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The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus . However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Decades of work in Aplysia californica established the general rule that principles of synaptic plasticity and their molecular mechanisms are evolutionarily conserved from mollusks to mammals. However, an exquisitely sensitive, activity-dependent homosynaptic mechanism that protects against the depression of neurotransmitter release in Aplysia sensory neuron terminals has, to date, not been uncovered in other animals, including mammals. Here, we discover that depression at a mammalian synapse that is implicated in habit formation and habit learning acceleration by ethanol, the fast-spiking interneuron (FSI) to medium spiny principal projection neuron (MSN) synapse of the dorsolateral striatum, is subject to this type of synaptic protection. We show that this protection against synaptic depression is calcium- and PDZ domain interaction-dependent. These findings support activity dependent protection against synaptic depression as an Aplysia-like synaptic switch in mammals that may represent a leveraging point for treating alcohol use disorders.

Summary New possibilities for treating posttraumatic stress disorder and anxiety disorders involving abnormal memories are emerging from analysis of persistent protein kinase activation and mechanisms of synapse-specific modification, known as synaptic tagging.

During the evolution of synapses, existing molecules were exapted to serve in specific synaptic roles. Recent increased availability of assembled transcriptomes from organisms that evolved before and after the appearance of the earliest synapses provides the opportunity to trace molecular adaptations important for development of fast synaptic transmission. We discuss issues that affect transcriptome assembly and phylogenetic analysis, and which therefore impact this analysis. We use relatively recent transcriptomes of pre-bilaterians to examine the molecular evolution of three types of critical synapse-specific proteins: vesicular transporters, synaptotagmins and ionotropic glutamate receptors. The results emphasize the fundamental difficulties in defining the specific point at which a protein “assumes” a synaptic function. Nevertheless, the analysis informs our understanding of several major evolutionary topics, including the evolution of synaptic vesicles and the identity of the first neurotransmitter used for fast, synchronous transmission. This analysis is also relevant for the current discussion of whether neuronal and synaptic function evolved separately, once in ctenophores and once in cnidarians and the main bilaterian lineage.

Habituation of a behavioral response to a repetitive stimulus enables animals to ignore irrelevant stimuli and focus on behaviorally important events. In Aplysia, habituation is mediated by rapid depression of sensory synapses, which could leave an animal unresponsive to important repetitive stimuli, making it vulnerable to injury. We identified a form of plasticity that prevents synaptic depression depending on the precise stimulus strength. Burst-dependent protection from depression is initiated by trains of 2–4 action potentials, and is distinct from previously described forms of synaptic enhancement. The blockade of depression is mediated by presynaptic Ca2+ influx and protein kinase C (PKC), and requires localization of PKC via a PDZ domain interaction with Aplysia PICK1. During protection from depression, PKC acts as a highly sensitive detector of the precise pattern of sensory neuron firing. Behaviorally, burst-dependent protection reduces habituation, enabling animals to maintain responsiveness to stimuli that are functionally important.

The most commonly cited descriptions of the behavioral characteristics of habituation come from two papers published almost 40 years ago [Groves, P. M., & Thompson, R. F. (1970). Habituation: A dual-process theory. Psychological Review, 77, 419-450; Thompson, R. F., & Spencer, W. A. (1966). Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychological Review, 73, 16-43]. In August 2007, the authors of this review, who study habituation in a wide range of species and paradigms, met to discuss their work on habituation and to revisit and refine the characteristics of habituation. This review offers a re-evaluation of the characteristics of habituation in light of these discussions. We made substantial changes to only a few of the characteristics, usually to add new information and expand upon the description rather than to substantially alter the original point. One additional characteristic, relating to long-term habituation, was added. This article thus provides a modern summary of the characteristics defining habituation, and can serve as a convenient primer for those whose research involves stimulus repetition.

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 μM 5-HT was unaffected by 100 μM spiperone, whereas it was effectively blocked by 100 μM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 μM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

We attempted to identify compounds that are effective in blocking the serotonin (5-hydroxytryptamine, 5-HT) receptor(s) that activate adenylyl cyclase (AC) in Aplysia CNS. We call this class of receptor 5-HTapAC. Eight of the 14 antagonists tested were effective against 5-HTapAC in CNS membranes with the following rank order of potency: methiothepin > metergoline ∼ fluphenazine > clozapine > cyproheptadine ∼ risperidone ∼ ritanserin > NAN-190. GR-113808, olanzapine, Ro-04-6790, RS-102221, SB-204070, and spiperone were inactive. Methiothepin completely blocked 5-HT stimulation of AC with a K b of 18 nM. Comparison of the pharmacological profile of the 5-HTapAC receptor with those of mammalian 5-HT receptor subtypes suggested it most closely resembles the 5-HT6 receptor. AC stimulation in Aplysia sensory neuron (SN) membranes was also blocked by methiothepin. Methiothepin substantially inhibited two effects of 5-HT on SN firing properties that are mediated by a cAMP-dependent reduction in S-K+ current: spike broadening in tetraethylammonium/nifedipine and increased excitability. Consistent with cyproheptadine blocking 5-HT stimulation of AC, cyproheptadine also blocked the 5-HT-induced increase in SN excitability. Methiothepin was less effective in blocking AC-mediated modulatory effects of 5-HT in electrophysiological experiments on SNs than in blocking AC stimulation in CNS or SN membranes. This reduction in potency appears to be due to effects of the high ionic strength of physiological saline on the binding of this antagonist to the receptor. Methiothepin also antagonized AC-coupled dopamine receptors but not AC-coupled small cardioactive peptide receptors. In conjunction with other pharmacological probes, this antagonist should be useful in analyzing the role of 5-HT in various forms of neuromodulation in Aplysia.

The synaptic connections of Aplysia sensory neurons (SNs) undergo dramatic homosynaptic depression (HSD) with only a few low-frequency stimuli. Strong and weak SN synapses, although differing in their probabilities of release, undergo HSD at the same rate; this suggests that the major mechanism underlying HSD in these SNs may not be depletion of the releasable pool of vesicles. In computational models, we evaluated alternative mechanisms of HSD, including vesicle depletion, to determine which mechanisms enable strong and weak synapses to depress with identical time courses. Of five mechanisms tested, only release-independent, stimulus-dependent switching off of release sites resulted in HSD that was independent of initial synaptic strength. This conclusion that HSD is a release-independent phenomenon was supported by empirical results: an increase in Ca(2+) influx caused by spike broadening with a K(+) channel blocker did not alter HSD. Once induced, HSD persisted during 40 min of rest with no detectable recovery; thus, release does not recover automatically with rest, contrary to what would be expected if HSD represented an exhaustion of the exocytosis mechanism. The hypothesis that short-term HSD involves primarily a stepwise silencing of release sites, rather than vesicle depletion, is consistent with our earlier observation that HSD is accompanied by only a modest decrease in release probability, as indicated by little change in the paired-pulse ratio. In contrast, we found that there was a dramatic decrease in the paired-pulse ratio during serotonin-induced facilitation; this suggests that heterosynaptic facilitation primarily involves an increase in release probability, rather than a change in the number of functional release sites.

Recent evidence implicates Ca2+/CaM-sensitive adenylyl cyclase (AC) as a molecular coincidence detector for temporally paired stimuli during associative learning. During conditioning in Aplysia, AC is optimally activated when Ca2+ influx, the cellular signal for the conditioned stimulus (CS), precedes binding of modulatory transmitter, the cellular signal for the unconditioned stimulus (US). This sequence preference of the AC for Ca2+-before-transmitter, parallels the CS-preceding-US pairing requirement of classical conditioning. In this study, we have examined the response of AC from rat cerebellum to brief exposures to Ca2+ and to transmitter in a perfused membrane assay. We observed modest synergism between Ca2+ and transmitter in activating AC. Activation was more effective when a Ca2+ stimulus immediately preceded a transmitter stimulus than when the two stimuli were delivered in the reverse order. Thus, rat cerebellar AC displayed a sequence preference for optimal activation by paired stimuli similar to that observed in Aplysia; this sequence dependence could contribute to the CS-US sequence requirement observed in most mammalian classical conditioning paradigms.

An important recent insight in a number of neurobiological systems is that during learning, individual dually regulated proteins with associative properties function as critical sites of stimulus convergence. During conditioning in Aplysia, the Ca2+ /calmodulin-sensitive adenylyl cyclase (AC) in mechanosensory neurons serves as a molecular site of interaction between Ca2+ and serotonin [5-hydroxytryptamine (5-HT)]-two signals that represent the CS and US in these cells. Conditioning requires that the CS and US be paired within a narrow time window and in the appropriate sequence. AC shows an analogous sequence preference: It is more effectively activated when a pulse of Ca2+ precedes a pulse of 5-HT than when the 5-HT precedes Ca2+. One mechanism that contributes to this sequence preference is that Ca2+/calmodulin binding to AC accelerates the rate of AC activation by receptor-Gs. We have identified two additional properties of AC activation that would cause pairing with Ca2+ preceding 5-HT to be more effective than simultaneous pairing or pairing with the reciprocal sequence: (1) Activation of Aplysia AC by a Ca2+ pulse rose with a delay compared with activation by a 5-HT pulse. (2) A late pulse of Ca2+, which arrived after 5-HT, acted, via calmodulin, to accelerate the decay of AC activation by receptor-Gs. Together, these activation properties of AC may contribute to the CS-US sequence requirement of classical conditioning.

Introduction: Lead exposure can cause learning disabilities, memory loss and severe damage to the nervous system. However, the exact mechanism by which lead causes learning disabilities is not fully understood. The effects of lead on calcium-regulated signaling pathways are difficult to study biochemically; with the traditional method of controlling the free calcium concentration with EGTA, the exact concentrations of free lead and calcium ions in solution are interdependent and prone to error because EGTA also buffers lead. Methods and Results: In our approach, we first reduced the free calcium concentration in the solution using calcium-binding resins before adding lead to buffers. The solution was sequentially treated with Chelex-100 ion exchange resin, followed by immobilized BAPTA resin. The final concentration of free calcium in the solution was measured with Fluo-3 indicator. Our protocol successfully produced buffers with free calcium levels below 15 nM, which is substantially below threshold for activation of calcium-dependent enzymes in signaling pathways (which is typically a few hundred nanomolar calcium, when determined in vitro ). Conclusion: This method provides an improved approach to study the effect of heavy metals on calcium-stimulated signaling pathways.

The facilitatory transmitters serotonin (5-HT) and the molluscan neuropeptides SCP A and SCP B both activate adenylyl cyclase in Aplysia mechanosensory neurons and produce multiple modulatory effects that contribute to increasing transmitter release from these cells. This enhancement of transmitter release from sensory neurons contributes to increased behavioral response during sensitization and classical conditioning in Aplysia . Recently, specific examples of modulation in these sensory neurons have been described that are more effectively initiated by 5-HT than by the SCPs. For example, in the present study, 5-HT produces 55% greater broadening of the normal sensory neuron action potential than did SCP B . These differences in the modulatory actions of the facilitatory transmitters have been interpreted as suggesting that 5-HT produces its modulatory effects at least partly via a cAMP-independent mechanism. However, we have found that the two types of facilitatory transmitters are not equally effective in activating adenylyl cyclase. In both whole CNS membranes and sensory neuron membranes, SCP B was less effective than 5-HT in stimulating adenylyl cyclase activity measured in steady state assays. Because electrophysiological experiments suggested that the response to the SCPs desensitizes rapidly, we further compared cyclase stimulation in perfused membrane assays that enable continuous monitoring of cyclase activity; however we observed that 5-HT was also more effective than SCP B in stimulating cyclase at the onset of transmitter exposure. We discuss the possibility that lower peak stimulation of cyclase by SCP B and a faster rate of desensitization could account for some of the differences between the SCPs and 5-HT in modulating sensory neurons.

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC- Apl A that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC- Apl A was stimulated by Ca 2+ /CaM. AC- Apl C is most similar to the Ca 2+ -inhibited AC5 and AC6 in mammals. Recombinant AC- Apl C was directly inhibited by Ca 2+ , independent of CaM. AC- Apl A and AC- Apl C are expressed in SNs, whereas AC- Apl B and AC- Apl D are not. Knockdown of AC- Apl A demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca 2+ -inhibited AC in SNs, together with a Ca 2+ /CaM-stimulated AC, would enhance the associative requirement for coincident Ca 2+ influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC- Apl A.

Enhancement of the defensive withdrawal reflex of Aplysia involves a prolongation of the action potentials of mechanosensory neurons, which contributes to facilitation of transmitter release from these cells. Recent reports have suggested that whereas cAMP-dependent modulation of K+ current increases sensory neuron excitability, a cAMP-independent decrease in K+ current may increase the action potential duration and, thus, facilitate transmitter release. We have tested this proposal using Walsh cAMP-dependent protein kinase inhibitor or activators of the cAMP cascade and found that cAMP plays a major role in the spike-broadening effects of facilitatory transmitter; however, broadening requires higher levels of activation of the cAMP-dependent kinase than does increasing excitability. A steeply voltage-dependent transient K+ current, termed IKV,early, and the slowly activating S-type K+ (S-K+) current are both reduced by activation of the cAMP cascade, although with different sensitivities to the second messenger, enabling excitability and spike duration to be regulated independently. Differences in cAMP sensitivity also suggested that the originally described S-K+ current actually consists of two independent components, a slowly activating component and a time-independent, "steady-state" current that is activated at rest.

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, we have quantified the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain with purified species-specific calmodulins. In all three species, adenylyl cyclase was activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity was dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raised the threshold for adenylyl cyclase activation by Ca2+ but also acted synergistically with Ca2+ to activate maximally adenylyl cyclase.

The protein kinase C (PKC) and the cAMP-dependent kinase (protein kinase A; PKA) pathways are known to play important roles in behavioral plasticity and learning in the nervous systems of a wide variety of species across phyla. We briefly review the members of the PKC and PKA family and focus on the evolution of the immediate upstream activators of PKC and PKA i.e., phospholipase C (PLC) and adenylyl cyclase (AC), and their conservation in gastropod mollusks, taking advantage of the recent assembly of the Aplysiacalifornica and Lottia gigantea genomes. The diversity of PLC and AC family members present in mollusks suggests a multitude of possible mechanisms to activate PKA and PKC; we briefly discuss the relevance of these pathways to the known physiological activation of these kinases in Aplysia neurons during plasticity and learning. These multiple mechanisms of activation provide the gastropod nervous system with tremendous flexibility for implementing neuromodulatory responses to both neuronal activity and extracellular signals.

This review focuses on synaptic depression at sensory neuron-to-motor neuron synapses in the defensive withdrawal circuit of Aplysia as a model system for analysis of molecular mechanisms of sensory gating and habituation. We address the following topics: Of various possible mechanisms that might underlie depression at these sensory neuron-to-motor neuron synapses in Aplysia, historically the most widely-accepted explanation has been depletion of the readily releasable pool of vesicles. Depletion is also believed to account for synaptic depression at long interstimulus intervals in a variety of other systems.Multiple lines of evidence now indicate that vesicle depletion is not an important contributing mechanism to synaptic depression at Aplysia sensory neuron-to-motor neuron synapses. More generally, it appears that vesicle depletion does not contribute substantially to depression that occurs with those stimulus patterns that are typically used in studying behavioral habituation.Recent evidence suggests that at these sensory neuron-to-motor neuron synapses in Aplysia, synaptic depression is mediated by an activity-dependent, but release-independent, switching of individual release sites to a silent state. This switching off of release sites is initiated by Ca2+ influx during individual action potentials. We discuss signaling proteins that may be regulated by Ca2+ during the silencing of release sites that underlies synaptic depression.Bursts of 2–4 action potentials in presynaptic sensory neurons in Aplysia prevent the switching off of release sites via a Ca2+- and protein kinase C (PKC)-dependent mechanism: “burst-dependent protection” from synaptic depression.This Ca2+- and PKC-regulated switch may explain the sensory gating that allows animals to discriminate which stimuli are innocuous and appropriate to ignore and which stimuli are more important and should continue to elicit responses.

Facilitation of the monosynaptic connection between siphon sensory neurons and gill and siphon motor neuron contributes to sensitization and dishabituation of the gill and siphon withdrawal reflex in Aplysia. The facilitatory transmitter serotonin (5-HT) initiates two mechanisms that act in parallel to increase transmitter release from siphon sensory neurons. 5-HT acts, at least partly through cAMP, to broaden the presynaptic action potential. 5-HT also initiates a second process that facilitates depressed sensory neuron synapses by a mechanism independent of changes in action potential duration. Recent experiments indicated that either of two protein kinases, cAMP-dependent protein kinase A and protein kinase C, are capable of effectively activating this second facilitatory mechanism, restoring synaptic transmission in depressed synapses. We have used the adenylyl cyclase inhibitor SQ 22,536 [9-(tetrahydro-2-furyl)adenine or THFA] to explore the contribution of cAMP to the reversal of synaptic depression. THFA effectively inhibited both adenylyl cyclase activity in vitro and known cyclase-mediated effects in intact sensory neurons. THFA also completely blocked facilitation of depressed synapses by 5-HT. These results suggest that adenylyl cyclase plays a critical role in the reversal of synaptic depression that contributes to dishabituation in this system.

We have characterized paired-pulse facilitation atAplysiasensory neuron-to-motoneuron synapses. This simple form of very short-term synaptic plasticity displayed an unusual feature: it decreased dramatically with repeated testing. Synaptic depression at these synapses and this use-dependent decrease in paired-pulse facilitation occurred independently of each other. Paired-pulse facilitation was inversely correlated with the size of the initial synaptic connection and was absent at stronger synapses. The use-dependent decrease in paired-pulse facilitation occurred at the same rate at large synapses as at small synapses, although the initial paired-pulse facilitation at large synapses was substantially smaller. Rates of synaptic depression were also independent of initial synaptic strength. Paired-pulse facilitation was blocked by presynaptic EGTA injection, but not by postsynaptic EGTA or BAPTA injection. These results indicate that presynaptic Ca2+influx plays a critical role in paired-pulse facilitation. However, the persistence of the decrease in paired-pulse facilitation for longer than 15 min suggests that Ca2+from the first paired action potential produces facilitation via a modulatory mechanism rather than by summating with Ca2+influx during the second paired action potential in activating the Ca2+binding sites that initiate exocytosis. This modulatory mechanism may not involve protein phosphorylation because paired-pulse facilitation was unaffected by the protein kinase inhibitors H7 and KN-62. These findings further suggest that release by the second paired action potential occurs at sites distinct from those that mediate release by the first action potential.

Ca2+/calmodulin-sensitive adenylyl cyclase plays a role in several forms of synaptic plasticity and learning. To understand how cellular signals from neuronal activity during behavioral stimuli might be integrated by adenylyl cyclase, we have characterized the response of type I adenylyl cyclase to transient Ca2+ stimuli. Stimulation by a several second Ca2+ stimulus is delayed, rising to a peak after the Ca2+ stimulus has ended. We attempted to identify the site of the persistent Ca2+ signal that enabled adenylyl cyclase stimulation to increase after free Ca2+ had declined. Free calmodulin itself displayed no persistent activation by Ca2+ and was unable to activate adenylyl cyclase if exposed to low Ca2+ solution

The molluscan neuropeptide FMRFamide has a number of inhibitory actions on the sensory neurons and motoneurons mediating the defensive gill and siphon withdrawal reflex pathway of Aplysia californica. Exogenous application of FMRFamide has a biphasic, dual-polarity effect on the majority of LFS siphon motoneurons, causing a transient depolarization followed by a prolonged hyperpolarization. FMRFamide induces this response in LFS neurons by causing an increase in multiple ionic currents, including a transient Na+ current, a slow prolonged Na+ current, a 4-aminopyridine (4-AP)-sensitive K+ current and a 4-AP-insensitive K+ current. We have found that a subset of LFS neurons exhibits an exclusively excitatory, biphasic response to FMRFamide, consisting of a transient depolarization followed by a prolonged depolarization of reduced magnitude. Over a period of 29 months, we consistently observed an increase in the incidence of the exclusively excitatory response during the summer months (June to September). From October to May, we observed an exclusively excitatory response to FMRFamide in 19 % of LFS neurons; yet, in the summer months, 51 % of LFS neurons exhibited this response pattern. We compared the ionic basis of the exclusively excitatory response to FMRFamide with the ionic mechanisms mediating the more frequently observed excitatory/inhibitory response. The exclusively excitatory response involves three of the same ionic components as the more typical excitatory/inhibitory response, including the activation of a transient Na+ current, a slow prolonged Na+ current and a 4-AP-insensitive K+ current. The principal difference between the two response types is that FMRFamide fails to activate a 4-AP-sensitive K+ current in those LFS neurons that exhibit an exclusively excitatory response to the peptide. In addition, LFS neurons with an exclusively excitatory response tend to show a coordinated increase in the magnitude of the inward current component of the FMRFamide response. Together, these changes during the summer months may enable this modulatory peptide to bring LFS neurons to suprathreshold levels of activity for eliciting a siphon withdrawal and should substantially alter the neuromodulatory effects of the peptide.

The molluscan neuropeptide FMRFamide has an inhibitory effect on transmitter release from the presynaptic sensory neurons in the neural circuit for the siphon withdrawal reflex. We have explored whether FMRFamide also acts postsynaptically in motor neurons in this circuit, focusing on the LFS motor neurons. FMRFamide typically produces a biphasic response in LFS neurons: a fast excitatory response followed by a prolonged inhibitory response. We have analyzed these postsynaptic actions and compared them with the mechanism of FMRFamide's inhibition of the presynaptic sensory neurons. The transient excitatory effect of FMRFamide, which desensitizes rapidly, is due to activation of a TTX- insensitive, Na(+)-dependent inward current. The late hyperpolarizing phase of the FMRFamide response results from activation of at least two K+ currents. One component of the hyperpolarizing response is active at rest and at more hyperpolarized membrane potentials, and is blocked by 5 mM 4-aminopyridine, suggesting that it differs from the previously described FMRFamide-modulated K+ currents in the presynaptic sensory neurons. In addition, FMRFamide increases a 4-aminopyridine-insensitive K+ current. Presynaptically, FMRFamide increases K+ conductance, acting via release of arachidonic acid. In the LFS motor neurons, application of arachidonic acid mimicked the prolonged, hyperpolarizing phase of the FMRFamide response; 4-bromophenacyl bromide, an inhibitor of phospholipase A2, selectively blocked this component of the FMRFamide response. Thus, FMRFamide may act in parallel pre- and post- synaptically to inhibit the output of the siphon withdrawal reflex circuit, producing this inhibitory effect via the same second messenger in the sensory neurons and motor neurons, though a number of the K+ currents modulated in these two types of neurons are different.

Activity-dependent facilitation is a mechanism of associative synaptic plasticity that contributes to classical conditioning in Aplysia. Previous studies of activity-dependent facilitation in the mechanosensory neurons of Aplysia suggested that the Ca2+ influx during paired spike activity enhances the transmitter-stimulated, cAMP- dependent, presynaptic facilitation in these cells. Moreover, paired activity was found to potentiate the activation of the adenylate cyclase by transmitter. It was therefore proposed that the Ca2+/calmodulin-sensitive cyclase may serve as a site of interaction between the inputs from the conditioned and unconditioned stimuli. These studies were carried out to test whether a Ca2+/calmodulin- sensitive adenylate cyclase in the Aplysia CNS has the properties necessary to mediate such an associative interaction. Three lines of evidence indicate that the same cyclase molecules that are sensitive to Ca2+/calmodulin are also stimulated by receptor to facilitatory transmitter via the stimulatory G-protein, Gs: First, calmodulin inhibitors reduced stimulation of the cyclase by facilitatory transmitter. When membranes had been preexposed to one of these inhibitors, trifluoperazine, the addition of exogenous calmodulin partially reversed the inhibition. Second, when Gs had been activated by GTP gamma S, so that it persistently activated the catalytic unit of the cyclase, stimulation of the cyclase by Ca2+ was greatly amplified, suggesting that the two inputs interact in activating a common population of the enzyme. Third, solubilized cyclase activity that bound to calmodulin-Sepharose in a Ca(2+)-dependent manner was stimulated by Gs, which had been partially purified from Aplysia CNS, as well as by Ca2+/calmodulin. Having demonstrated dual activation of the cyclase, we have explored the dependence of cyclase activation on the temporal pattern of Ca2+ and transmitter addition. Optimal activation required that a pulse of Ca2+ temporally overlap the addition of facilitatory transmitter. These several results suggested that the dually regulated adenylate cyclase might underlie the temporal requirements for effective classical conditioning in this system.

Evidence suggests that the Ca2+/calmodulin-sensitive adenylyl cyclase may play a key role in neural plasticity and learning in Aplysia, Drosophila, and mammals. This dually-regulated enzyme has been proposed as a possible site of stimulus convergence during associative learning. This commentary discusses the evidence that is required to demonstrate that a protein in a second messenger cascade actually functions as a molecular site of associative integration. It also addresses the issue of how a dually-regulated protein could contribute to the temporal pairing requirements of classical conditioning: that relationship between stimuli display both temporal contiguity and predictability.

To a prey, a salient visual characteristic of an approaching predator is ‘looming’, a figure of average subtended visual angle increasing as some function of time. If the approach is at constant velocity, two important characteristics of the looming figure are an ever increasing velocity (i.e. acceleration) of sideways motion of edges away from the centroid of the figure, and the opposed motion of opposite edges. These characteristics make looming objects distinct from translation of single objects across the visual field, so that the visual looming characteristics may have value in warning of approaching predators. In the locust, a visual neurone, the DCMD (descending contralateral movement detector) in the ventral nerve cord forms an important input to the interneurones triggering the jump response in the locust (Pearson, Heitler & Steeves, 1980; Pearson & Robertson, 1981). Thus it is of considerable interest to determine whether the DCMD responds preferentially to looming visual stimuli.

SummaryFor decades, the marine snail Aplysia has proven to be a powerful system for analyzing basic neurobiological mechanisms, particularly cellular and molecular mechanisms of neural plasticity. Three new findings on Aplysia may be relevant for the understanding and treatment of chronic human disorders. This research on this simple molluscan nervous system may lead to new therapeutic approaches for spinal cord injury, Fragile X syndrome, and genetic learning deficits more generally.

The nervous systems of a broad variety of animals have evolved the ability to recognize predictive relationships between events if these occur repeatedly in a temporally contiguous manner. Recent evidence suggests that, in the simplest cases, learning about these predictive relationships, such as occurs during conditioning, may be mediated by molecular interactions within individual nerve cells. Studies of conditioning in both Aplysia and Drosophila indicate that the enzyme adenylate cyclase may serve as a molecular site of convergence between two signals: Ca 2+ influx, the signal from the conditioned stimulus, and transmitter, the signal from the unconditioned stimulus. We review recent evidence that this dually regulated enzyme may have the necessary properties for it to play such an associative role. Recent analyses of associative LTP in the hippocampus reveal a second molecular mechanism for associative synaptic plasticity, which could also contribute to the learning of predictive relationships. In a fashion analogous to the dual regulation of adenylate cyclase, effective activation of the NMDA-gated Ca 2+ conductance requires temporal pairing of two signals: transmitter binding and postsynaptic depolarization.

In studying the classical conditioning of the siphon withdrawal reflex in Aplysia, we have identified a neuronal mechanism that plays an important role in this conditioning: activity-dependent presynaptic facilitation. This review describes our analysis of the cellular basis of this associative mechanism. During the conditioning of the withdrawal reflex, the unconditioned stimulus, a tail shock, produces presynaptic facilitation of synaptic transmission from the siphon sensory neurons in the conditioned stimulus pathway. The facilitation is enhanced if a sensory neuron has fired action potentials just prior to receiving facilitatory input, as occurs during training when the conditioned stimulus precedes the unconditioned stimulus. This activity-dependent enhancement of presynaptic facilitation provides a mechanism for the temporal specificity in conditioning of the reflex. Activity-dependent facilitation appears to involve the same cyclic AMP (cAMP)-dependent cascade that underlies presynaptic facilitation in these neurons in the absence of paired spike activity. Our evidence suggests that it is the transient elevation of intracellular Ca2+ that is responsible for the enhancement of the facilitation response by paired spike activity. Moreover, our preliminary results indicate that Ca2+/calmodulin is able to potentiate the activation of adenylate cyclase in Aplysia neurons by facilitatory transmitter. Thus, the dual activation of the calmodulin-dependent cyclase by Ca2+ and transmitter may give this enzyme an important associative role in learning. In the conclusion, the possible phylogenetic generality of this associative mechanism is discussed as well as its possible role in activity-dependent processes in neuronal development.

Grasshoppers, like many poikilotherms, are generally more active at warmer body temperatures. In particular, they jump more frequently when warm. To determine the neuronal basis of this increase in jumping activity, we investigated the effects of temperature on the properties of identified central neurons known to be involved in the control of the jump; these included the fast extensor tibiae (FETi) motoneuron and the C, G, and M interneurons. Heating did not result in a reduction in the current or voltage threshold for action potentials; in most cases, there was an increase in the current threshold with heating. At higher temperatures, the frequency-current relations of interneurons and motoneurons had steeper slopes. With strong current pulses, increasing the temperature resulted in an increase in the initial peak firing frequencies of central neurons and usually also in their steady state firing frequencies. A second temperature effect favoring increased CNS activity in warm grasshoppers was increased afferent input from the periphery. In a broad variety of sensory receptors, there was a dramatic increase in their sensitivity to sensory stimuli at both threshold and suprathreshold intensities. Various identified central neurons differed in the way in which some of their properties were influenced by temperature. The C and G interneurons showed a striking similarity in the unusual way in which their repetitive firing properties were influenced by heating. Since these neurons are sibling progeny of a single neuroblast, this shared physiological property is correlated with their developmental history.

For much of this century, a central goal for both psychologists and neurobiologists has been to understand the mechanisms underlying associative learning. Of the various forms of associative learning, classical conditioning is perhaps the simplest and is thought to be the prototypical way in which an animal learns a predictive relationship (Pavlov, 1927; Kamin, 1969; Rescorla and Wagner, 1972). In classical conditioning, an animal alters its response to one stimulus as a result of the temporal pairing of this stimulus with a second event. During training, the animal comes to know that the first stimulus, called the conditioned stimulus, signals the occurrence of the second stimulus, the unconditioned stimulus. Because of its simplicity, many of the efforts to analyze cellular mechanisms of associative learning have focused on classical conditioning.

We have found that two endogenous neuropeptides in Aplysia, the small cardioactive peptides SCPA and SCPB, facilitate synaptic transmission from siphon mechano-sensory neurons and enhance the defensive withdrawal reflex that these sensory neurons mediate. Single-channel recording revealed that these peptides close a specific K+ channel, the S channel, which is sensitive to cAMP. Moreover, the peptides increase cAMP levels in these sensory neurons. This reduction in K+ current slows the repolarization of the action potential in these cells, which increases transmitter release. In these actions, the SCPs resemble both noxious sensitizing stimuli, which enhance the reflex, and serotonin. Bioassay of HPLC fractions of abdominal ganglion extracts and immunocytochemistry indicate that both the SCPs and serotonin are present in the ganglion and are found in processes close to the siphon sensory neurons, suggesting that these transmitters may be involved in behavioral sensitization. Recent evidence suggests that one group of identified facilitatory interneurons, the L29 cells, does not appear to contain either the SCPs or serotonin but may use yet another facilitatory transmitter. Thus, it appears that several transmitters can converge to produce presynaptic facilitation in the sensory neurons of the defensive withdrawal reflex. All of the transmitters studied here, the SCPs and serotonin, act via an identical molecular cascade: cAMP-dependent closure of the S-K+ channel, broadening of the presynaptic action potential, and facilitation of transmitter release.

The Ca2+/calmodulin (CaM)-activated adenylate cyclase has been implicated as playing an important associative role in classical conditioning in both Aplysia and Drosophila. Studies of the cyclase in mammalian cerebral cortex have suggested that Ca2+/CaM sensitivity is confined to a subpopulation of total cyclase activity. We investigated the properties of cyclase from Aplysia, rat, and bovine central nervous system membranes by using CaM-Sepharose chromatography. Although only a minority of total cyclase activity bound to the CaM column, both bound and unbound fractions of cyclase from all three species showed comparable stimulation by Ca2+ in the presence of CaM. When solubilized bovine membranes were first depleted of most of their endogenous CaM by prior chromatography, binding to the CaM column was substantially increased and Ca2+ stimulation of the unbound fraction was somewhat reduced. However, this reduction in Ca2+ sensitivity resulted from the loss of Ca2+ sensitivity during prior chromatography, rather than from the more efficient separation of Ca2+-sensitive and -insensitive forms. This finding, together with the fact that we never observed any enrichment for Ca2+ sensitivity in the bound fraction over the level in the starting preparation, suggests that the vast majority of the cyclase present in solubilized central nervous system membranes is Ca2+/CaM-sensitive.

In 1949, D. O. Hebb proposed a novel mechanism for producing changes in the strength of synapses that could account for associative learning. According to Hebb , the strength of a synapse might increase when the use of that synapse contributes to the generation of action potentials in a postsynaptic neuron. Thus, an essential feature of this postulate is that action potentials must occur in both a postsynaptic cell and a presynaptic cell for associative synaptic changes to occur. We have directly tested Hebb 's postulate in Aplysia at identified synapses which are known to exhibit a temporally specific increase in efficacy during a cellular analogue of differential conditioning. We find that the mechanism postulated by Hebb is neither necessary nor sufficient to produce the associative change in synaptic strength that underlies conditioning in Aplysia. In contrast, impulse activity in the presynaptic cell must be paired with facilitatory input, supporting the hypothesis that the temporal specificity of classical conditioning in Aplysia can be accounted for by activity-dependent amplification of presynaptic facilitation.

Publisher Summary This chapter summarizes the evidence that a conventional transmitter, serotonin, and the small cardioactive peptides—SCP A and SCP B —can modulate a specific K + channel by means of cyclic adenosine monophosphate. Although the information is still fragmentary, there is the possibility that a third class of transmitter, yet unidentified, works by the same mechanism. In modulating this K + channel, each of these transmitters also regulates transmitter release at a specific set of synapses involved in two short-term forms of memory, each lasting minutes to hours: the memory for short-term behavioral sensitization and for classical conditioning. The specific set of connections modulated during short-term sensitization also undergoes a prolonged modulation lasting days and weeks, which contributes to long-term memory for sensitization. The ion channel gated by transmitters consists of at least three functional components: (1) a receptor or recognition site, (2) a channel, and (3) a gate. The receptor recognizes the transmitter and instructs the gate to open or close the channel. The channel conducts ions only when it is gated open.

During short-term sensitization, a simple form of nonassociative learning in Aplysia, the presentation of a single brief noxious stimulus results in enhancement of the defensive withdrawal reflex lasting minutes to tens of minutes. This behavioral plasticity involves presynaptic facilitation of synaptic transmission from the mechanosensory neurons that mediate the reflex to their central target cells. This facilitation is due to cAMP-dependent protein phosphorylation. To determine whether the time course of presynaptic facilitation might be due to a persistent increase in activity of adenylate cyclase (EC 4.6.1.1) itself, persistence of the transmitter, or yet other processes, we developed a perfused-membrane method to analyze the time course of activation of adenylate cyclase by transient stimuli. After stimulation by a pulse of stimulatory transmitter, activation of adenylate cyclase decayed within 60 sec. This finding indicates that the enzyme does not remain persistently active in the absence of transmitter and suggests that short-term retention is likely to be due to other mechanisms. Possible additional mechanisms include continued activation of the cyclase by transmitter, cellular factors extrinsic to the cyclase that prolong the time course of its activation, and persistence of processes downstream from the cyclase.