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An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V max of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.

Mathematical models are useful for providing a framework for integrating data and gaining insights into the static and dynamic behavior of complex biological systems such as networks of interacting genes. We review the dynamic behaviors expected from model gene networks incorporating common biochemical motifs, and we compare current methods for modeling genetic networks. A common modeling technique, based on simply modeling genes as ON–OFF switches, is readily implemented and allows rapid numerical simulations. However, this method may predict dynamic solutions that do not correspond to those seen when systems are modeled with a more detailed method using ordinary differential equations. Until now, the majority of gene network modeling studies have focused on determining the types of dynamics that can be generated by common biochemical motifs such as feedback loops or protein oligomerization. For example, these elements can generate multiple stable states for gene product concentrations, state-dependent responses to stimuli, circadian rhythms and other oscillations, and optimal stimulus frequencies for maximal transcription. In the future, as new experimental techniques increase the ease of characterization of genetic networks, qualitative modeling will need to be supplanted by quantitative models for specific systems.

A training protocol was developed to classically condition feeding behavior inAplysia californicausing tactile stimulation of the lips as the conditional stimulus (CS) and food as the unconditional stimulus (US). Paired training induced a greater increase in the number of bites to the CS than unpaired training or US-only stimulation. Memory for classical conditioning was retained for at least 24 hr. The organization of the reinforcement pathway that supports classical conditioning was analyzed in additional behavioral experiments. No evidence was found for the contribution to appetitive reinforcement of US-mediating pathways originating in the lips of the animals. Bilateral lesions of the anterior branch of the esophageal nerve, which innervates parts of the foregut, however, were found to attenuate classical conditioning. Thus, it appears likely that reinforcement during appetitive classical conditioning of feeding was mediated by afferent pathways that originate in the foregut. The companion paper (Lechner et al., 2000) describes two neurophysiological correlates of the classical conditioning.

Feeding behavior inAplysia californicacan be classically conditioned using tactile stimulation of the lips as conditional stimulus (CS) and food as unconditional stimulus (US) [Lechner et al., 2000 (companion paper)]. Conditioning resulted in an increase in the number of CS-evoked bites that persisted for at least 24 hr after training. In this study, neurophysiological correlates of classical conditioning training were identified and characterized in anin vitropreparation of the cerebral and buccal ganglia. Stimulation of a lip nerve (AT4), which mediates mechanosensory information, resulted in a greater number of buccal motor patterns (BMPs) in ganglia isolated from animals that had received paired training than in ganglia from control animals. The majority of the evoked BMPs were classified as ingestion-like patterns. Intracellular recordings from pattern-initiating neuron B31/32 revealed that stimulation of AT4evoked greater excitatory input in B31/32 in preparations from animals that had received paired training than from control animals. In contrast, excitatory input to buccal neuron B4/5 in response to stimulation of AT4was not significantly increased by paired training. Moreover, correlates of classical conditioning were specific to stimulation of AT4. The number of spontaneously occurring BMPs and the intrinsic properties of two buccal neurons (B4/5 and B31/32) did not differ between groups. These results suggest that appetitive classical conditioning of feeding resulted in the pairing-specific strengthening of the polysynaptic pathway between afferent fibers and pattern-initiating neurons of the buccal central pattern generator.

The dynamical behavior of individual neurons and of neural circuits emerges from the interactions among multiple nonlinear processes at the molecular, cellular, synaptic and network levels. Thus, characterizing the dynamical performance of a complex system of nonlinear elements is fundamental to an understanding of neural function. This chapter illustrates how some of the concepts and analytical techniques of nonlinear dynamical systems were applied to computational and experimental analyses of single cell and multicellular neuronal oscillators. The results of these studies provided additional insights into how neurons and neural circuits might exploit nonlinear dynamics at the cellular level to generate and control oscillatory patterns of electrical activity and to process and store information.

To examine the capability of genetic regulatory systems for complex dynamic activity, we developed simple kinetic models that incorporate known features of these systems. These include autoregulation and stimulus-dependent phosphorylation of transcription factors (TFs), dimerization of TFs, crosstalk, and feedback. The simplest model manifested multiple stable steady states, and brief perturbations could switch the model between these states. Such transitions might explain, for example, how a brief pulse of hormone or neurotransmitter could elicit a long-lasting cellular response. In slightly more complex models, oscillatory regimes were identified. The addition of competition between activating and repressing TFs provided a plausible explanation for optimal stimulus frequencies that give maximal transcription. Such optimal frequencies are suggested by recent experiments comparing training paradigms for long-term memory formation and examining changes in mRNA levels in repetitively stimulated cultured cells. In general, the computational approach illustrated here, combined with appropriate experiments, provides a conceptual framework for investigating the function of genetic regulatory systems.

In order to assess the relative contributions to pattern-generation of the intrinsic properties of individual neurons and of their connectivity, we examined a ring circuit composed of four complex physiologically based oscillators. This circuit produced patterns that correspond to several quadrupedal gaits, including the walk, the bound, and the gallop. An analysis using the phase response curve (PRC) of an uncoupled oscillator accurately predicted all modes exhibited by this circuit and their phasic relationships – with the caveat that in certain parameter ranges, bistability in the individual oscillators added nongait patterns that were not amenable to PRC analysis, but further enriched the pattern-generating repertoire of the circuit. The key insights in the PRC analysis were that in a gait pattern, since all oscillators are entrained at the same frequency, the phase advance or delay caused by the action of each oscillator on its postsynaptic oscillator must be the same, and the sum of the normalized phase differences around the ring must equal to an integer. As suggested by several previous studies, our analysis showed that the capacity to exhibit a large number of patterns is inherent in the ring circuit configuration. In addition, our analysis revealed that the shape of the PRC for the individual oscillators determines which of the theoretically possible modes can be generated using these oscillators as circuit elements. PRCs that have a complex shape enable a circuit to produce a wider variety of patterns, and since complex neurons tend to have complex PRCs, enriching the repertoire of patterns exhibited by a circuit may be the function of some intrinsic neuronal complexity. Our analysis showed that gait transitions, or more generally, pattern transitions, in a ring circuit do not require rewiring the circuit or any changes in the strength of the connections. Instead, transitions can be achieved by using a control parameter, such as stimulus intensity, to sculpt the PRC so that it has the appropriate shape for the desired pattern(s). A transition can then be achieved simply by changing the value of the control parameter so that the first pattern either ceases to exist or loses stability, while a second pattern either comes into existence or gains stability. Our analysis illustrates the predictive value of PRCs in circuit analysis and can be extended to provide a design method for pattern-generating circuits.

Operant conditioning is characterized by the contingent reinforcement of a designated behavior. Previously, feeding behavior inAplysiahas been demonstrated to be modified by operant conditioning, and a neural pathway (esophageal nerve; E n.) that mediates some aspects of reinforcement has been identified. As a first step toward a cellular analysis of operant conditioning, we developed anin vitrobuccal ganglia preparation that expressed the essential features of operant conditioning. Motor patterns that represented at least two different aspects of fictive feeding (i.e., ingestion-like and rejection-like motor patterns) were elicited by tonic stimulation of a peripheral buccal nerve (n.2,3). Three groups of preparations were examined. In a contingent-reinforcement group, stimulation of E n. was contingent on the expression of a specific type of motor pattern (i.e., either ingestion-like or rejection-like). In a yoke-control group, stimulation of E n. was not contingent on any specific pattern. In a control group, E n. was not stimulated. The frequency of the reinforced pattern increased significantly only in the contingent-reinforcement group. No changes were observed in nonreinforced patterns or in the motor patterns of the control and yoke-control groups. Contingent reinforcement of the ingestion-like pattern was associated with an enhancement of activity in motor neuron B8, and this enhancement was specific to the reinforced pattern. These results suggest that the isolated buccal ganglia expressed an essential feature of operant conditioning (i.e., contingent reinforcement modified a designated operant) and that this analog of operant conditioning is accessible to cellular analysis.

The synaptic connections between the sensory neurons ofAplysiaand their follower neurons have been used as a model system for examining the cellular mechanisms contributing to neuronal and synaptic plasticity. Recent studies suggest that at least two protein kinases, protein kinase A (PKA) and protein kinase C (PKC), contribute to serotonin (5-HT)-induced short-term facilitation. The interaction between these two kinase cascades has not been examined, however. Using electrophysiological and biochemical approaches, we examined possible interactions between PKA and PKC cascades. The results indicated that prolonged activation of PKC by preincubation with phorbol esters attenuated PKA-mediated actions of 5-HT, including increases in sensory neuron excitability and spike broadening in the presence of tetraethylammonium (TEA) and nifedipine. Although phorbol esters also attenuated increases in excitability by an analog of cAMP and small cardioactive peptide B (SCPB), the degree of attenuation was smaller. In addition, phorbol esters did not attenuate broadening of TEA spikes by the cAMP analog and SCPB. Thus, phorbol esters appeared specifically to attenuate aspects of the 5-HT activation of the cAMP/PKA cascade. Measurements of cAMP levels with radioimmunoassays revealed that phorbol esters did not attenuate 5-HT-induced cAMP synthesis, however. Finally, the results indicated that phorbol esters themselves induced a small but significant increase in excitability as well as an increase in the level of cAMP. Our results suggest that there is crosstalk between the PKC and PKA cascades. The mechanisms by which phorbol esters specifically attenuate 5-HT-induced activation of the cAMP/PKA cascade are not known, however.

Nakanishi, Keiko, Fan Zhang, Douglas A. Baxter, Arnold Eskin, and John H. Byrne. Role of calcium-calmodulin–dependent protein kinase II in modulation of sensorimotor synapses in Aplysia. J. Neurophysiol. 78: 409–416, 1997. The Ca2+-calmodulin–dependent protein kinase II (CaMKII) inhibitor, {1-[N,O - bis(5 - isoquinolinesulfonyl) - N - methyl - L - tyrosyl] - 4 - phenylpiper azine} (KN-62), was used to investigate the role of CaMKII in synaptic transmission and serotonin (5-HT)-induced facilitation in Aplysia. Application of KN-62 (10 μM) by itself increased the amplitude of excitatory postsynaptic potentials (EPSPs) at sensorimotor synapses in pleural-pedal ganglia. Moreover, in the presence of KN-62, 5-HT–induced short-term facilitation was attenuated. Application of KN-62 by itself slightly increased the duration of action potentials in isolated sensory neuron somata but did not block spike broadening produced by 5-HT. KN-62 had no effect on excitability of isolated sensory neuron somata nor did it block 5-HT–induced enhancement of excitability. These results indicate that the attenuation of short-term facilitation by KN- 62 is not due to modulation of the membrane currents contributing to 5-HT–induced spike broadening or enhancement of excitability. Rather, these data are consistent with the hypothesis that CaMKII contributes to the regulation of sensorimotor connections and that it has a role in spike-duration–independent processes contributing to short-term facilitation.

Sugita, Shuzo, Douglas A. Baxter, and John H. Byrne. Differential effects of 4-aminopyridine, serotonin, and phorbol esters on facilitation of sensorimotor connections in Aplysia. J. Neurophysiol. 77: 177–185, 1997. 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 μM) 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 paralleled that of synaptic facilitation. Thus spike broadening via the reduction of IK,Vcan directly contribute to synaptic facilitation. The relationship between spike broadening induced by 5-HT (10 μM) 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.

The mechanisms underlying short-term presynaptic facilitation, the enhancement of transmitter release from sensory neurons in Aplysia, induced by serotonin (5-HT), can be divided into two categories: (1) changes in ionic conductances leading to spike broadening and enhancement of Ca2+ influx; and (2) actions on the machinery for transmitter release that are independent of spike broadening and the resulting increases in Ca2+ influx. Spike broadening and the associated enhancement of excitability are induced by the modulation of K+ conductances in the sensory neuron. The cellular mechanisms that contribute to the enhancement of release that is independent of spike broadening are not known and may involve vesicle mobilization or other steps in exocytotic release. These two facilitatory actions of 5-HT are mediated by at least two second-messenger-activated protein kinase systems, protein kinase A (PKA) and protein kinase C (PKC). These two second-messenger cascades overlap in their contributions to synaptic facilitation. However, their relative contributions to enhancement of transmitter release are not simply synergistic but are state- and time-dependent. The state dependence is a reflection of the synapse's previous history of activity. When the synapse is rested (and not depressed), a brief pulse of 5-HT (lasting from 10 sec to 5 min) produces its actions primarily through PKA via both spike broadening-dependent and -independent mechanisms. The broadening primarily involves the modulation of a voltage-dependent K+ current, IKV, with a small contribution by a voltage-independent K+ current, IKS. By contrast, the enhancement of excitability is mediated primarily by the modulation of IKS. As the synapse becomes depressed with repeated activity, the contribution of PKC becomes progressively more important. As is the case with PKA, PKC produces its action both by broadening the spike via modulation of IKV and by a spike broadening-independent mechanism. In addition to being state-dependent, the mechanisms of facilitation are time-dependent. There are differences in the response to 5-HT when it is given briefly to produce short-term facilitation or when the exposure is prolonged. When exposure is brief (< or = 5 min), PKA dominates. When exposure is prolonged (10-20 min), PKC becomes dominant as it is with depressed synapses. Thus, synaptic plasticity appears to be expressed in several overlapping time domains, and the transition between very short-term facilitation and various intermediate duration phases seems to involve interactive processes between the kinases.

The peptide neurotransmitter Phe-Met-Arg-PheNH 2 (FMRFamide) increases outward K + currents and promotes dephosphorylation of many phosphoproteins in Aplysia sensory neurons. We examined FMRFamide-induced current responses in sensory neurons injected with thiophosphorylated protein phosphate inhibitor-1 and inhibitor-2 (I-1 and I-2), two structurally different vertebrate protein phosphatase-1 (PP1) inhibitors to define a role for PP1 in the physiological actions of FMRFamide. Thiophosphorylated I-1 and I-2 both reduced the amplitude of outward currents elicited by FMRFamide by 50-60% and were as effective as microcystin-LR, which inhibited both PP1 and protein phosphatase-2A in Aplysia neuronal extracts. These data suggested that of the two major neuronal protein serine/threonine phosphatases, FMRFamide utilized primarily PPI to open serotonin-sensitive K + (S-K + ) channels. Earlier studies showed that a membrane-associated phosphatase regulated S-K + channels in cell-free patches from sensory neurons. Utilizing its unique substrate specificity and inhibitor sensitivity, we have characterized PP1 as the principal protein phosphatase associated with neuronal plasma membranes. Two protein phosphatase activities (apparent M r values of 170,000 and 38,000) extracted from crude membrane preparations from the Aplysia nervous system were shown to be isoforms of PP1. These biochemical and physiological studies suggest that PP1 is preferentially associated with neuronal membranes and that its activity may be required for the induction of outward K + currents in the Aplysia sensory neurons by FMRFamide

In a computational model of the bursting neuron R15, we have implemented proposed mechanisms for the modulation of two ionic currents (IR and ISI) that play key roles in regulating its spontaneous electrical activity. The model was sufficient to simulate a wide range of endogenous activity in the presence of various concentrations of serotonin (5-HT) or dopamine (DA). The model was also sufficient to simulate the responses of the neuron to extrinsic current pulses and the ways in which those responses were altered by 5-HT or DA. The results suggest that the actions of modulatory agents and second messengers on this neuron, and presumably other neurons, cannot be understood on the basis of their direct effects alone. It is also necessary to take into account the indirect effects of these agents on other unmodulated ion channels. These indirect effects occur through the dynamic interactions of voltage-dependent and calcium-dependent processes.

Neurons on the rostral edge of the ventral surface of the right pleural ganglion were identified as elements of the circuit mediating the defensive tail withdrawal reflex of Aplysia. These neurons produced IPSPs in tail sensory neurons and were classified into two groups, RPI4 and RPI5, according to their affinity for an antibody directed against FMRFamide. RPI4 was not FMRFamide immunoreactive, and RPI5 was. RPI4 and RPI5 were found to have different electrophysiological profiles. The summated IPSPs in sensory neurons produced by RPI4 developed more rapidly and had a shorter duration than those produced by RPI5. In addition, RPI4 produced IPSPs in the tail motor neurons, whereas RPI5 did not. Both RPI4 and RPI5 received excitatory synaptic inputs from stimulation of the pleural-abdominal connective as well as peripheral nerves P8 and P9, which innervate the tail and posterior part of the animal's body. These inputs were sufficient to elicit spikes. In RPI4, the excitatory synaptic inputs were followed by short and transient hyperpolarization, whereas in RPI5, the excitatory synaptic inputs were followed by slow and long-lasting hyperpolarization. Excitatory inputs elicited in RPI4 by stimulation of peripheral nerves appeared to be mediated, at least in part, by activation of tail sensory neurons. Intracellular stimulation of sensory neurons produced EPSPs in RPI4 that appeared to be monosynaptic. These results suggest that inhibitory interneurons underlying the circuit of the tail withdrawal reflex may play roles in mediating or modulating neuronal responses to tail stimulation. By inhibiting tail sensory and motor neurons, these interneurons may reduce the effectiveness of an animal's response to stimulation of the tail.

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.

1. The tail withdrawal reflex is mediated by a monosynaptic circuit composed of tail sensory and motor neurons, but there appear to be additional neuronal elements that also contribute to the reflex. A newly identified interneuron, called LP117, was located in the pleural ganglion. This neuron formed a parallel excitatory pathway between sensory and motor neurons. The distinguishing feature of LP117 was its ability to elicit a long-lasting (5-100 s) excitatory postsynaptic potential (EPSP) in the motor neuron. 2. Intracellular labeling of LP117 revealed axons projecting to the cerebral and abdominal as well as the pedal ganglia. Simultaneous intracellular recordings confirmed the widely divergent output of LP117 to tentacle motor neurons in the cerebral ganglion, as well as to gill, siphon, and ink motor neurons in the abdominal ganglion. 3. Also receiving input were abdominal neurons L29, which excites LFs motor neurons and facilitates LE sensory neurons, and L25, which is part of the pattern-generating network underlying respiratory pumping. Thus LP117 appears to be a neural element important for the conduction of information about tail stimulation to ganglia that are not innervated by tail sensory neurons themselves. Moreover, the divergent outputs suggest that LP117 is an element of a neural circuit underlying defensive arousal. 4. LP117 produced slow EPSPs in several motor neurons. The long time course of the EPSP could prolong the burst in the motor neuron produced by LP117 itself as well as increase the effectiveness of coincident synaptic input. This suggests that an important function of this interneuron is to extend the duration of the response to tail stimulation in the motor neuron. This could account for the relatively long time course of the motor neuron response to tail stimulation compared with that of the sensory neuron. 5. Sensitization is a form of nonassociative learning that produces changes in the amplitude and duration of reflex responses. It seems unlikely that all of these changes can be attributed to enhanced amplitude of the sensory-motor synapse, however. Therefore LP117 may itself be a site of plasticity for reflexes elicited by tail stimulation.

Electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia, an in vitro analogue of long-term behavioral training in Aplysia, produced changes in the synthesis of specific proteins in pleural sensory neurons. The changes in incorporation of [35S]methionine into proteins occurring 24 h after electrical stimulation (late) were determined and compared with changes occurring immediately after stimulation (early). Eight proteins were affected 24 h after electrical stimulation. Three of these proteins were also affected immediately after electrical stimulation. Two of the proteins affected late are components of the cytoskeleton. One protein was identified as actin. The other protein was purified from preparative 2D-gels and partial amino acid sequences of 3 peptides derived from this protein were determined. The peptide sequences were found to be identical to those of an Aplysia intermediate filament protein.

The peptides released from the bag cells (bag cell peptides, BCPs) in Aplysia californica are responsible for eliciting or modulating a repertoire of behaviors preceding and accompanying egg laying. Our interest was in determining to what extent behaviors that can occur simultaneously with egg laying are modulated. To address this issue, we examined the effects of an extract (bag cell extract, BCE) that contained BCPs, as well as egg-laying hormone (ELH), an identified BCP, on both the tail-siphon withdrawal reflex and the ability of that reflex to exhibit sensitization, a simple form of nonassociative learning. In addition, we examined the ability of BCE, as well as the individual BCPs, to modulate a component of the circuitry of the reflex. Behavioral experiments indicated that BCE inhibited the baseline withdrawal reflex and suppressed long-term sensitization of the reflex while leaving short-term sensitization unaffected. These effects were not observed in animals receiving an extract of a control ganglion or in animals receiving ELH, and therefore may be attributed to the actions of one or more BCPs, but presumably not to ELH alone. Electrophysiological experiments revealed that BCE also attenuated the monosynaptic EPSP elicited in tail motor neurons by tail sensory neurons. In subsequent experiments in which the BCPs were individually applied, only one BCP, beta-BCP, significantly attenuated the EPSPs. This effect was independent of narrowing of the presynaptic action potential. Taken together, these results suggest a novel behavioral modification attributed to BCPs, inhibition of the tail-siphon withdrawal reflex, and suggest a possible locus of action and peptide candidate for this effect. In addition, these experiments raise the possibility that BCPs may exert a long-lasting effect (> 24 hr), selectively blocking a simple form of long-term memory.

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.

We have identified an inhibitory neuron (RPL4) in the right pleural ganglion ofAplysia, which produced hyperpolarization of the sensory and motor neurons involved in the tail withdrawal reflex. Activation of RPL4 significantly reduced the amplitude of excitatory postsynaptic potentials produced in tail motor neurons by action potentials triggered in sensory neurons. This example of heterosynaptic inhibition was due, at least in part, to an increase in membrane input conductance in the motor neuron. Since the synaptic strength of the sensory-to-motor neuron connection has been associated with the strength of the tail withdrawal reflex, RPL4 may contribute to modulation of that reflex.

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.

Neurons have a multiplicity of ionic conductance mechanisms, the interactions of which determine in part the response of a neuron to chemical and electrical synaptic interactions, firing patterns, excitability, membrane potential and action-potential waveform. Several papers published in the past year have provided important new information on the role that ionic conductance mechanisms play in determining the electrophysiological properties of neurons, and how subtle differences can contribute to considerable variability of response.

Publisher Summary This chapter summarizes progress that has been made toward a mechanistic analysis of learning in three invertebrates: Aplysia, Hermissenda, and Drosophila. The selection was somewhat arbitrary because numerous invertebrates have exhibited both associative and nonassociative learning. The selection was guided, at least in part, by recent progress made on mechanistic analyses in these three invertebrates. Invertebrates are attractive animals for the study of learning and memory because of the relative simplicity of their central nervous systems. The central nervous systems of many invertebrates contain only several thousand cells compared with the billions of cells in the vertebrate nervous system. Despite the small number of cells, an invertebrate ganglion can control a variety of different behaviors, and a given behavior such as a withdrawal response may be mediated by 100 neurons or less. Moreover, many neurons are relatively large and can be uniquely identified, permitting one to examine the functional properties of an individual cell and to relate those properties to a specific behavior mediated by the cell. The cellular analysis of nonassociative and associative learning has progressed in a number of invertebrate preparations. The most extensive analysis of nonassociative learning has been conducted on cellular studies of habituation and sensitization.

Background The management of intermittent claudication is usually non-operative. Most surgeons accept, however, that intervention may be warranted for those with debilitating symptoms. Such interventions are limited to above-knee bypass or balloon angioplasty. However, the authors have performed almost 4500 bypasses for limb salvage, with careful audit of results, and suggest that, in very carefully selected cases, tibial bypass can also be performed for intermittent claudication. The present study aimed to review the results of infrainguinal bypass in this unit; to compare the results for above-knee, below-knee popliteal and tibial bypass in claudicants using either in situ or excised vein as the conduit; and to examine the indications and long-term follow-up for those undergoing tibial bypass for claudication. Methods A database has been maintained prospectively in this unit since 1986. Details of all patients undergoing infrainguinal bypass for claudication were retrieved. In-hospital charts were also reviewed and cross-referenced with the computer database. In addition to demographic details, data were extracted on operative indication and procedure, postoperative complications, return to desired level of activity and long-term graft patency. Results From 1987 to 1997, 409 infrainguinal reconstructions were performed for intermittent claudication (9 per cent of all infrainguinal reconstructions). Of these 73 per cent were in men and 27 per cent in women with a mean age of 64 (range 24–91) years. The operative procedures comprised 165 above-knee popliteal grafts, 150 bypasses to the below-knee popliteal artery and 94 to tibial arteries. There were no operative deaths. However, one of 165 patients who had an above-knee popliteal graft underwent an amputation. Primary and secondary patency rates at 4 years were 62 and 64 per cent for above-knee popliteal bypass, 77 and 81 per cent for below-knee popliteal bypass, and 86 and 90 per cent for tibial bypass. Cumulative survival was 93 per cent at 4 years. Conclusion Tibial bypass for severely disabling claudication can be performed with minimal morbidity and mortality rates, and is at least as durable as more commonly performed bypasses. These data indicate that concern over limb loss, death and limited life span may be exaggerated.

The connections between neurons (synapses) can adapt to changing circumstances of nervous transmissions, a phenomenon known as activity-dependent plasticity. Moreover, such plasticity is itself regulated, but little is known about the mechanisms or behavioural consequences of this metaplasticity. New work on the mollusc Aplysia, involving study of the neural circuits that respond to stimulation of the siphon and tail, unveils connections and feedback systems that provide a handle on the regulation of metaplasticity.

Many neurological disorders are based on mutations in 1 or more genes. To understand and optimally treat these disorders, it is necessary to understand the functions and regulation of the genes involved. It has become apparent that intuitive descriptions of gene regulation are often insufficient. A mathematical description adds precision and detail. Therefore, a mathematical description is important for networks of genes that underlie development, synaptic plasticity, and other complex biological processes. A mathematical description is also important to represent the combined effects of multiple genes that contribute to the phenotype of complex neurological disorders. In such gene networks, it is common for some of the gene products to regulate the expression of other network members. Also, the expression of all or some network members is commonly coregulated. Gene product proteins that regulate transcription are termed transcription factors (TFs), and many genes are activated by multiple TFs.

We have improved and extended an equivalent circuit model of the bursting neuron R15 in Aplysia and have simulated the neuromodulatory effects of serotonin (5-HT) and dopamine (DA), incorporating previously proposed mechanisms. The model produces dynamic model behavior consistent with the observed behavior of R15 in vitro , demonstrating the sufficiency of the proposed mechanisms. Responses of the model to extrinsic stimuli reveal that some of the seemingly paradoxical results can be attributed to the increase in conductance of two opposing currents, providing insight into the complex effects of the neuromodulation of multiple currents by a single neurotransmitter. These simulation results also suggest that neuromodulatory agents and second messengers act not only directly upon channel conductances, but also indirectly through the subsequent regulation of intracellular Ca 2+ concentrations. A nullcline and bifurcation analysis of reduced fast and slow subsystems of the model is employed to investigate the mathematical mechanism of bursting and neuromodulation. The slow processes define a steady-state manifold upon which the burst trajectory lies upon during the silent phase of the burst. The active (spiking) phase of the burst occurs as the slow system variables sweep into an oscillatory branch of the solution space of the fast subsystem via a homoclinic transition. The neuromodulatory effects of 5-HT and DA are examined by analyzing their effects upon the slow subsystem. Changes in behavioral modes (silence, bursting, beating) of the model are related to the occurence of subcritical Hopf bifurcations in the slow subsystem. Theses bifurcations are caused by the modulation of the shape of both the nullclines and manifold of the slow subsystem by 5-HT and DA.

1. The release of ink occurs selectively to long-lasting stimuli (18). There is a good correspondence between the features of the behavior and the firing pattern of the ink motor cells since the initial synaptic input is ineffective in firing the ink gland motor neurons. The features of the firing pattern of these cells have in turn been attributed to a fast K+ current that shunts the initial excitatory input and a late buildup of synaptic input. The purpose of the present paper is to examine these features quantitatively using a kinetic model of the ink gland motor neurons. 2. A modified Hodgkin-Huxley (13) model was developed, which includes seven membrane currents: a ) a fast inward Na+ current, 6) a slow inward Ca2+ current, c) a fast outward K+ current, d) a slow (delayed) outward K+ current, e) a synaptic current, f) a leakage current, and g) a capacitative current. The three ink gland motor neurons are electrically coupled to each other. The model also includes the features of this electronic coupling. 3. The Na+ current underlies the spike initiation, while the delayed K+ current accounts for spike repolarization. The fast transient K+ current also contributes to the repolarization process and slows the rate of rise of the membrane potential during the interspike interval. The slow Ca2+ current makes only a small contribution to the generation of the action potential, but contributes to the rise of the membrane potential during the interspike interval. 4. A depolarizing current pulse to one ink gland motor neuron yields an initial rapid depolarization followed by a slower component, which after a several-second period results in a burst of action potentials. The activation and inactivation sequence of a fast transient K+ current contributes to the cell’s selective response to long-lasting depolarizing current pulses. If the suprathreshold current pulse is preceded by a small subthreshold pulse, spikes are elicited immediately with the application of the suprathreshold pulse, since the subthreshold pulse inactivates the transient outward current. 5. The ink gland motor neurons also show a selective response to long-lasting trains of synaptic input. A train of synaptic input causes an initial large depolarization, but the initial synaptic input is ineffective in firing the cell. As a result, there is a severalsecond silent period or pause before an accelerating burst of spikes is produced. The fast transient K+ current shunts the initial synaptic input, preventing the cell from reaching spike threshold. Over a several-second period this current inactivates and the synaptic current is more effective in firing the cell. 6. While the fast outward current is inactivating, the synaptic current builds up and contributes to the accelerating burst of spikes.

Progress in unraveling the mysteries of the mind has been slow, but recent work indicates that a complete mechanistic understanding of at least simple forms of learning will soon be in hand. For example, it is becoming evident that associative learning is due to changes in existing neural circuits and that the acquisition of a learned behavior is due to the activation of second messenger systems in nerve cells.

Carew and Kandel (1977) found that weak stimuli to the head or siphon fail to elicit the release of ink. When paired with each other, however, the second of the two leads to the release of ink. The present paper quantifies and simulates the neural events which underlie this short-term modification of the behavior. Noxious stimuli to the intact animal were mimicked by delivering trains of electrical stimuli to the connectives (conditioning input) and siphon nerve (test input) which drive the ink gland motor neurons located within the abdominal ganglion. Estimates of the synaptic conductance and equilibrium potential during the conditioning and test inputs were then made and used to drive a previously developed Hodgkin-Huxley model of the ink motor neurons. The experimental and simulated results are in good agreement. Activation of one stimulus pathway augments or facilitates the ability of the other pathway to fire the ink motor neurons. The behavioral modification is causally related to a sustained synaptic current activated by the conditioning stimulus.

1. There is general agreement that the gill-withdrawal reflex elicited by weak tactile stimuli (less than 2 g to the siphon skin) is mediated almost entirely by the central nervous system (CNS) (13, 15, 18, 23). However, there was disagreement concerning the effects of moderate intensity (2--4 g) stimuli. Kupfermann et al. (18) found that the CNS mediates approximately 94% of the reflex elicited by moderate-intensity stimuli, whereas Peretz et al. (23) found that in this stimulus range, the amplitude of the reflex was, on average, unaltered when the CNS was removed. 2. To resolve this difference we first carried out pilot experiments in collaboration with B. Peretz, J. W. Jacklet, and K. Lukowiak using isolated mantle preparations, and then performed a systematic study using both the isolated mantle and intact animals. The main difference that seemed to account for the discrepancy in the results was the magnitude of the reflex response that was selected for study. Previous studies from this laboratory used a minimum-response criterion whereby only brisk and clearly observable responses of at least 35% maximum were examined. By contrast, Peretz et al. (23) examined all responses, even those that were extremely small. In addition, the two groups used different methods of stimulation so that stimulus intensities could not really be compared. 3. By comparing the effects of moderate-intensity stimuli in experiments with and without a response criterion in isolated mantle preparations, we found that when a minimal response-amplitude criterion is imposed the CNS mediates 90--95% of the gill-withdrawal reflex, whether it is elicited by the "tapper" stimulus used by Peretz et al. (23) or by the servo-controlled probe previously used in this laboratory. On the other hand, when no minimal response criterion is used and small responses are also examined, the response to the probe is still significantly reduced by 85% when the CNS is removed, whereas the reflex response to the tapper is more variable, sometimes increasing and other times decreasing with deganglionation. 4. We have also tested, in intact animals, the role of the CNS in mediating gill withdrawal. Water-jet stimuli were delivered to the siphon to elicit the reflex, as in previous behavioral studies. As was the case with probe stimulation in the isolated mantle preparation, in intact animals the CNS mediates 90% of the reflex evoked by moderate-intensity stimuli when a minimal response-amplitude criterion is imposed. 5. Our experiments indicate that, using response criteria and methods of stimulation, one can reliably study the reflex as predominantly mediated by the CNS.

Bilateral clusters of sensory neurons in the pleural ganglia of Aplysia contain cells involved in a defensive tail withdrawal reflex. These cells exhibit heterosynaptic facilitation in response to noxious skin stimulation that can be mimicked by the application of serotonin. Recently it has been shown that this facilitation can be selectively amplified by the application of a classical conditioning procedure to individual sensory neurons. We now report that an analog of this classical conditioning paradigm produces a selective amplification of the cAMP content of isolated sensory neuron clusters. The enhancement is achieved within a single trial and appears to be localized to the sensory neurons. These results indicate that a pairing-specific enhancement of cAMP levels may be a biochemical mechanism for associative neuronal modifications and perhaps learning.

1. Opaline release in Aplysia provides a simple model system for examining the biochemical and electrophysiological mechanisms underlying glandular secretion and its modulation. The opaline gland is a large multivesicular structure, which is innervated by at least three large identified motor neurons located within the right pleural ganglion (28). In this paper we have investigated the roles of dopamine (DA), acetylcholine (ACh), and serotonin (5-HT) in this simple neural system. 2. DA infusion produces a gland contraction that is similar to the response produced by neural activity in the previously identified opaline motor neurons. 3. The gland response to DA infusion is not affected by blocking synaptic transmission in the gland, suggesting that DA acts directly on muscle cells surrounding the gland and not through interposed interneurons. 4. In addition to its effect of producing contractions of the gland, DA enhances the size of subsequent neurally evoked gland contractions and increases the size of excitatory junctional potentials (EJPs) recorded from the opaline gland. Thus DA may have an additional modulatory role in opaline release. 5. DA antagonists such as fluphenazine and haloperidol inhibit the gland response to DA and also block the gland contraction produced by neural activity in the identified motor neurons. In addition, the DA antagonists reversibly block the EJPs recorded from the gland cells. Compounds known to block the effects of ACh or 5-HT have no effect on the dopamine-induced gland contractions, the contractions produced by firing the motor neurons, or the EJPs evoked by motor neuron stimulation. These results suggest that DA may be the neurotransmitter used by the identified opaline motor neurons. 6. ACh produces a decrease in pressure recorded from the lumen of the opaline gland that can be blocked by hexamethonium. 7. While 5-HT does not directly produce a contraction, treatment of the gland with the transmitter increases the size of subsequent gland responses produced either by DA infusion or activity in the opaline motor neurons. This enhancement has a relatively slow onset and long duration and persists for more than 15 min after the serotonin is washed out. In 60% of the experiments 5-HT also increased the size of the EJPs recorded from the opaline gland. The results suggest a modulatory role for serotonin in opaline secretion similar to the one it plays in other neural systems in Aplysia.

Respiratory pumping in Aplysia californica is a relatively stereotyped behavioral pattern with three components: (1) withdrawal of gill, siphon and mantle shelf; (2) closing of parapodia; (3) heart inhibition accompanied by a decrease in vasomotor tone. This phasic behavior is triggered by a central burst-generating network of interneurons in the abdominal ganglion. During respiratory pumping, motor neurons innervating the several effector organs receive a burst of either excitatory or inhibitory synaptic input which has previously been attributed to an unidentified central command cell called Interneuron II. Several of these motor cells are also concomitantly release from tonic synaptic input, which is opposite in sign to that which they receive from Interneuron II. This tonic input has been attributed to an unidentified cell called Interneuron XI. In this paper we identify and describe some of the neurons which contribute to the burst generating network; specifically, we focus on the neurons that produce the synaptic action attributed to Interneurons II and XI. The synaptic actions attributed to Interneuron XI are produced by a single, spontaneously active neuron, cell L24. This cell is a multi-action interneuron: it produces inhibitory synaptic potentials in some follower motor neurons, excitatory synaptic potentials in other follower cells, and a conjoint excitatory-inhibitory synaptic action onto gill motor neuron L7. At low frequency, L24 is excitatory to L7. With high frequency firing of L24, the synaptic potential produced in L7 converts from excitatory to inhibitory. In contrast to Interneuron XI, which is a single cell, the synaptic potentials previously attributed to Interneuron II are actually produced by a cluster of at least 3 respiratory command cells which we call L25, L26 and L27. Each of these cells accounts for only a limited portion of the synaptic input that drives the motor neurons during respiratory pumping. For most motor neurons innervated by both the respiratory command cells and Interneuron XI, the two synaptic inputs are opposite in sign. Mutually inhibitory connections between Interneuron XI and some of the central respiratory command cells ensure that the synaptic potentials from these two sources are constrained to occur at different times. Thus, centrally commanded synaptic inhibition or excitation of these motor neurons is made more effective by simultaneous disexcitation or disinhibition of Interneuron XI input. In addition to their role in generating respiratory pumping, L24 and L26 also contribute to the mediation of the defensive gill and siphon withdrawal reflex.

1. Repeated stimulation of the siphon skin results in short-term habituation of the reflex contractions of the gill (38). The habituation, in turn, is correlated with a depression of the excitatory postsynaptic potentials (EPSPs) in motor neurons from mechanoreceptor sensory neurons (SN) (7, 16). The present study was undertaken to examine the parametric features of the synaptic depression and gain insights into the mechanisms underlying the reduced transmitter release. 2. Single sensory neuron action potentials were repeatedly elicited with depolarizing current pulses while the amplitude of the resultant EPSPs in the motor neuron was monitored. Synaptic depression varies as a complex function of interstimulus interval (ISI). At an ISI of 1 s, depression is rapid and reaches a plateau at 36% of control. In contrast, the depression at an ISI of 100 s is less pronounced, showing a gradual decay to 65% of control with the 10th EPSP. Surprisingly, there are no significant differences in time course or magnitude of depression across a broad range of intermediate ISIs (3, 10, and 30 s), although depression at these ISIs is intermediate between the 1 and 100 s ISIs. 3. There is also a complex relationship between spike interval and the depression of the second of two EPSPs. Thus, depression of the second of two EPSPs or depression of a train of EPSPs is not a monotonic function of spike interval. Indeed, the data suggest that there may be a slight underlying facilitatory process with short spike intervals. 4. The results also indicate that the recovery of synaptic depression following a train of 10 stimuli is not constant. Shorter spike intervals produce more rapid recovery. 5. These data are inconsistent with a classical depletion model (33) for synaptic depression and indicate that either a single complex function of time and ISI or multiple functions underlie synaptic depression and its recovery at the sensory neuron synapse.

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.

1. Using combined morphological and electrophysiological techniques, we have identified motor neurons in the right pleural ganglion of Aplysia californica that contribute to the release of opaline from the opaline gland. 2. Three pleural ganglion neurons were found to meet the requirements for identification as opaline gland motor neurons by a) sending processes in nerve P5, which innervates the gland; b) producing contractions of the gland in the absence of central synaptic activity; and c) producing excitatory junctional potentials (EJPs) in cells making up the opaline gland itself. The neurons can be reliably located and have been designated PLR1, PLR2, and PLR3. 3. When gland contraction is measured by the change in luminal pressure, the gland response is a graded function of low-frequency spike activity in the motor neurons. 4. Presumptive EJPs recorded from opaline gland cells are reversibly decreased in size by high extracellular Mg2+ and reversibly increased in size by raising the concentration of extracellular Ca2+. These results suggest that the presumptive EJPs are chemically mediated. The presumptive EJPs show facilitation and posttetanic potentiation. 5. The identified opaline motor neurons may constitute a significant portion of the motor input to the opaline gland via nerve P5 since hyperpolarization of the cells prevents the opaline gland response elicited by right connective stimulation in vitro. 6. We have compared the properties of the opaline motor neurons with the previously identified properties of the ink motor neurons (6--9, 19). Like the ink motor neurons, the opaline motor neurons have high resting potentials, are electrically coupled, and have no spontaneous spike activity. They also receive a slow and long-lasting evoked depolarizing synaptic input, which appears to be mediated by a decreased conductance mechanism. The firing pattern of the opaline motor neurons produced by synaptic input shows the same delayed bursting pattern previously described for the ink motor neurons. 7. The biophysical properties and synaptic input to the ink motor neurons have been shown to affect the features of inking behavior (4, 6--9, 19). The opaline motor neurons share some of these biophysical characteristics and mediate a defensive behavior similar to ink release. Further comparisons of these behaviors and their underlying neural circuits may provide a better understanding of the extent to which cellular biophysical properties and patterns of synaptic input influence the features of the behaviors that individual neurons mediate.

1. The release of ink in response to a noxious stimulus is a relatively stereotyped behavior produced by strong and long-lasting stimuli. The purpose of this series of papers is to determine the quantitative extent to which the known voltage- and time-dependent ionic conductance mechanisms and synaptic influences can account for the ink gland motor neurons' firing pattern and, thus, the features of the behavior. 2. Four voltage- and time-dependent ionic currents have been analyzed. These include a fast transiet Na+-mediated inward current, a slower Ca2+-mediated inward current, a fast transient K+-mediated outward current, and a slower delayed outward current also mediated by K+ ions. 3. The current-voltage (I-V) relationships, equilibrium potentials, and steady-state activation and inactivation characteristics appear qualitatively similar to comparable currents observed in other gastropod neurons. 4. The recovery from inactivation of the delayed outward current has two time constants, one comparable to the inactivation time constant and the other more than an order of magnitude larger. The fast transient K+ current also appears to have a similar slow recovery from inactivation. 5. The synaptic current contributing to the firing pattern of the ink motor cells is a complex function of time. Initially, the synaptic conductance is high and the equilibrium potential near 0 mV. But, with time there is a gradual decrease in synaptic conductance and shift in the equilibrium potential to more depolarized levels.

Electrical stimulation of the connectives presynaptically inhibits the PSP from cell L10 to the left upper quadrant cells (LUQC). The present report describes the properties of some of the individual neurons contributing to this response. Action potentials produced in a cluster of cells in the abdominal ganglion reduce the amplitude of the L10-LUQC PSP for periods greater than 30 sec. At least some of their inhibitory action is mediated by a slow hyperpolarization of L10 which results in a decreased transmitter release. In other cases, however, the inhibition is produced with no significant alteration of L10 membrane potential, indicating that additional mechanisms may also be present. The neurons producing these effects are approximately 75 microns in diameter and are located on the left ventral surface of the ganglion. They have axons in the connectives and are thus activated by stimuli previously utilized to produce presynaptic inhibition. They appear to be some of the same cells that produce a slow inhibition of ink motoneuron L14; one of these has been identified as L32. The identification of these cells allows for the further biochemical, biophysical and morphological analysis of the events underlying presynaptic inhibition.

Post-tetanic potentiation (PTP) lasting several minutes is produced by high frequency intracellular activation of mechanosensory neurons. Control procedures suggest that the PTP involves a homosynaptic mechanism, restricted to the active synapses. The capacity for PTP appears to be a general property of mechanosensory neurons in this animal since it is found in separate sensory populations innervating the tail, head, and siphon of Aplysia .

The marine mollusc Aplysia is one of several experimental preparations that neuroscientists are using to help understand the mechanisms underlying learning and memory. Simple behavioral modifications such as sensitization and classical conditioning can be related to changes in the ability of previously formed synaptic connections to release neurotransmitter substance. The modifications of transmitter release in turn are regulated by the intracellular second messengers Ca 2+ and cAMP. The second messenger systems may act separately in the case of non-associative learning (sensitization) but their specific interactions may underly forms of associative learning such as classical conditioning.