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Many neurons exhibit regular firing that is limited to the duration and intensity of depolarizing stimuli. However, some neurons exhibit all-or-nothing plateau potentials that, once elicited, can lead to prolonged activity that is independent of stimulus intensity or duration. To better understand this diversity of information processing, we compared the voltage-gated and Ca 2+ -gated currents of three identified neurons from hermaphroditic Aplysia californica Two of these neurons, B51 and B64, generated plateau potentials and a third neuron, B8, exhibited regular firing and was incapable of generating a plateau potential. With the exception of the Ca 2+ -gated potassium current ( I KCa ), all three neuron types expressed a similar array of outward and inward currents, but with distinct voltage-dependent properties for each neuron type. Inhibiting voltage-gated Ca 2+ channels with Ni + prolonged the plateau potential, indicating I KCa is important for plateau potential termination. In contrast, inhibiting persistent Na + ( I NaP ) blocked plateau potentials, empirically and in simulations. Surprisingly, the properties and level of expression of I NaP were similar in all three neurons, indicating that the presence of I NaP does not distinguish between regular-firing neurons and neurons capable of generating plateau potentials. Rather, the key distinguishing factor is the relationship between I NaP and outward currents such as the delayed outward current ( I D ), and I KCa We then demonstrated a technique for predicting complex physiological properties such as plateau duration, plateau amplitude, and action potential duration as a function of parameter values, by fitting a curve in parameter space and projecting the curve beyond the tested values. SIGNIFICANCE STATEMENT Plateau potentials are intrinsic properties of neurons that are important for information processing in a wide variety of nervous systems. We examined three identified neurons in Aplysia californica with different propensities to generate a plateau potential. No single conductance was found to distinguish plateau generating neurons. Instead, plateau generation depended on the ratio between persistent Na + current ( I NaP ), which favored plateaus, and outward currents such as I KCa , which facilitated plateau termination. Computational models revealed a relationship between the individual currents that predicted the features of simulated plateau potentials. These results provide a more solid understanding of the conductances that mediate plateau generation., (Copyright © 2023 the authors.)

Despite numerous studies examining the mechanisms of operant conditioning (OC), the diversity of OC plasticity loci and their synergism have not been examined sufficiently. In the well-characterized feeding neural circuit of Aplysia , in vivo and in vitro appetitive OC increases neuronal excitability and electrical coupling among several neurons leading to an increase in expression of ingestive behavior. Here, we used the in vitro analog of OC to investigate whether OC reduces the excitability of a neuron, B4, whose inhibitory connections decrease expression of ingestive behavior. We found OC decreased the excitability of B4. This change appeared intrinsic to B4 because it could be replicated with an analog of OC in isolated cultures of B4 neurons. In addition to changes in B4 excitability, OC decreased the strength of B4's inhibitory connection to a key decision-making neuron, B51. The OC-induced changes were specific without affecting the excitability of another neuron critical for feeding behavior, B8, or the B4-to-B8 inhibitory connection. A conductance-based circuit model indicated that reducing the B4-to-B51 synapse, or increasing B51 excitability, mediated the OC phenotype more effectively than did decreasing B4 excitability. We combined these modifications to examine whether they could act synergistically. Combinations including B51 synergistically enhanced feeding. Taken together, these results suggest modifications of diverse loci work synergistically to mediate OC and that some neurons are well suited to work synergistically with plasticity in other loci. SIGNIFICANCE STATEMENT The ways in which synergism of diverse plasticity loci mediate the change in motor patterns in operant conditioning (OC) are poorly understood. Here, we found that OC was in part mediated by decreasing the intrinsic excitability of a critical neuron of Aplysia feeding behavior, and specifically reducing the strength of one of its inhibitory connections that targets a key decision-making neuron. A conductance-based computational model indicated that the known plasticity loci showed a surprising level of synergism to mediate the behavioral changes associated with OC. These results highlight the importance of understanding the diversity, specificity and synergy among different types of plasticity that encode memory. Also, because OC in Aplysia is mediated by dopamine (DA), the present study provides insights into specific and synergistic mechanisms of DA-mediated reinforcement of behaviors., (Copyright © 2022 the authors.)

A key issue in neuroscience is understanding the ways in which neuromodulators such as dopamine modify neuronal activity to mediate selection of distinct motor patterns. We addressed this issue by applying either low or high concentrations of l-DOPA (40 or 250 μM) and then monitoring activity of up to 130 neurons simultaneously in the feeding circuitry of Aplysia using a voltage-sensitive dye (RH-155). l-DOPA selected one of two distinct buccal motor patterns (BMPs): intermediate (low l-DOPA) or bite (high l-DOPA) patterns. The selection of intermediate BMPs was associated with shortening of the second phase of the BMP (retraction), whereas the selection of bite BMPs was associated with shortening of both phases of the BMP (protraction and retraction). Selection of intermediate BMPs was also associated with truncation of individual neuron spike activity (decreased burst duration but no change in spike frequency or burst latency) in neurons active during retraction. In contrast, selection of bite BMPs was associated with compression of spike activity (decreased burst latency and duration and increased spike frequency) in neurons projecting through specific nerves, as well as increased spike frequency of protraction neurons. Finally, large-scale voltage-sensitive dye recordings delineated the spatial distribution of neurons active during BMPs and the modification of that distribution by the two concentrations of l-DOPA.

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.

Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches., (Copyright © 2015 the authors 0270-6474/15/351617-10$15.00/0.)

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.)

Mutations in the gene encoding CREB-binding protein (CBP) cause deficits in long-term plasticity, learning, and memory. Here, long-term synaptic facilitation (LTF) at Aplysia sensorimotor synapses in cell culture was used as a model system to investigate methods for overcoming deficits in LTF produced by a CBP knockdown. Injecting CBP-siRNA into individual sensory neurons reduced CBP levels and impaired LTF produced by a standard protocol of five 5-min pulses of serotonin (5-HT) delivered at 20 min interstimulus intervals. A computational model, which simulated molecular processes underlying LTF induction, predicted a rescue protocol of five pulses of 5-HT at non-uniform interstimulus intervals that overcame the consequences of reduced CBP and restored LTF. These results suggest that complementary empirical and computational studies can identify methods for ameliorating impairments of learning attributable to molecular lesions.

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.

Although classical and operant conditioning are operationally distinct, it is unclear whether these two forms of learning are mechanistically distinct or similar. Feeding behavior of Aplysia provides a useful model system for addressing this issue. Both classical and operant appetitive behavioral training enhance feeding, and neuronal correlates have been identified. Behavioral training was replicated by in vitro analogs that use isolated ganglia. Moreover, a single-cell analog of operant conditioning was developed using neuron B51, a cell important for the expression of the conditioned behavior. Here, a single-cell analog of classical conditioning was developed. Acetylcholine (ACh) mediated the conditioned stimulus (CS)-elicited excitation of B51 in ganglia and mimicked the CS in the single-cell analog of classical conditioning. Pairing ACh with dopamine, which mediates the unconditioned stimulus in ganglia, decreased the excitability of B51, and increased the CS-elicited excitation of B51, similar to results following both in vivo and in vitro classical training. Finally, a D1 dopamine receptor (D1R) agonist failed to support classical conditioning in the cellular analog, whereas D1R mediates reinforcement in operant conditioning.

Accumulating evidence suggests that the transcriptional activator cAMP response element-binding protein 1 (CREB1) is important for serotonin (5-HT)-induced long-term facilitation (LTF) of the sensorimotor synapse in Aplysia. Moreover, creb1 is among the genes activated by CREB1, suggesting a role for this protein beyond the induction phase of LTF. The time course of the requirement for CREB1 synthesis in the consolidation of long-term facilitation was examined using RNA interference techniques in sensorimotor cocultures. Injection of CREB1 small-interfering RNA (siRNA) immediately or 10 h after 5-HT treatment blocked LTF when measured at 24 and 48 h after treatment. In contrast, CREB1 siRNA did not block LTF when injected 16 h after 5-HT treatment. These results demonstrate that creb1 expression must be sustained for a relatively long time to support the consolidation of LTF. In addition, LTF is also accompanied by a long-term increase in the excitability (LTE) of sensory neurons (SNs). Because LTE was observed in the isolated SN after 5-HT treatment, this long-term change was intrinsic to that element of the circuit. LTE was blocked when CREB1 siRNA was injected into isolated SNs immediately after 5-HT treatment. These data suggest that 5-HT-induced CREB1 synthesis is required for consolidation of both LTF and LTE.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

The transcription factor cAMP response element (CRE)-binding protein (CREB) plays an essential role in the induction of many forms of long-term synaptic plasticity. Levels of CREB1, the Aplysia homolog of CREB, show sustained elevations for several hours after the induction of long-term synaptic facilitation (LTF). Furthermore, CREB1 binds to the promoter of its own gene. These results suggest the existence of a CREB1-positive feedback loop that contributes to the consolidation of LTF. In the present study, we provide a detailed, quantitative characterization of the dynamics of CREB1 mRNA and protein as well as CREB1 phosphorylation after LTF induction. Injections of CRE oligonucleotides prevented the increase in CREB1 in response to 5-HT, corroborating the existence of the CREB1 feedback loop. This loop probably sustains CRE-dependent gene transcription, which remains elevated for at least 12 h after LTF induction. LTF is blocked by injection of CREB1 antibody after the induction phase, suggesting that the CREB1-positive feedback is required for consolidation of LTF.

Both short- and long-term sensitization of withdrawal reflexes of Aplysia are attributable at least in part to facilitation of the sensorimotor synapse. Previously, short-term synaptic facilitation has been associated with spike broadening and no change in temporal dynamics of burst transmission. In the present study, we examined whether long-term sensitization (LTS) is also associated with spike broadening and whether long-term synaptic facilitation is accompanied by changes in temporal dynamics. The results indicate that the temporal dynamics of the sensorimotor synapse are preserved after long-term facilitation. However, in contrast to short-term sensitization, LTS was accompanied by spike narrowing. The spike narrowing was observed both in centrally triggered spikes in isolated ganglia and in peripherally triggered spikes in reduced tail preparations. In addition, in reduced tail preparations, fewer spike failures in the afferent discharge of sensory neurons occurred in response to tail stimulation after ipsilateral LTS. Collectively, the results reveal that long-term sensitization affects the spike waveform of sensory neurons and enhances the sensory neuron responses to peripheral stimuli, but does not modify the synaptic dynamics of homosynaptic depression.

In Aplysia, long-term facilitation (LTF) at sensorimotor synapses of the pleural-pedal ganglia is mediated by an increase in the release of a neurotransmitter, which appears to be glutamate. Glutamate uptake also is increased in sensory neurons 24 hr after the induction of long-term sensitization (Levenson et al., 2000b). The present study investigated whether the same signaling pathways were involved in the long-term increase in glutamate uptake as in the induction of LTF. Thus, roles for cAMP, PKA (cAMP-dependent protein kinase), MAPK (mitogen-activated protein kinase), and tyrosine kinase in the regulation of glutamate uptake were tested. We found that 5-HT increased cAMP and activated PKA in sensory neurons. Exposure of pleural-pedal ganglia to analogs of cAMP or forskolin increased glutamate uptake 24 hr after treatments. Inhibitors of PKA (KT5720), MAPK (U0126 and PD98059), and tyrosine kinase (genistein) blocked the long-term increase in glutamate uptake produced by 5-HT. In addition, bpV, a tyrosine phosphatase inhibitor, facilitated the ability of subthreshold levels of 5-HT to increase glutamate uptake. Inhibition of PKC, which is not involved in LTF, had no effect on the long-term increase in glutamate uptake produced by 5-HT. Furthermore, activation of PKC by phorbol-12,13-dibutyrate did not produce long-term changes in glutamate uptake. The results demonstrate that the same constellation of second messengers and kinases is involved in the long-term regulation of both glutamate release and glutamate uptake. These similarities in signaling pathways suggest that regulation of glutamate release and uptake during formation of long-term memory are coordinated through coregulation of these two processes.

The Aplysia sensorimotor synapse is a key site of plasticity for several simple forms of learning. Plasticity of this synapse has been extensively studied, albeit primarily with individual action potentials elicited at low frequencies. Yet, the mechanosensory neurons fire high-frequency bursts in response to even moderate tactile stimuli delivered to the skin. In the present study, we extend this analysis to show that sensory neurons also fire bursts in the range of 1-60 Hz in response to electrical stimuli similar to those used in behavioral studies of sensitization. Intracellular stimulation of sensory neurons to fire a burst of action potentials at 10 Hz for 1 sec led to significant homosynaptic depression of postsynaptic responses. The depression was transient and fully recovered within 10 min. During the burst, the steady-state depressed phase of the postsynaptic response, which was only 20% of the initial EPSP of the burst, still contributed to firing the motor neuron. To explore the functional contribution of transient homosynaptic depression to the response of the motor neuron, computer simulations of the sensorimotor synapse with and without depression were compared. Depression allowed the motor neuron to produce graded responses over a wide range of presynaptic input strength. In addition, enhancement of synaptic transmission throughout a burst increased motor neuron output substantially more than did preferential enhancement of the initial phase of a burst. Thus, synaptic depression increased the dynamic range of the sensorimotor synapse and can, in principle, have a profound effect on information processing.

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.

Biophysical, biochemical, and morphological studies have implicated sensory neurons as key sites of plasticity in the formation and retention of the memory of long-term sensitization in Aplysia californica. This study examined the effects of different sensitization training protocols on the structure of sensory neurons mediating the tail-siphon withdrawal reflex. A 4 d training period produced a robust localized outgrowth in these sensory neurons observed 24 hr after the end of training. These changes are consistent with previous results in siphon sensory neurons (Bailey and Chen, 1988a). In contrast, 1 d of sensitization training, which has been shown to effectively induce long-term behavioral sensitization and synaptic facilitation (Frost et al., 1985; Cleary et al., 1998), is not associated with morphological changes in tail sensory neurons at either 24 hr or 4 d after training. Similarly, a single treatment with the growth factor TGF-beta, which also induced facilitation, did not alter sensory neuron morphology. The different effectiveness of the two protocols was not simply a reflection of the number of stimuli presented, because a 1 d massed training protocol did not produce sensitization 24 hr after training, nor did it induce neuronal outgrowth. These results suggest that extensive sensitization training is required to induce neuronal outgrowth in tail sensory neurons, indicating that the memory of long-term sensitization induced by 1 d of training is mechanistically different from that induced by 4 d of training. Moreover, the induction of a form of long-term sensitization associated with neuronal outgrowth does not appear to be a function of the amount of stimulation but does appear to be dependent on the temporal spacing of the stimulation over multiple days.

Transforming growth factor beta1 (TGF-beta1) induces long-term synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has acute effects as well. Treatment of pleural-pedal ganglia with TGF-beta1 for 5 min activated mitogen-activated protein kinase (MAPK) and stimulated the phosphorylation of synapsin in a MAPK-dependent manner. This phosphorylation appeared to modulate synapsin distribution in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-beta1-treated sensory neurons showed a significant reduction in the number of these puncta, an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-beta1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-beta1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.

Both positive and negative feedback loops of transcriptional regulation have been proposed to be important for the generation of circadian rhythms. To test the sufficiency of the proposed mechanisms, two differential equation-based models were constructed to describe the Neurospora crassa and Drosophila melanogaster circadian oscillators. In the model of the Neurospora oscillator, FRQ suppresses frq transcription by binding to a complex of the transcriptional activators WC-1 and WC-2, thus yielding negative feedback. FRQ also activates synthesis of WC-1, which in turn activates frq transcription, yielding positive feedback. In the model of the Drosophila oscillator, PER and TIM are represented by a "lumped" variable, "PER." PER suppresses its own transcription by binding to the transcriptional regulator dCLOCK, thus yielding negative feedback. PER also binds to dCLOCK to de-repress dclock, and dCLOCK in turn activates per transcription, yielding positive feedback. Both models displayed circadian oscillations that were robust to parameter variations and to noise and that entrained to simulated light/dark cycles. Circadian oscillations were only obtained if time delays were included to represent processes not modeled in detail (e.g., transcription and translation). In both models, oscillations were preserved when positive feedback was removed.

Feeding behavior in Aplysia californica can be classically conditioned using tactile stimulation of the lips as conditional stimulus (CS) and food as unconditional stimulus (US) [ (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 an in vitro preparation of the cerebral and buccal ganglia. Stimulation of a lip nerve (AT(4)), 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 AT(4) evoked 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 AT(4) was not significantly increased by paired training. Moreover, correlates of classical conditioning were specific to stimulation of AT(4). 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.

A training protocol was developed to classically condition feeding behavior in Aplysia californica using 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 () describes two neurophysiological correlates of the classical conditioning.

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.

Previously, an analog of operant conditioning was developed using the buccal ganglia of Aplysia, the probabilistic occurrences of a specific motor pattern (i.e., pattern I), a contingent reinforcement (i.e., stimulation of the esophageal nerve), and monotonic stimulation of a peripheral nerve (i.e., n.2,3). This analog expressed a key feature of operant conditioning (i.e., selective enhancement of the probability of occurrence of a designated motor pattern by contingent reinforcement). In addition, the training induced changes in the dynamical properties of neuron B51, an element of the buccal central pattern generator. To gain insights into the neuronal mechanisms that mediate features of operant conditioning, the present study identified a neuronal element that was critically involved in the selective enhancement of pattern I. We found that bursting activity in cell B51 contributed significantly to the expression of pattern I and that changes in the dynamical properties of this cell were associated with the selective enhancement of pattern I. These changes could be induced by an explicit association of reinforcement with random depolarization of B51. No stimulation of n.2,3 was required. These results indicate that the selection of a designated motor pattern by contingent reinforcement and the underlying neuronal plasticity resulted from the association of reinforcement with a component of central neuronal activity that contributes to a specific motor pattern. The sensory stimulus that allows for occurrences of different motor acts may not be critical for induction of plasticity that mediates the selection of a motor output by contingent reinforcement in operant conditioning.

Previously, an analog of operant conditioning in Aplysia was developed using the rhythmic motor activity in the isolated buccal ganglia. This analog expressed a key feature of operant conditioning, namely a selective enhancement in the occurrence of a designated motor pattern by contingent reinforcement. Different motor patterns generated by the buccal central pattern generator were induced by monotonic stimulation of a peripheral nerve (i.e., n.2,3). Phasic stimulation of the esophageal nerve (E n.) was used as an analog of reinforcement. The present study investigated the neuronal mechanisms associated with the genesis of different motor patterns and their modifications by contingent reinforcement. The genesis of different motor patterns was related to changes in the functional states of the pre-motor neuron B51. During rhythmic activity, B51 dynamically switched between inactive and active states. Bursting activity in B51 was associated with, and predicted, characteristic features of a specific motor pattern (i.e., pattern I). Contingent reinforcement of pattern I modified the dynamical properties of B51 by decreasing its resting conductance and threshold for eliciting plateau potentials and thus increased the occurrences of pattern I-related activity in B51. These modifications were not observed in preparations that received either noncontingent reinforcement (i.e., yoke control) or no reinforcement (i.e., control). These results suggest that a contingent reinforcement paradigm can regulate the dynamics of neuronal activity that is centrally programmed by the intrinsic cellular properties of neurons.

Although in vitro analyses of long-term changes in the sensorimotor connection of Aplysia have been used extensively to understand long-term sensitization, relatively little is known about the ways in which the connection is modified by learning in vivo. Moreover, sites other than the sensory neurons might be modified as well. In this paper, several different biophysical properties of sensory neurons, motor neurons, and LPl17, an identified interneuron, were examined. Membrane properties of sensory neurons, which were expressed as increased excitability and increased spike afterdepolarization, were affected by the training. The biophysical properties of motor neurons also were affected by training, resulting in hyperpolarization of the resting membrane potential and a decrease in spike threshold. These results suggest that motor neurons are potential loci for storage of the memory in sensitization. The strength of the connection between sensory and motor neurons was affected by the training, although the connection between LPl17 and the motor neuron was unaffected. Biophysical properties of LPl17 were unaffected by training. The results emphasize the importance of plasticity at sensory-motor synapses and are consistent with the idea that there are multiple sites of plasticity distributed throughout the nervous system.

Operant conditioning is characterized by the contingent reinforcement of a designated behavior. Previously, feeding behavior in Aplysia has 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 an in vitro buccal 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 of Aplysia and 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.

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.