modulated by DA or 5-HT. For example, bath application of DA decreased the excitability of cells B4/5 and B34, which in turn may have contributed to the DA-induced increase in ingestion-like BMPs. In summary, bite-like movements were induced by DA in the semi-intact preparation, and neural correlates of these DA-induced effects were manifest as an increase in ingestion-like BMPs in the isolated ganglia. Swallow-like movements were induced by 5-HT in the semi-intact preparation. Neural correlates of these 5-HT-induced effects were not evident in isolated buccal ganglia, however.

Serotonergic modulation of the sensory neurons that mediate the gill- and tail-withdrawal reflexes of Aplysia is a useful model system for studies of neuronal plasticity that contributes to learning and memory. The effects of serotonin (5-HT) are mediated, in part, via two protein kinases (protein kinase A, PKA, and protein kinase C, PKC), which in turn, modulate at least four membrane currents, including a S ("serotonin-sensitive") K(+) current (I(K, S)), a steeply voltage-dependent K(+) current (I(K-V)), a slow component of the Ca(2+)-activated K(+) current (I(K,Ca-S)), and a L-type Ca(2+) current (I(Ca-L)). The present study investigated how the modulation of these currents altered the spike duration and excitability of sensory neurons and examined the relative contributions of PKA- and PKC-mediated effects to the actions of 5-HT. A Hodgkin-Huxley type model was developed that described the ionic conductances in the somata of sensory neurons. The descriptions of these currents and their modulation were based largely on voltage-clamp data from sensory neurons. Simulations were preformed with the program SNNAP (Simulator for Neural Networks and Action Potentials). The model was sufficient to replicate empirical data that describes the membrane currents, action potential waveform and excitability as well as their modulation by application of 5-HT, increased levels of adenosine cyclic monophosphate or application of active phorbol esters. In the model, modulation of I(K-V) by PKC played a dominate role in 5-HT-induced spike broadening, whereas the concurrent modulation of I(K,S) and I(K,Ca-S) by PKA primarily accounted for 5-HT-induced increases in excitability. Finally, simulations indicated that a PKC-induced increase in excitability resulted from decreases of I(K,S) and I(K,Ca-S), which was likely the indirect result of cross-talk between the PKC and PKA systems. The results provide several predictions that warrant additional experimental investigation and illustrate the importance of considering indirect as well as direct effects of modulatory agents on the modulation of membrane currents.

Serotonin (5-hydroxytryptamine, 5-HT) modulates the behavior and physiology of both vertebrate and invertebrate animals. Effects of injections of 5-HT and the morphology of the serotonergic system of Aplysia indicate that 5-HT may have a humoral, in addition to a neurotransmitter, role. To study possible humoral roles of 5-HT, we measured 5-HT in the hemolymph. The concentration of 5-HT in the hemolymph was approximately 18 nM, a value close to previously reported thresholds for eliciting physiological responses. The concentration of 5-HT in the hemolymph expressed a diurnal rhythm. In addition, electrical stimulation that leads to long-term sensitization significantly increased levels of 5-HT in the hemolymph during training, 1.5 hr after training, and 24 hr after training. Moreover, levels of 5-HT in the hemolymph were significantly correlated with the magnitude of sensitization. The half-life of an increase in 5-HT in the hemolymph was approximately 0.5 hr. Therefore, the persistent increase of 5-HT in the hemolymph 24 hr after sensitization training indicates that training caused a long-lasting increase in the release of 5-HT. This long-lasting increase in 5-HT in the hemolymph was blocked by treatment with an inhibitor of protein synthesis during training. Based on the levels of 5-HT in the hemolymph and its regulation by environmental events, we propose that 5-HT has a humoral role in regulation of the behavioral state of Aplysia. In support of this hypothesis, we found that increasing levels of 5-HT in the hemolymph led to significant alterations in feeding behavior. Increasing levels of 5-HT during the daytime when they were normally low increased the latency to assume feeding posture from daytime to nighttime values.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Protein phosphorylation appears to play important roles in the mechanisms responsible for presynaptic facilitation in Aplysia. To screen for phosphoproteins that may be involved in facilitation, we previously examined protein phosphorylation in pleural sensory neurons as a function of different durations (2 min, 25 min and 1.5 h) of serotonin treatments. Different durations of serotonin had unique effects on the phosphorylation of different sets of proteins. To determine the functions of these phosphoproteins, we have begun to obtain their amino acid sequences using protein microsequencing techniques. We report here partial sequencing of 2 such proteins. One protein (S6), whose phosphorylation was affected by 2 min treatments with serotonin, appeared to be an intermediate filament protein. Another protein (L55), whose phosphorylation was affected by 1.5-h treatments with serotonin, appeared to be a calmodulin-like Ca(2+)-binding protein. Although the exact cellular functions for S6 and L55 are not known, obtaining partial sequences of these proteins sets the stage for future studies that will examine their regulation and their specific roles in facilitation.

Long-term sensitization training, or procedures that mimic the training, produces long-term facilitation of sensory-motor neuron synapses in Aplysia. The long-term effects of these procedures require mRNA and protein synthesis (Montarolo et al., 1986; Castellucci et al., 1989). Using the techniques of differential display reverse transcription PCR (DDRT-PCR) and ribonuclease protection assays (RPA), we identified a cDNA whose mRNA level was increased significantly in sensory neurons by treatments of isolated pleural-pedal ganglia with serotonin for 1.5 hr or by long-term behavioral training of Aplysia. The effects of serotonin and behavioral training on this mRNA were mimicked by treatments that elevate cAMP. The aplysia mRNA increased by serotonin and behavioral training was 41-45% identical to a developmentally regulated gene family which includes Drosophila tolloid and human bone morphogenetic protein-1 (BMP-1). Both tolloid and BMP-1 encode metalloproteases that might activate TGF-beta (transforming growth factor beta)-like molecules or process procollagens. Aplysia tolloid/BMP-1-like protein (apTBL-1) might regulate the morphology and efficacy of synaptic connections between sensory and motor neurons, which are associated with long-term sensitization.

Serotonergic modulation of sensory neurons in Aplysia and their synaptic connections with follower cells has been used extensively as a model system with which to study mechanisms underlying neuronal plasticity. Serotonin (5-HT)-induced facilitation of sensorimotor connections is due to at least two processes: a process related to the broadening of presynaptic action potentials and a spike-duration-independent (SDI) process that may involve mobilization of transmitter. We have examined the relationship between spike broadening and synaptic facilitation of relatively nondepressed sensorimotor connections in the intact pleural-pedal ganglia. Previously, 5-HT-induced spike broadening in the sensory neuron was shown to be primarily due to the modulation of a voltage-dependent K+ current (Ik.v). Low concentrations (20-30 microM) of 4-aminopyridine (4-AP) were used to rather selectively block Ik.v. 4-AP increased spike duration in the sensory neuron and the excitatory postsynaptic potential (EPSP) in the motor neuron. The temporal development of 4-AP-induced spike broadening closely parallel that of synaptic facilitation. Thus spike broadening via the reduction of Ik.v can directly contribute to synaptic facilitation. The relationship between spike broadening induced by 5-HT (10 microM) and enhancement of the EPSP was also analyzed. We found that components of 5-HT-induced synaptic facilitation preceded the development of 5-HT-induced spike broadening. The comparison between the results of 4-AP and 5-HT revealed that the SDI processes made an important contribution to the rapid development of 5-HT-induced synaptic facilitation and that spike broadening made an important contribution to its maintenance. The SDI process and a slowly developing component of 5-HT-induced spike broadening are mediated, at least in part, by the activation of protein kinase C (PKC). Application of phorbol 12,13-diacetate (PDAc), an activator of PKC, partially mimicked the effects of 5-HT on spike duration and the EPSP. PDAc-induced enhancement of the EPSP preceded the slower development of PDAc-induced spike broadening. Like 5-HT, PDAc enhanced the EPSP via both spike broadening and the SDI processes. In addition, a 15-min exposure to PDAc occluded 5-HT-induced enhancement of the EPSP, suggesting that PKC and 5-HT engage similar or overlapping mechanisms. On the basis of these results and others, we propose a time-dependent hypothesis for the 5-HT-induced synaptic facilitation of nondepressed synapses, in which multiple second-messenger/protein kinase systems mediate the actions of 5-HT via both spike-duration-dependent and SDI processes.

1. Previous computational studies of models of neuron R15 in Aplysia have indicated that several distinct modes of electrical activity may coexist at a given set of parameters, that this multistability can be modulated by transmitters such as serotonin (5-HT) and that brief perturbations of the membrane potential can induce persistent changes in the mode of electrical activity. To test these predictions, the responses of R15 neurons to injections of brief (1.5 s) current pulses were recorded intracellularly in the absence and presence of 5-HT. 2. In the absence of 5-HT, brief perturbations induced abrupt transitions in the electrical activity from bursting to beating. Such transitions were observed in approximately 20% of the cases. The duration of beating activity varied from several seconds to tens of minutes. In the presence of low concentrations (1 microM) of 5-HT, both the probability of mode transitions and the duration of induced beating activity increased significantly. 3. These results indicate that at least two stable modes of electrical activity can coexist in R15 neurons and that this bistability can be regulated by 5-HT. In general, these conclusions agree with the results from analyses of mathematical models of R15. Although the function of these dynamic properties in R15 is speculative, our results, interpreted on the background of the model, support the notion that nonlinear dynamical properties of individual neurons can endow them with richer forms of information processing than has generally been appreciated.

1. In the pleural mechanoafferent sensory neurons of Aplysia, serotonin (5-HT)-induced spike broadening consists of at least two components: a cAMP and protein kinase A (PKA)-dependent, rapidly developing component and a protein kinase C (PKC)-dependent, slowly developing component. Voltage-clamp experiments were conducted to identify currents that are modulated by PKC and thus may contribute to the slowly developing component of 5-HT-induced spike broadening. 2. We compared the effects of phorbol esters, activators of PKC, on membrane currents with those of 5-HT. Bath application of 5-HT had complex modulatory effects on currents elicited by voltage-clamp pulses to potentials > 0 mV. The kinetics of both activation and inactivation of the membrane currents were slowed by 5-HT. This led to a decrease in an outward current at the beginning of the voltage-clamp pulse and an increase at the end of the pulse. Previous work has shown that these effects represent, in part, the modulation of a large, voltage-dependent K+ current (IK,V) by 5-HT. 3. Active phorbol esters mimicked some of the actions of 5-HT on membrane currents in that they slowed activation and inactivation kinetics of current responses to voltage-clamp pulses more positive than 0 mV. This led to a decrease in an outward current at the beginning of the pulse and an increase at the end of the pulse. Because inactive phorbols did not mimic the actions of 5-HT, the effects of active phorbol esters appeared to be PKC specific. In addition, preexposure of the sensory neurons to active phorbol esters appeared to occlude the modulatory actions of 5-HT on IK,V. Thus it is likely that modulation of IK,V by 5-HT is mediated, at lease in part, by PKC. 4. To further characterize which currents were modulated by PKC, low concentrations of tetraethylammonium (TEA, 2 mM) were used to block Ca(2+)-activated K+ current (IK,Ca). Low TEA partially blocked the phorbol ester-induced increase of the outward current at the end of voltage-clamp pulses. These results agreed with previous reports that activation of PKC enhanced a fast component of IK,Ca in these sensory neurons. Such an enhancement would lead to an increase in outward current that should be blocked by low TEA. Low TEA, however, did not affect phorbol ester-induced decrease of the outward current at the beginning of pulse, where the predominant current is IK,V, which is less sensitive to TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

A form of associative plasticity in Aplysia, activity-dependent neuromodulation, involves the convergence of neuronal activity and the effects of a modulatory transmitter. To investigate the role of protein synthesis in associative plasticity, we examined the effects of a biochemical analogue of activity-dependent neuromodulation on the level of incorporation of labeled amino acid into proteins. To mimic associative training, abdominal ganglia were exposed to paired treatments of a depolarizing agent, elevated potassium, and a modulatory transmitter, serotonin. The effects of elevated potassium and serotonin applied alone were also examined. At least two proteins (nos. 9 and 17) were affected in a nonadditive way by the paired procedure. Incorporation of label into protein 9 was increased by the paired procedure but was not affected by either elevated potassium or serotonin. Incorporation of label into protein 17 was significantly affected by elevated potassium or serotonin, but the effect of the paired procedure was significantly less than the summed effects of elevated potassium and serotonin applied alone. These results indicate that changes in protein synthesis may be important in the induction of associative plasticities. Amino acid sequences of two peptides derived from protein 9 were obtained. Then, a partial cDNA clone for protein 9 was obtained by performing PCR with degenerate primers corresponding to portions of the sequences of the two peptides. The sequence of protein 9 is related to sequences previously reported for a family of genes comprising the stringent starvation protein of Escherichia coli, auxin-induced proteins of plants, and glutathione S-transferases of a number of organisms.

Potassium currents in pleural sensory neurons of Aplysia were studied under control conditions and in the presence of serotonin (5-HT). Using pharmacological techniques we isolated a current that we refer to as IK,V. Although it is not known whether IK,V represents a distinct type of membrane channel, we described its properties using a Hodgkin-Huxley type model. The effects of 5-HT on IK,V were complex. 5-HT decreased by 50% the steady-state magnitude (Iss) of IK,V in response to a voltage-clamp pulse from -50 mV to +20 mV. In addition, 5-HT significantly slowed both activation kinetics (the time constant of activation was increased by 29% at +20 mV) and inactivation kinetics (the time constant of inactivation was increased by 518% at +20 mV). Mathematical descriptions of IK,V in control conditions and in the presence of 5-HT were used to estimate the relative contribution of serotonergic modulation of IK,V to the total 5-HT-induced modulation of membrane currents. Effects of 5-HT on IK,V account for more than 87% of the 5-HT-induced reduction in outward current during the first 20 ms of a voltage-clamp pulse to +20 mV. This result implies that 5-HT exerts many of its effects on spike width in sensory neurons via modulation of IK,V. Effects of 5-HT on IK,V are consistent with a model in which the maximal conductance underlying the current is decreased by 50%, and the rate constants between open and closed states of both the activation and inactivation processes are diminished in magnitude across all membrane potentials.

1. Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning. Earlier studies have concluded that serotonin (5-HT) is a key modulatory transmitter and that it exerts its short-term actions via cAMP-dependent activation of protein kinase A. Subsequently, it has become clear that other kinase systems such as protein kinase C (PKC) also may be involved in the actions of 5-HT. 2. Application of phorbol esters, which activate PKC, produced a slowly developing spike broadening but had little effect on excitability (a process known to be primarily cAMP dependent). Moreover, the effects of phorbol esters and 5-HT on spike duration were not additive, suggesting that they may share some common mechanisms. 3. The protein kinase inhibitor staurosporine suppressed both 5-HT-induced slowly developing spike broadening and, under certain conditions, facilitation of transmitter release. Staurosporine did not inhibit 5-HT-induced enhancement of excitability. The effectiveness of staurosporine on spike broadening was dependent on the time at which spike broadening was examined after application of 5-HT. Staurosporine appeared to have little effect on spike broadening 3 min after application of 5-HT, whereas it inhibited significantly 5-HT-induced spike broadening at later times. The staurosporine-insensitive component of 5-HT-induced spike broadening may be mediated by cAMP. 4. The results suggest that the activation of PKC plays a key role in components of both 5-HT-induced spike broadening and facilitation of synaptic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

An in vitro analogue of long-term behavioral training in Aplysia was developed to simulate the intensity and timing of shocks delivered to the body wall of animals during sensitization training. The in vitro training analogue consisted of electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia. We found that the in vitro training analogue led to long-term (24 h) changes in the membrane currents of sensory neurons; these changes were similar to those produced by behavioral training. Using two dimensional polyacrylamide gel electrophoresis, we examined early effects on protein synthesis in pleural sensory neurons induced by the training analogue. Incorporation of amino acid into 5 proteins was affected at the end of training. Incorporation of amino acid into some of these proteins was also affected by serotonin (5-HT) and by an analogue of cyclic adenosine monophosphate (cAMP). These results suggest that the effects on protein synthesis that are produced by the in vitro training analogue are mediated, at least in part, by release of 5-HT and an increase in the level of cAMP in the sensory neurons.

1. The electrophysiological properties of sensory neurons that mediate withdrawal reflexes of Aplysia can be modulated by a variety of neurotransmitters. We compared the known excitatory actions of serotonin (5-HT) with the actions of FMRFamide (Phe-Met-Arg-Phe-NH2) and myomodulin (Pro-Met-Ser-Met-Leu-Arg-Leu-NH2) on the durations of action potentials and excitability. In addition, with the use of voltage-clamp and pharmacological separation techniques, we characterized the membrane currents that were modulated by each of the three agents. 2. Application of 5-HT produced an increase in the duration of action potentials and an enhancement of excitability in somata of the tail sensory neurons. FMRFamide and myomodulin reversed these excitatory effects and decreased the duration of action potentials and excitability. These results indicated that FMRFamide and myomodulin exerted inhibitory effects on the electrophysiological properties of the sensory neurons. properties of the sensory neurons. 3. FMRFamide appeared to modulate three K+ currents. The first current, which was increased by FMRFamide, had properties closely resembling those of the S-K+ current (IK,S). These properties include slow activation, little inactivation, and relative insensitivity to the K+ channel blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA). The second current, which was reduced by FMRFamide, had kinetic and pharmacological properties similar to those of a component of the Ca(2+)-activated K+ current (IK,Ca). Finally, at large depolarizations, FMRFamide appeared to increase a third current that was attenuated by 4-AP, suggesting that FMRFamide also modulated the delayed or voltage-dependent K+ current (IK,V). 4. Myomodulin appeared to modulate two of the currents modulated by FMRFamide, because it increased both IK,S and IK,V. Unlike FMRFamide, however, myomodulin did not appear to modulate IK,Ca. 5. Arachidonic acid mimicked the modulation of IK,S, IK,Ca, and IK,V by FMRFamide. Because myomodulin did not modulate IK,Ca, it appears that a second messenger other than arachidonic acid or its metabolites mediates the modulatory effects of myomodulin. 6. These results indicate that both FMRFamide and myomodulin can inhibit the tail sensory neurons by increasing IK,S. FMRFamide, but not myomodulin, also reduces IK,Ca, which suggests that under some conditions FMRFamide may also have excitatory actions. Finally, these results suggest that the effects of FMRFamide and myomodulin may be mediated by different second-messenger systems.

Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia.

Microcystin-LR (MCYST-LR), a hepatotoxin produced by cyanobacteria, inhibited purified protein phosphatases (PrPs) from rabbit skeletal muscle and the enzymes from Aplysia with an IC50 of approximately 10(-10) M. MCYST-LR also prolonged both serotonin- (5-HT) and cyclic adenosine monophosphate-induced inward currents in sensory neurons of Aplysia. These results, which are consistent with inhibition of Aplysia PrPs, indicate that MCYST-LR may be a useful probe to elucidate the function of PrPs in neural tissues.

1. In somata of sensory neurons in the pleural ganglia of Aplysia californica, serotonin (5-HT) modulates at least three K+ currents: the S K+ current (IK,S), a slow component of the Ca2(+)-activated K+ current (IK,Ca), and the delayed or voltage-dependent K+ current (IK,V). The modulation of IK,S and the slow component of IK,Ca by 5-HT has been shown previously to be mediated via adenosine 3',5'-cyclic monophosphate (cAMP). To determine whether the modulation of IK,V by 5-HT also is mediated via cAMP, we used two-electrode voltage-clamp techniques to compare the modulation of membrane current by cAMP and 5-HT. 2. Current responses were elicited by brief (200 ms) voltage-clamp pulses before and after the bath application of analogues of cAMP. At all voltage-clamp potentials examined (-40-30 mV), analogues of cAMP reduced the amplitude of the current response. The properties of the cAMP-sensitive component of membrane current were revealed by computer subtraction of current responses elicited in the presence of the analogue of cAMP from current responses elicited before application of the analogue. The characteristics of the resulting cAMP difference current (IcAMP) suggested that cAMP modulated a component of membrane current that was relatively voltage independent, did not inactivate, and was active over a wide range of membrane potentials. In addition, the current-voltage (I-V) relationship of the cAMP difference current had a positive slope. These properties of the cAMP difference current were consistent with those of IK,S but did not indicate that IK,V was modulated by cAMP. 3. The cAMP-independent modulation of membrane current by 5-HT was examined by eliciting current responses first in the presence of an analogue of cAMP and again after the addition of 5-HT to the bath, which still contained the analogue. The presence of the analogue of cAMP occluded further modulation of IK,S by 5-HT. However, the analogue of cAMP did not occlude the modulation of IK,V by 5-HT. This cAMP-independent effect of 5-HT on membrane current was revealed by computer subtraction of current responses elicited in the presence of 5-HT from current responses elicited before the application of 5-HT (the analogue of cAMP was present throughout). The resulting cAMP-independent 5-HT difference current (I5-HT) was highly voltage dependent, had complex kinetics, and its I-V relationship had a negative slope at membrane potentials above 0 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The properties of membrane currents that were modulated by serotonin (5-HT) were investigated with two-electrode voltage-clamp techniques in sensory neuron somata isolated from the pleural ganglion of Aplysia californica. The modulatory effects of 5-HT were revealed by computer subtraction of current responses elicited in the presence of 5-HT from current responses elicited prior to the application of 5-HT. The complexities of the resulting 5-HT difference currents (I5-HT) suggested that 5-HT modulated more than one component of membrane current. 2. The 5-HT difference currents appeared to have at least two distinct components. One component was clearly evident at membrane potentials more negative than -10 mV was relatively voltage independent and did not inactivate. A second component was activated at membrane potentials more positive than -10 mV, had complex kinetics, and was highly voltage dependent. In an attempt to identify the membrane currents that were modulated by 5-HT, we compared the pharmacologic sensitivity of I5-HT to that of previously described K+ currents. 3. The two components of I5-HT had different sensitivities to agents that block K+ currents. The relatively voltage-independent component of I5-HT was not blocked by 2 mM 4-aminopyridine (4-AP) and was relatively insensitive to tetraethylammonium (TEA) (estimated Kd of 92 mM). In contrast, the voltage-dependent component of I5-HT was blocked by 4-AP (2 mM) and moderate concentrations of TEA (estimated Kd of 5 mM). 4. The K+ current blockers that were used to examine I5-HT were also used to examine voltage-activated membrane currents. Externally applied TEA blocked the delayed or voltage-dependent K+ current (IK.V) with an estimated dissociation constant (Kd) of 8 mM and a membrane current similar to the Ca2+-activated K+ current (IK.Ca) with an estimated Kd of 0.4 mM. In addition, externally applied 4-AP (2 mM) blocked IK.V. Thus TEA and 4-AP were equipotent in blocking both IK.V and the voltage-dependent component of I5-HT. 5. The suggestion that I5-HT contained multiple components was supported further by examining the modulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) that mediates some actions of 5-HT on membrane currents in these cells. cAMP difference currents (IcAMP) were similar to the relatively voltage-independent component of I5-HT. The subsequent addition of 5-HT to solutions already containing cAMP resulted in 5-HT difference currents similar to the voltage-dependent component of I5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)

Micropressure ejection of serotonin (5-hydroxytryptamine, 5-HT) produced excitatory responses in the L14 ink motor neurons of Aplysia that depended on the site of application. Ejection of 5-HT onto the cell body produced a slow response that showed variability in voltage sensitivity between preparations. In contrast, ejection of 5-HT onto the neuropil underneath the cell body produced a response whose amplitude was consistently a linear function of the holding potential, reversing near the predicted potassium equilibrium potential. Subsequent analyses focused on this second response. The neuropil response induced by 5-HT had a linear current-voltage relationship (reversing at ca. -80 mV), was associated with a decrease in input conductance, and was sensitive to changes in the concentration of extracellular K+. Serotonin application in artificial seawater (ASW) containing 30 mM K+ produced a response that reversed close to the altered Nernst potential for K+. The 5-HT response did not appear to be due to secondary activation of interneurons or to depend primarily on extracellular Ca2+, since ejection of 5-HT onto cells bathed in ASW containing 30 mM Co2+ produced responses comparable to, although somewhat attenuated from, those observed in ASW. Serotonin responses similar to those produced in ASW were obtained after perfusing the ganglion with ASW containing Co2+, 4-aminopyridine (4-AP), and tetraethylammonium (TEA). This suggests that the 5-HT-sensitive current is separate from the Ca2+-activated, fast, and delayed rectifying K+ currents. The 5-HT response appeared to be mediated by changes in levels of cAMP. Bath application of the phosphodiesterase inhibitors IBMX (3-isobutyl-1-methylxanthine) or Ro 20-1724, or the adenylate cyclase activator forskolin mimicked the 5-HT response by producing a slow inward current associated with a decrease in membrane conductance. Alteration of cellular cAMP metabolism modulated the response to 5-HT. Exposure of the ganglion to low concentrations of either Ro 20-1724 or forskolin potentiated the 5-HT response. Higher concentrations of these agents largely blocked the response to subsequent 5-HT applications. Bath application of the 8-bromo derivative of either cAMP or cGMP produced a slow inward current associated with a decrease in membrane conductance in cells voltage clamped at the resting potential. Responses to 5-HT were blocked, however, after exposure to 8-bromo-cAMP, but not to 8-bromo-cGMP. These results suggest that 5-HT produces a voltage-independent decrease in a steady-state potassium conductance that may be mediated by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

To identify proteins that may be involved in the induction of long-term changes in the nervous system, we investigated whether specific proteins in pleural sensory neurons of Aplysia were affected by procedures that mimic those used to produce long-term sensitization. Using two-dimensional PAGE, we found that exposure to serotonin (5-hydroxytryptamine, 5-HT) for 2 or 3 hr appeared to increase incorporation of labeled amino acids into one protein (P9) and decrease incorporation into two other proteins (P19 and P20). These effects of 5-HT were observed whether the labeled amino acid was leucine or methionine. The same proteins that were affected by 5-HT were also altered by the adenylate cyclase activator forskolin and by the 8-bromo and 8-benzylthio analogs of cAMP. Addition of Co2+ to 5-HT did not seem to affect the action of 5-HT on P9 and P20, but it did seem to block the effect of 5-HT on P19. However, the effect of analogs of cAMP on P9, P19, and P20 was not altered by inclusion of Co2+. A phorbol ester that activates protein kinase C did not appear to affect the proteins that were modified by 5-HT, but phorbol ester did appear to increase the amount of labeled amino acids incorporated into another protein (P24). To investigate the specificity of these effects for pleural ganglion neurons, we examined the effect of 3- and 6-hr treatments of 5-HT on proteins in the abdominal ganglion. 5-HT affected at least nine proteins in the abdominal ganglion. One of these proteins (P9) appeared to be the same as one altered by 5-HT in the pleural sensory neurons. However, the occurrence of some proteins and some effects of 5-HT were specific for one ganglion or the other. The identified proteins that were affected by both 5-HT and changes in cAMP may be involved in the induction of long-term changes in the nervous system of Aplysia.

Recent evidence suggests that serotonin (5-HT) and a neuropeptide, small cardioactive peptideB (SCPB), exert parallel modulatory actions on neuronal and muscular function in Aplysia via changes in the levels of cAMP. We have examined the effects of both of these neurotransmitters on the adenylate cyclase activity in membrane homogenates of pleural ganglia. The results indicated that 5-HT and SCPB enhance the production of cAMP via a direct activation of adenylate cyclase. In addition, we found that the increase in cyclase activity produced by 5-HT could be selectively blocked by the S2-serotonergic antagonists, ketanserin and ritanserin. Thus, specific 5-HT receptors are present which appear to be distinct from those that mediate the effects of SCPB on the activity of adenylate cyclase.

While recent evidence indicates a role for serotonin (5-HT) in modulating the defensive tail-withdrawal reflex in Aplysia, little information exists concerning the specificity of these 5-HT effects. As a first-step in addressing this issue we have examined the dose-response relationship for one aspect of the 5-HT modulation (enhancement of cAMP levels in isolated clusters of sensory neurons) and compared the effects of 5-HT with three potential neurotransmitters: tryptamine, FMRFamide (Phe-Met-Arg-Phe-NH2) and small cardioactive peptideB (SCPB). Cyclic adenosine monophosphate (cAMP) levels were enhanced as a graded function of the concentration of 5-HT with an EC50 of 14 microM. At a concentration of 5 microM, both 5-HT and SCPB produced nearly identical increases in the cAMP content of sensory neurons. In contrast, 5 microM tryptamine or 5 microM FMRFamide had little or no effect on cAMP levels. We also examined the effects of these agents on membrane currents and membrane conductance. Both 5-HT and SCPB produced an inward current associated with a decrease in input conductance. Tryptamine had little or no effect, while FMRFamide produced a response opposite to that of 5-HT and SCPB; an outward current associated with an increase in membrane conductance.

A modified glyoxylic acid technique was used to examine central and peripheral nervous tissues in Aplysia californica. In addition to confirming the distribution of catecholamines and serotonin in the central nervous system, the method demonstrated the presence of monoamines in the opaline gland and bag cell clusters where they may act as transmitters. In conjunction with electrophysiological techniques this method may be useful to identify other monoamine-containing neurons in Aplysia.

An important mechanism that contributes to sensitization in Aplysia is heterosynaptic facilitation of the synaptic connections between sensory neurons (SNs) and motor neurons (MNs). Heterosynaptic facilitation, in turn, is associated with broadening of the spike in the SN. Spike broadening is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture, but broadening in synaptic terminals has only been inferred. Intracellular recordings were made from somata of SNs and from somata of follower MNs. Additional recordings were made from the axons of SNs as they enter the neuropil in the pedal ganglion. Serotonin (5-HT) broadened action potentials in axons of SNs and enhanced excitatory postsynaptic potentials (EPSPs) in the MNs, even after the axons of SNs were surgically separated from their somata. These results indicate that both heterosynaptic facilitation and spike broadening in the axon are due to the local action of 5-HT and can occur independently of modulation of membrane properties in the soma.

Facilitation of synaptic connections between sensory neurons and motor neurons mediating the tail-withdrawal reflex in Aplysia is produced by sensitizing stimuli. These effects can be mimicked by perfusing the pleural and pedal ganglia of a semi-intact preparation with serotonin (5-HT). In addition to the synaptic facilitation, 5-HT produces a depolarization associated with an increase in input resistance in the sensory neurons. The 5-HT-induced changes appear to be mediated by an elevation in cAMP levels. To examine further the role of cAMP in mediating the 5-HT response we utilized the potent cyclase activator forskolin. Forskolin mimics the 5-HT response and blocks the response to subsequent 5-HT applications indicating that both 5-HT and forskolin act through a common saturable mechanism. Voltage clamp and ion substitution experiments indicate that the 5-HT response is due, at least in part, to a decrease in a resting K+ conductance.

1. The modulation of membrane currents by serotonin (5-HT) was studied in isolated clusters of tail sensory neurons. Serotonin was applied by micropressure ejection onto the somata of sensory neurons voltage-clamped at fixed holding potentials. The range of holding potentials tested in this study was selected to produce a steady-state Ca2+-activated K+ current (IK,Ca). Serotonin induced an inward shift in the holding current associated with a decrease in slope conductance. 2. Intracellular injection of adenosine 3',5'-cyclic monophosphate (cAMP) mimicked the response to 5-HT and induced an inward current associated with a decrease in slope conductance. The responses to 5-HT and cAMP had similar voltage dependencies and both responses were due to an apparent decrease in K+ current. Responses to cAMP were markedly reduced when generated at the peak of a response to 5-HT. The nonsummation of the maximal current responses indicated that 5-HT and cAMP utilize a common, saturable mechanism. 3. In contrast to the consistent decrease in steady-state K+ conductance elicited by cAMP, injection of guanosine 3',5'-cyclic monophosphate (cGMP) produced variable responses. In most cells, cGMP induced outward shifts in holding current that were associated with an increase in slope conductance. 4. Several lines of evidence indicated that IK,Ca contributed to the holding current at the level of membrane potentials that were examined. Inward shifts in holding current associated with a decrease in slope conductance were produced in the presence of agents that block Ca2+ channels, such as Co2+, Cd2+ or Ni2+ and by replacement of extracellular Ca2+ with Ba2+. Reducing the concentration of cytoplasmic Ca2+ through intracellular injection of EGTA had similar effects. Furthermore, inward shifts in holding current were produced by 5 mM tetraethylammonium chloride (TEA), which is known to block IK,Ca in neurons of Aplysia. This concentration of TEA also attenuated the outward current produced in response to direct intracellular injection of Ca2+. 5. Serotonin appears to modulate the IK,Ca that contributes to the steady-state holding current. The same manipulations that block the steady-state IK,Ca (see above) also attenuated the response to 5-HT. Furthermore, K+ currents activated by intracellular injection of Ca2+ were attenuated by 5-HT. 6. These results indicate that the changes in holding current produced by 5-HT are mediated, at least in part, by cAMP. In addition, it appears that 5-HT modulates a steady-state calcium-activated K+ current in addition to the previously described S-current (40, 58) and delayed K+ current (8, 9).(ABSTRACT TRUNCATED AT 250 WORDS)