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It is well established that the hippocampal formation is critically involved in the acquisition of trace memories, a paradigm in which the conditioned (CS) and unconditioned stimuli (US) are separated by a temporal gap (Solomon et al., 1986). The structure is reportedly not critical for the acquisition of delay memories, where the CS and the US overlap in time (Berger & Orr, 1983; Schmaltz & Theios, 1972). Based on these results, it is often stated that the hippocampus is involved in "filling the gap" or otherwise associating the two stimuli in time. However, in addition to the presence of a temporal gap, there are other differences between trace and delay conditioning. The most apparent difference is that animals require many more trials to learn the trace task, and thus it is inherently more difficult than the delay task. Here, we tested whether the hippocampus was critically involved in delay conditioning, if it was rendered more difficult such that the rate of acquisition was shifted to be analogous to trace conditioning. Groups of rats received excitotoxic lesions to the hippocampus, sham lesions or were left intact. Using the same interstimulus intervals (ISI), control animals required more trials to acquire the trace than the delay task. As predicted, animals with hippocampal lesions were impaired during trace conditioning but not delay conditioning. However, when the delay task was rendered more difficult by extending the ISI (a long delay task), animals with hippocampal lesions were impaired. In addition, once the lesioned animal learned the association between the CS and the US during delay conditioning, it could learn and perform the trace CR. Thus, the role of the hippocampus in classical conditioning is not limited to learning about discontiguous events in time and space; rather the structure can become engaged simply as a function of task difficulty., (Copyright 2001 Elsevier Science.)

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca(2+) increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca(2+), co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA(2) stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 microM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 microM). Furthermore, direct stimulation of PLA(2) by melittin (10 microM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 microM) and light (to elevate intracellular Ca(2+)), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 microM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for > or =90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 microm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca(2+) to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca(2+) may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.

The observation that retrieval returns a stable memory into a labile state cannot be readily explained by any simple version of consolidation theory. This finding has been interpreted as evidence for the need to reconsolidate a memory after reactivating it. However, as we discuss in this commentary, other behavioural observations indicate that even this modification to consolidation theory may be insufficient to describe the dynamic properties of memory.

The hypothesis that an individual's capacity for learning might be predicted or influenced by basal levels of synaptic efficacy has eluded empirical tests, owing in part to the inability to compare between animals single identified synaptic responses in the mammalian brain. To overcome this limitation, we have focused our analysis on the invertebrate Hermissenda, whose nervous system is composed of identifiable cells and synaptic interactions. Hermissenda were exposed to paired presentations of light and rotation such that the light came to elicit a learned defensive motor response. An animal's rate of learning was strongly correlated with the amplitude of the synaptic potential evoked in that animal's visual (light sensitive) receptors in response to stimulation of presynaptic vestibular (rotation sensitive) hair cells. In naive animals, strong correlations between the amplitude of both inhibitory and excitatory synaptic potentials were observed between synapses distributed throughout an animal's nervous system, and this conservation of synaptic efficacy was largely attributable to a common influence on transmitter release. These observations suggest that basal synaptic efficacy may be uniformly regulated throughout a nervous system, and provide direct evidence that the basal efficacy of synaptic transmission predicts, and possibly contributes to, individual differences between animals in their capacity to learn.

Descriptions of conditioned response generation in Hermissenda stipulate that the synaptic interaction between type B and A photoreceptors should be enhanced after associative pairings of light and rotation. Although evidence from several laboratories has confirmed this assumption, the mechanism underlying this synaptic facilitation has not been elucidated. Here we report that in vitro conditioning (i.e., light paired with stimulation of vestibular hair cells) modifies the kinetics of presynaptic action potentials in the B photoreceptor in a manner sufficient to account for this synaptic facilitation. After paired training, we observed an increase in the duration of evoked action potentials and a decrease in the amplitude of the spike afterhyperpolarization in the B-cell. As previously reported, paired training also enhanced the excitability (i.e., input resistance and evoked spike rate) of the B photoreceptor. In a second experiment, simultaneous recordings were made in type B and A photoreceptors, and paired training was found to produce an increase in the amplitude of the IPSP in the A photoreceptor in response to an evoked spike in the B-cell. Importantly, there was no change in the initial slope of the postsynaptic IPSP in the A photoreceptor, suggesting that spike duration-independent mechanisms of neurotransmitter exocytosis or postsynaptic receptor sensitivity did not contribute to the observed synaptic facilitation. Perfusion of 4-aminopyridine (4-AP) mimicked a known effect of behavioral conditioning in that it specifically reduced the amplitude of the transient voltage-dependent K(+) current (I(A)) in the B-cell, but in addition, produced action potential broadening and synaptic facilitation that was analogous to that observed after in vitro conditioning. Finally, the effect of 4-AP on B-cell action potentials and on the postsynaptic IPSP in the A-cell was occluded by previous paired (but not unpaired) training, suggesting that the prolongation of the B-cell action potential by a reduction of I(A) was sufficient to account for the observed synaptic facilitation. The occlusion of the effects of 4-AP by paired training was not attributable to a saturation of the capacity of the B-cell for transmitter exocytosis, because it was observed that tetraethylammonium (TEA)-induced inhibition of the delayed voltage-dependent K(+) current induced both spike broadening and synaptic facilitation regardless of training history. Collectively, these results demonstrate that training-induced facilitation at B-cell synapses is attributable to the effects of a reduction of a presynaptic K(+) conductance on action potential kinetics and suggest another critical similarity between the cellular basis for learning in Hermissenda and other invertebrate systems.

The neurophysiological basis for context conditioning is conceptually problematic because neurophysiological descriptions of activity-dependent (associative) forms of neuronal plasticity uniformly assume that a specific temporal relationship between signals is necessary for memory induction. In the present experiments, this problem is addressed empirically by presenting, as a temporally diffuse contextual signal, a stimulus that results in known neural modifications following punctate (temporally contiguous) pairings with an aversive unconditioned stimulus. Hermissenda were trained to discriminate between adjoining contexts that were distinguished only in that one was lit and one was dark. Thirty unsignaled rotations were presented during each of three 15-min sessions in one of the two (lit or dark) contexts. Prior to training, animals displayed a slight preference for the lit context. After exposure to unsignaled rotation, animal's preferences shifted strongly to the dark context if unsignaled rotations were presented in the light, and tended (nonsignificantly) to the lit context if unsignaled rotations were presented in the dark. The B photoreceptors of the Hermissenda eye undergo several forms of activity-dependent facilitation (e.g., an increase in neuronal input resistance and evoked spike frequency) following pairings of punctate light (CS) and presynaptic vestibular stimulation (US). Similar facilitation in the B photoreceptor was observed following in vitro training that mimicked context conditioning in which presynaptic vestibular stimulation was presented repetitively during a continuous 7.5-min light. Subsequently, Ca(2+)-imaging experiments were conducted with Fura-2AM. It was determined that intracellular Ca(2+), the CS-induced second messenger critical for the induction of activity-dependent facilitation, was elevated in the B photoreceptor throughout the 7.5-min light presentation. These results indicate that activity-dependent facilitation within similar neural structures can underlie learning about both temporally diffuse contextual stimuli and temporally punctate CS-US pairings. These results suggest that a common mechanism may underlie learning about diffuse contextual stimuli as well as punctate-conditioned stimuli, provided that the stimuli are processed similarly in each type of conditioning arrangement. Consequently, the expression of different responses to contextual and discrete stimuli are likely to reflect a higher property of the neural network, and do not necessarily arise from unique underlying mechanisms., (Copyright 1999 Academic Press.)

Using Hermissenda as subjects, massed-trials training deficits were examined. Associative pairings of light and rotation induced a progressively greater conditioned foot contraction in response to light as the intertrial interval (ITI) was extended (up to 8 min). In contrast, a short ITI (30 s) produced no evidence of learning. In a corresponding in vitro conditioning experiment that mimicked training of the intact animal, facilitation of neuronal excitability in the animal's B photoreceptors paralleled the results obtained in vivo. Imaging of intracellular Ca2+ using Fura-2 indicated that Ca2+ levels remained elevated during short ITIs. This Ca2+ accumulation appears to induce activation of protein phosphatases because normal facilitation of the B photoreceptors was induced with a short ITI if training occurred in the presence of a phosphatase inhibitor. These results suggest that intracellular Ca2+ and protein phosphatases contribute interactively to the kinetics of memory formation and provide evidence that an accumulation of intracellular Ca2+ across training trials may impede memory formation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction.

Long-term potentiation (LTP) is operationally defined as a long-lasting increase in synaptic efficacy following high-frequency stimulation of afferent fibers. Since the first full description of the phenomenon in 1973, exploration of the mechanisms underlying LTP induction has been one of the most active areas of research in neuroscience. Of principal interest to those who study LTP, particularly in the mammalian hippocampus, is its presumed role in the establishment of stable memories, a role consistent with "Hebbian" descriptions of memory formation. Other characteristics of LTP, including its rapid induction, persistence, and correlation with natural brain rhythms. provide circumstantial support for this connection to memory storage. Nonetheless, there is little empirical evidence that directly links LTP to the storage of memories. In this target article we review a range of cellular and behavioral characteristics of LTP and evaluate whether they are consistent with the purported role of hippocampal LTP in memory formation. We suggest that much of the present focus on LTP reflects a preconception that LTP is a learning mechanism, although the empirical evidence often suggests that LTP is unsuitable for such a role. As an alternative to serving as a memory storage device, we propose that LTP may serve as a neural equivalent to an arousal or attention device in the brain. Accordingly, LTP may increase in a nonspecific way the effective salience of discrete external stimuli and may thereby facilitate the induction of memories at distant synapses. Other hypotheses regarding the functional utility of this intensely studied mechanism are conceivable; the intent of this target article is not to promote a single hypothesis but rather to stimulate discussion about the neural mechanisms underlying memory storage and to appraise whether LTP can be considered a viable candidate for such a mechanism.

An incremental increase in the excitability (i.e., input resistance, evoked spike frequency) of B photoreceptors in Hermissenda accompanied successive pairings of light and presynaptic stimulation of vestibular hair cells (simulating light-rotation pairings in an intact animal). Analysis of protein kinase C (PKC) in the Hermissenda's photoreceptors indicated a training-induced incremental reduction of PKC in cytosolic compartments, a tendency toward an increase in membrane compartments, and a small decrease in total enzyme activity (possibly owing to a downregulation or conversion of PKC to a calcium-independent state). Neither the biophysical or biochemical effects were observed in Hermissenda exposed to unpaired light and rotation or in those trained in the presence of the selective PKC inhibitor NPC-15437 (which had no effect on synaptic interactions or light-induced generator potentials). These results suggest that the intracellular redistribution of a protein kinase contributes critically to the kinetics of new learning.

With the invertebrate Hermissenda as subjects, variability in acquisition of a learned association between light and rotation was correlated with the magnitude of the unconditioned responses elicited by these stimuli. Moreover, learning was facilitated by increasing stimulus intensity. In the isolated nervous system, pairings of light and mechanical stimulation of the animal's vestibular hair cells resulted in an increase in the excitability of B photoreceptors (an in vitro index of learning) that was strongly correlated with the strength of the synaptic interaction between the hair cells and the photoreceptors and weakly correlated with the magnitude of the light response in the photoreceptors. Because these in vitro results are not attributable to motor or motivational variables, they suggest that the efficacy of synaptic integration between sensory systems and sensory transduction is the primary determinant of the variability in learning.

A series of experiments is described that elucidates the sources of Ca2+ that contribute to activity-dependent neuronal facilitation in Hermissenda B photoreceptors during associative conditioning. In an in vitro preparation, pairings of a 4-s light with a 3-s mechanical stimulation of presynaptic hair cells increased the input resistance and elicited spike rate (i.e., excitability) of the B photoreceptors in the Hermissenda eye, indicative of a Ca(2+)-dependent process that is analogous to associative conditioning in the intact animal. This increase in excitability was reduced but not eliminated when hyperpolarizing current was applied to the B cell during the pairings, suggesting that voltage-dependent influx of Ca2+ contributed only a portion of the total calcium signal necessary for facilitation. Moreover, no increase in excitability was observed when a comparable current-induced depolarization of the photoreceptor was substituted for light-induced depolarization. In other experiments, Ca(2+)-dependent inactivation of a light-induced Na+ current was used as an index of intracellular Ca2+ concentration. It was determined that light caused a large increase in intracellular Ca2+ concentration regardless of whether the photoreceptor was allowed to freely depolarize in response to light or was voltage clamped at its resting membrane potential. Current-induced depolarization produced a smaller increase, while presynaptic stimulation had no measurable effect. Intracellular injections of either heparin, an antagonist of intracellular Ca2+ release, or EGTA, a general Ca2+ chelator, induced comparable reductions of light-induced Ca2+ accumulation. Finally, intracellular injections of heparin blocked the pairing-induced increases in B cell excitability as effectively as injections of EGTA. Taken as a whole, these data suggest that Ca2+ release from intracellular stores may be sufficient for the induction of facilitation in this preparation, while Ca2+ influx through voltage-dependent channels may have an additive effect and provide further evidence for the ubiquitous role of Ca2+ in learning-related forms of neuronal plasticity.

A G-protein dependent serotonin (5-HT) receptor on B photoreceptors of Hermissenda crassicornis was investigated. Microapplication of 5-HT to the soma region, but not to the terminal branches, resulted in a rapid, biphasic depolarization with a slow time course of dissipation. The 5-HT-induced depolarization increased at hyperpolarized potentials, and exhibited a strong and complex sensitivity to external K+, but not Na+ or Ca2+. The 5-HT response, but not a cholinergically mediated intraphotoreceptor interaction, was abolished by intracellular injection of the G protein antagonist, GDP[beta S], although the response was unaffected by pretreatment with pertussis toxin. These results are discussed in terms of known 5-HT receptor subtypes, and the potential role of this receptor in activity-dependent forms of plasticity exhibited by these cells.

In behaving Hermissenda, a preparatory conditioned response developed across repeated pairings of light (conditioned stimulus; CS) and rotation (unconditioned stimulus; US) with intertrial intervals (ITIs) of 60 and 120 s, but not 30 s. Likewise, contiguous in vitro stimulation of the visual and vestibular receptors, an analog of behavioral conditioning, resulted in an increase in the input resistance (i.e., excitability, a correlate of conditioning) of the B photoreceptors of the Hermissenda's eye, but only with ITIs greater than 60 s. Calcium signaling in the B cell, critical to the induction of this neuronal plasticity, was attenuated with shorter ITIs owing to (a) a reduction of the light-induced generator potential and hence voltage-dependent Ca2+ influx during the light CS, (b) a depression of the Ca2+ current that persisted throughout shorter ITIs, and (c) a steady-state inactivation of the Ca2+ current as a result of a sustained depolarization persisting from the previous trial. These results are consistent with a 2-process theory of associative learning in which a primary process (Ca2+ influx) may be opposed by a secondary process (depression of the Ca2+ current) during short ITIs.

The neuronal modifications that underlie associative memory in Hermissenda have their origins in a synaptic interaction between the visual and vestibular systems, and can be mimicked by contiguous in vitro stimulation of these converging pathways. At the offset of vestibular stimulation (i.e., hair cell activity), the B photoreceptors are briefly released from synaptic inhibition resulting in a slight depolarization (2-4 mV). If contiguous pairings of light-induced depolarization and presynaptic vestibular activity occur in close temporal succession, this depolarization "accumulates" and has been hypothesized to culminate in a sustained rise in intracellular Ca2+ and a resultant Ca(2+)-mediated phosphorylation of K+ channels as well as an associated increase in input resistance. Here we demonstrate that this cumulative depolarization is neither necessary nor sufficient for the biophysical modifications of the B cell membrane indicative of memory formation. Consistent with several recent reports of one-trial learning in Hermissenda, one pairing of light with mechanical stimulation of the vestibular hair cells resulted in a rise in neuronal input resistance across the B cell membrane that was attenuated by a prepairing iontophoretic injection of the Ca2+ chelator EGTA (25 mM), indicating that this potentiation was Ca2+ dependent. However, the use of a single pairing negates the possibility of an accumulation of depolarization across trials. In a subsequent experiment, B photoreceptors underwent a cumulative depolarization, and a coincident rise in input resistance, during multiple pairings of light and hair cell stimulation. However, if the B photoreceptor was voltage clamped at its initial resting potential before and after each pairing, thus eliminating the cumulative depolarization, the rise in resistance not only persisted, but was enhanced. Moreover, if unpaired light presentations were followed by a current-induced depolarization (to mimic cumulative depolarization), no increase in input resistance was detected. To assess directly the effect of a cumulative depolarization on the voltage-dependent Ca2+ current, an analysis of the inward current on the B cell soma membrane was conducted. It was determined that (1) the inward current may undergo a partial inactivation during sustained depolarization, (2) the peak current was depressed during repetitive depolarizations, and (3) the peak current underwent a steady-state inactivation, such that it was reduced when elicited from holding potentials more positive than -60 mV. The analysis of this current suggests that pairings of light and presynaptic activity would reduce voltage-dependent Ca2+ influx when those pairings are conducted at depolarized membrane potentials, such as during cumulative depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural correlates of associative memory and "forgetting" were observed 1, 6, and 14 days after acquisition of a conditioned response (CR) in the marine snail Hermissenda. Behavioral expression of a light-rotation association, as indexed by contraction of the animal's foot in response to light, dissipated throughout the 14-day interval such that a CR was observed 1 and 6 days after conditioning but was absent 14 days later. In relation to naive or pseudoconditioned animals, membrane resistance (inversely related to neuronal membrane conductance and directly related to excitability) of the isolated Type B photoreceptor (B cell) was elevated in conditioned animals on Days 1 and 6, whereas no elevation was detectable on Day 14. However, both the behavioral response and the elevated membrane resistance in conditioned animals were hypersensitive to light-rotation pairings (i.e., exhibited "savings") on Day 14, which is indicative of a latent memory trace. In a second experiment, a current-induced depolarization of the B cell after 14 retention days resulted in an increase in input resistance of the B cell membrane in previously conditioned animals but a weaker, transient rise in resistance in B cells from animals exposed to the nonassociative control procedure 14 days earlier. This effect was Ca(2+)-dependent because no rise in resistance was observed if Ca2+ was removed from the extracellular bath. These results indicate that modification of membrane conductance (i.e., elevated resistance), although apparently critical for the behavioral expression of the memory, is not essential for the maintenance of the latent memory trace, whereas Ca2+ hypersensitivity may be a principal contributor to the storage of a latent memory trace and memory reactivation.

Caudal hair cell impulses cause postsynaptic inhibition of ipsilateral type B photoreceptors in the snail Hermissenda. This inhibition is shown to be GABAergic according to a number of criteria. HPLC, mass spectrophotometric, and immunocytochemical techniques demonstrated the presence of GABA in the hair cells and their axons. GABA agonists and antagonists mimic and block the synaptic effect in a manner consistent with endogenous GABAergic transmission. Other properties, including I-V relations, conductance changes and reversal potentials, are comparable for exogenous GABA responses and endogenous effects of the hair cell impulses. This inhibitory synapse has been found to undergo a long-lasting transformation into an excitatory synapse if GABA release is paired with post-synaptic depolarization. GABA, via GABAA and GABAB receptors in the B cell, causes the opening of calcium sensitive chloride and potassium channels that leads to the post-synaptic hyperpolarization. GABA also induces a long-lasting intracellular calcium elevation at the terminal branches of the B cell that greatly outlasts the voltage responses. Synaptic transformation induced by pairings is caused by a decrease in both GABA induced chloride and potassium conductances in the post-synaptic B cell, as well as a significant prolongation of the intracellular calcium accumulation in the B cell's terminal axonal branches.

The temporal convergence of neuronal signals is commonly considered as a likely prerequisite for enhanced neuronal excitability underlying the induction of associative memories. Here we report that transmitter application on presynaptic terminals of the Hermissenda Type B photoreceptors, when paired with depolarization, results in a potentiation of the excitability of the B-cell which derives from an increase in input resistance across the B-cell soma membrane. Pressure microapplication of gamma-aminobutyric acid (GABA) (12.5 microM) on the terminal branches of the Hermissenda Type B photoreceptors results in the fast (less than 1 s) activation of an inward Cl- conductance, characterized by a decrease in neuronal membrane resistance and an accompanying hyperpolarization (3-6 mV) of the B-cell. A slower effect of GABA, characterized by a slight depolarization (2-4 mV) and increase in resistance was observed approximately 2 min after GABA application. Following bath application of the Cl- channel blocker picrotoxin (100 microM), this increase in resistance was observed within 20 s of GABA application, suggesting that it was normally masked by the faster Cl- conductance. The magnitude of the resistance increase in response to GABA was enhanced when the B-cell was held at depolarized membrane potentials (-40 to -20 mV), but was eliminated if Ca2+ was removed from the extracellular bath, or if the non-specific protein kinase inhibitor H7 (100 microM) was added to the extracellular bath. In a final experiment, GABA application was paired with a transient (10 s) depolarization of the B-cell (to -20 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Addiction has long been thought to include both metabolic and psychological dependence. Psychological dependence must involve long-term memory of behavioral patterns in response to specific experimental contexts. Mammalian memory, and more specifically, human memory, is largely associative. Animal models of associative memory have been provided by Pavlovian conditioning of the snail Hermissenda crassicornis and the rabbit. Striking parallels have been observed in the intrinsic molecular and biophysical transformations which accompany acquisition of the conditioned response in these different animals. In brief, associated stimuli cause elevation of Ca2+ and diacylglycerol, translocation of protein kinase C, phosphorylation of a membrane-associated G-protein, reduction of K+ currents, modification of axonal transport and structural alterations of neuronal branches. These changes can be understood and modelled as a plausible basis for memory acquisition during conditioning as well as more cognitively relevant learning such as spatial maze learning for which related neuronal alterations have recently been found. Identification of memory-specific molecular steps may help target pharmacologic agents for amelioration of learned aspects of psychiatric syndromes such as those of drug dependence.

We have found changes in the morphology of photoreceptor somata from the mollusc Hermissenda that are produced by application of 12,13-phorbol dibutyrate (PDBU), an activator of PKC, in combination with elevated intracellular Ca2+ levels. The changes in morphology were expressed as rapid and repetitive outgrowths and additionally as more general changes in shape of the soma. Application of 4 alpha-PMA, a phorbol ester which does not activate PKC, did not produce these changes. The functional integrity of the photoreceptors in these dissociated eye preparations was maintained throughout the period of incubation with PDBU according to standard electrophysiological criteria. It has previously been shown that classical conditioning produced a reduction of dendritic volume in the type B photoreceptor of Hermissenda, a specific locus for associative memory storage. These changes in dendritic morphology were correlated with increased resistance across the cell membrane caused by learning-induced reductions of outward somatic K+ currents. Such conditioning-specific reductions of somatic K+ currents appear to depend on the phosphorylation of a 20-kDa G-protein (CP20) mediated by the Ca2+ and phospholipid-dependent kinase, protein kinase C (PKC). Thus PKC activity may be important in structural changes of the synaptic region of specific neurons involved in associative memory. The results of the present study suggest that the effects of PKC activation may also include structural changes in the soma of these same neurons.

In two experiments with the nudibranch mollusk Hermissenda, distinct characteristics of conditioned and unconditioned responses to high-speed orbital rotation were examined. In Experiment 1, two principle unconditioned responses to rotation were identified, namely, reduced rate of locomotion and contraction of the foot. The magnitude of the foot contraction increased throughout a 20-s period of rotation, whereas locomotion was reduced immediately after the onset of the rotation and was maintained at this constant low rate throughout the stimulus presentation. These divergent response patterns suggest that the two responses emerge independently. In Experiment 2, a classical conditioning procedure was employed in which a light (CS) was paired with the rotation (US) employed in Experiment 1. In a subsequent test, it was found that the light had acquired the capability to evoke both foot contraction and decreased locomotion. Although the magnitude of these conditioned responses was reduced relative to the corresponding unconditioned response, the patterns of responding were virtually identical; that is, foot contraction developed gradually whereas locomotion decreased immediately. In contrast, animals that received unpaired presentations of the light and rotation, light alone, or no prior exposure to those stimuli exhibited foot extension in response to the light. These results illustrate a transfer of some of the response-evoking properties of the US to the CS as a result of conditioning, as well as the emergence of two independent conditioned responses. Moreover, these results suggest modulation of at least two distinct motor pathways as a function of learning.

Conditioned suppression of photokinesis by the marine mollusc Hermissenda was examined in 3 experiments. In each experiment, groups of animals received light (the conditioned stimulus, CS) that was paired with high-speed orbital rotation (the unconditioned stimulus, UCS), light and rotation explicitly unpaired, or no exposure to these stimuli. Twenty-four hours after training, all animals were tested for suppression of photokinesis in the presence of the light. To establish the effectiveness of our conditioning procedure, in Experiment 1 individual groups of animals received either 50, 100, or 150 CS-UCS pairings. Fifty pairings resulted in a marginal suppression of photokinesis, whereas 100 and 150 pairings produced strong suppression. In Experiment 2, the delay between CS onset and UCS onset was varied between 1 and 10 s. The 10-s interstimulus interval (ISI) did not support conditioning, whereas 1-s and 2-s ISIs were effective. As predicted by the current understanding of Hermissenda's neural network, in Experiment 3 it was found that CS-UCS pairings in which the CS preceded the onset of the UCS and terminated with the offset of the UCS evoked stronger conditioned suppression than either a CS that preceded the UCS and terminated with its onset or a CS that was paired in simultaneous compound with the UCS. This result indicates that CS-UCS contiguity as well as the forward ISI act additively to establish the CS-UCS association. In none of the 3 experiments were any differences observed between groups that were untreated and that received the CS and UCS unpaired. In total, these experiments suggest strong similarities in the temporal characteristics of associative learning in Hermissenda and vertebrate species.

Neural and behavioral correlates of an associative memory in Hermissenda were examined during induction and/or formation of the memory. Hermissenda received either light (conditioned stimulus or CS) and rotation (unconditioned stimulus or US) paired (i.e., Pavlovian conditioning), light and rotation unpaired (pseudoconditioning), or no exposure to light and rotation. Following 9 pairings in a 6 min session, conditioned animals exhibited a contraction of the foot in response to a test CS presented 2 min after the last conditioning trial, whereas pseudoconditioned and untreated animals exhibited a foot extension to the same CS. In addition, both an associative and a nonassociative reduction in light-induced locomotion was observed. To examine neural correlates of this learning within minutes of acquisition, the isolated nervous system of the Hermissenda (containing the visual and vestibular organs) was trained with stimulus conditions identical to those used for the intact animal. Prior isolation and preparation of the nervous system permitted immediate intracellular recording following the final conditioning trial. Relative to pseudoconditioned and untreated animals, the B photoreceptors in conditioned nervous systems were found to have elevated input resistance (inversely related to K+ channel conductance and positively related to excitability) and exhibited increased steady-state depolarization in response to the light CS, as well as a prolonged depolarization after the CS offset. These neural correlates of the associative memory were attenuated if the protein kinase inhibitor H7 was present in the extracellular bath during conditioning, demonstrating in the reduced preparation that antagonism of protein kinase activity blocks the induction of membrane alterations of identified neurons that correlate with memory storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Using conditioned suppression of barpressing to investigate the stability of a conditioned stimulus-unconditioned stimulus (CS-US) association, we gave water-deprived rats either a few pairings of the CS with a strong footshock US or many pairings with a weak footshock US so that barpress suppression in response to the CS was equated. Experiment 1 established training parameters that yielded this equivalence. Specifically, rapid acquisition to a preasymptotic level of responding with strong shock produced suppression comparable to the asymptotic level reached more slowly with weak shock. Experiment 2 showed that although equivalent performance was obtained from extensive conditioning with a weak shock or limited conditioning with strong shock, only extensive conditioning with weak shock resulted in retarded acquisition of an association between that same CS and a footshock level perceived as midway between the two initial training shock intensities as implied by asymptotic performance in Experiment 1. Experiment 3 demonstrated that the observed retardation in animals given many conditioning trials with weak shock was CS specific. Collectively, these findings indicate that the malleability of learned behavior is not simply a function of initial associative strength but is dependent on path during initial acquisition.

With the use of parameters intended to maximize the potential to observe hyperalgesia, the possibility was examined that hyperalgesia might be the immediate response to aversive stimulation, whereas analgesia is delayed (Matzel & Miller, 1987). Consistent with the later prediction, analgesia in rats, as assessed by latency to paw lick in response to thermal stimulation, increased as a function of the delay between a tailshock (Experiment 1) or footshock (Experiment 3) and the test of pain sensitivity. However, in neither case was a hyperalgesic response observed at shock offset. In Experiment 2, the strength of the analgesic response was found to increase as a direct function of both the time since the tailshock and tailshock intensity over the limited ranges examined, but no hyperalgesia was observed immediately after either low- or high-intensity shock. In Experiment 4, the opiate antagonist naloxone was found to attenuate both a weak immediate and stronger delayed analgesia, results suggesting a common underlying mechanism. This mitigates the likelihood that differential behavioral responses at short and long delays following shock were obscuring hyperalgesia at the time of shock offset or were summating with an analgesic response at the long delay to create the impression of enhanced analgesia. In total, these experiments provide evidence that opioid analgesia mediates a compensatory process that increases over time, but they provide no evidence that pain sensitivity increases above baseline levels immediately following an aversive event. These data are discussed in relation to preparatory models of endogenous analgesic functioning and the role of endogenous opioids in learning.

Three experiments examined the development of conditioned analgesia in rats exposed to stimuli that had previously been paired with footshock. In Experiment 1, tailflick latencies increased if the tailflick test for analgesia was immediately preceded by 90 sec of exposure to a context in which unsignaled shock had previously been administered. This analgesia was blocked by the opiate antagonist naloxone administered prior to exposure to the context on the test day. Experiment 2 determined that 90 and 300 sec of exposure to the conditioning context immediately prior to testing evoked comparable analgesia as indexed by increased latencies to pawlick in response to thermal stimulation (hotplate). However, no analgesia was evident in animals exposed to the aversive context for 5 sec immediately prior to the hotplate test relative to animals not exposed to that context. In Experiment 3, a 5-sec exposure to the aversive context produced analgesia comparable to a 90-sec exposure if an 85-sec delay intervened between the 5-sec exposure and the hotplate test. These results suggest that brief exposure to stimuli previously paired with shock can activate the endogenous opioid system, but the analgesic action of these opioids is delayed. Implications for the role of endogenous opioids in learning are discussed.

After training in a double alley for 100% food reward in each of two goalboxes, 14 male Long-Evans hooded rats were run on a partial reinforcement schedule with a target rat present in either the first goalbox or the second runway. The incidence of attack was objectively recorded in both locations. Significantly higher levels of aggression were recorded during nonreward trials than during reward trials in the goalbox, or during either nonreward or reward trials in the runway. The results are interpreted as support for the hypothesis that, in general, aggression is elicited by frustration, but that under certain conditions alternative responses to frustration may preclude aggression.

In two experiments we examined factors that contribute to retarded emergence of conditioned responding to a conditioned stimulus (CS) trained in a context in which unsignaled unconditioned stimuli (USs) had previously been administered. In both experiments water-deprived rats were used in a conditioned lick suppression task to measure the conditioned response elicitation potential of the CS and the training context. From Experiment 1 we determined that nonreinforced exposure to the excitatory context after US preexposure and prior to CS-US pairings in that context eliminated the conditioned response deficit observed on a subsequent test of the CS. The recovery from the US preexposure deficit was nearly as great in animals that received nonreinforced exposure to the excitatory training context after the CS-US pairings but prior to the ultimate test of the CS. From Experiment 2 we determined that the recovery induced by contextual deflation after CS training was specific to deflation of the context in which the CS was trained as opposed to another excitatory context. In total, these experiments suggest that context-US associations partially mask the expression of a learned CS-US association. These results are discussed in terms of recent models of conditioned response generation.

Robbins (1988) reported data that he viewed as inconsistent with Miller and Schachtman's (1985a) comparator hypothesis of conditioned response generation. Here we explain why we do not find his experiments a compelling test of the comparator hypothesis. We also briefly review other studies that tested the same predictions of the comparator hypothesis that Robbins examined. We conclude that there is considerable evidence that following excitatory or inhibitory conditioning with a target conditioned stimulus (CS) and unconditioned stimulus (US), extinction of other cues that were present during CS training ordinarily increases excitatory responding and decreases inhibitory responding to the CS. However, consistent with Robbins's conclusion, there is scant evidence that after CS-US training, enhancing the associative value of other cues that were present during CS training influences excitatory or inhibitory responding to the CS. The implications of these conclusions for the comparator hypothesis as an explanation of differences in acquired behavior and as a heuristic tool are considered.