Contextual Pavlovian conditioning in the crab Chasmagnathus

In contextual conditioning, a complex pattern of information is processed to associate the characteristics of a particular place with incentive or aversive reinforcements. This type of learning has been widely studied in mammals, but studies of other taxa are scarce. The context-signal memory (CSM) paradigm of the crab Chasmagnathus has been extensively used as a model of learning and memory. Although initially interpreted as habituation, some characteristics of contextual conditioning have been described. However, no anticipatory response has been detected for animals exposed to the training context. Thus, CSM could be interpreted either as an associative habituation or as contextual conditioning that occurs without a context-evoked anticipatory response. Here, we describe a training protocol developed for contextual Pavlovian conditioning (CPC). For each training trial, the context (conditioned stimulus, CS) was discretely presented and finished together with the unconditioned stimulus (US). In agreement with the CSM paradigm, a robust freezing response was acquired during the 15 training trials, and clear retention was found when tested with the US presentation after short (2 and 4 h) and long (1–4 days) delays. This CPC memory showed forward but not simultaneous presentation conditioning and was context specific and protein synthesis dependent. Additionally, a weak CPC memory was enhanced during consolidation. One day after training, CPC was extinguished by repeated CS presentation, while one presentation induced a memory labilisation–reconsolidation process. Finally, we found an anticipatory conditioned response (CR) during the CS presentation for both short-term (4 h) and long-term memory (24 h). These findings support the conditioning nature of the new paradigm.     

Effect of naloxone pretreatment on habituation in the crab Chasmagnathus granulatus

On sudden presentation of a passing shadow, the crab Chasmagnathus granulatus reacts with an escape response that habituates after repeating the same stimulus. In a first series of experiments, a range of naloxone (NX) doses (0.8, 2.4, 3.2, and 6.2 micrograms/g) was injected into crabs 15 min before one 15-trial habituation session. An enhancing effect of 3.2 micrograms NX/g on responsiveness appeared over trials, that cannot be explained either by a ceiling effect or by a delay in peak drug action. Two doses below 3.2 microgram/g and one dose above had no significant effect. Results from a second series of experiments showed that the 3.2 microgram NX/g effect vanishes after 15 trials (1 h after injection). The hypothesis that crab's habituation involves the action of an endogenous opioid mechanism is put forward to account for the naloxone pretreatment effect.     

Differential roles of hippocampal metabotropic glutamate receptors 1 and 5 in inhibitory avoidance learning

Group I metabotropic glutamate receptors (mGlu1 and 5) have been implicated in synaptic plasticity and learning and memory. However, much of our understanding of how these receptors in different brain regions contribute to distinct memory stages in different learning tasks remains incomplete. The present study investigated the effects of the mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and mGlu1 receptor antagonist, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzene-acetic acid (LY 367385) in the dorsal hippocampus on the consolidation and extinction of memory for inhibitory avoidance learning. Male, Sprague-Dawley rats were trained in a single-trial step-down inhibitory avoidance task. MPEP, LY 367385 or saline were infused bilaterally into the CA1 region immediately after training or immediately after the first retention test which was given 24h after training. Rats receiving MPEP (1.5 or 5.0 microg/side) or LY 367385 (0.7 or 2.0 microg/side) infusion exhibited a dose-dependent decrease in retention when tested 24h later. MPEP was ineffective while LY 367385 significantly attenuated extinction when injected after the first retention test using an extinction procedure. These findings indicate a selective participation of hippocampal group I mGlu receptors in memory processing in this task.     

Extinction memory in the crab Chasmagnathus: recovery protocols and effects of multi-trial extinction training

A decline in the frequency or intensity of a conditioned behavior following the withdrawal of the reinforcement is called experimental extinction. However, the experimental manipulation necessary to trigger memory reconsolidation or extinction is to expose the animal to the conditioned stimulus in the absence of reinforcement. Recovery protocols were used to reveal which of these two processes was developed. By using the crab contextual memory model (a visual danger stimulus associated with the training context), we investigated the dynamics of extinction memory in Chasmagnathus. Here, we reveal the presence of three recovery protocols that restore the original memory: the old memory comes back 4 days after the extinction training, or when a weak training is administered later, or once the VDS is presented in a novel context 24 h after the extinction session. Another objective was to evaluate whether the administration of multi-trial extinction training could trigger an extinction memory in Chasmagnathus. The results evince that the extinction memory appears only when the total re-exposure time is around 90 min independently of the number of trials employed to accumulate it. Thus, it is feasible that the mechanisms described for the case of the extinction memory acquired through a single training trial are valid for multi-trial extinction protocols. Finally, these results are in agreement with those reports obtained with models phylogenetically far apart from the crab. Behind this attempt is the idea that in the domain of studies on memory, some principles of behavior organization and basic mechanisms have universal validity.     

Enhanced inhibitory avoidance learning produced by post-trial injections of substance P into the basal forebrain

The effects of injections of the neuropeptide substance P or the GABA agonist muscimol on performance of a step-down inhibitory avoidance task were examined. Immediately after the training trial, rats with chronically implanted cannulas were injected with 100 or 10 ng of substance P or 500 or 50 ng of muscimol into the region of the nucleus basalis magnocellularis. Control groups included vehicle-injected rats, a sham-operated group, a substance P 5-h delay group, and a substance P no-footshock group. Rats injected with 100 ng of substance P exhibited longer step-down latencies when tested 24 h later than did vehicle-injected rats. The retention latencies for rats in the substance P 5-h delay group did not differ from those of vehicle-injected animals, indicating that proactive effects on performance were not responsible for the effect. In contrast to injections of SP, injections of 500 or 50 ng of muscimol disrupted performance. However, in the absence of a delayed-injection control group, proactive effects cannot be ruled out.     

Differential activity profile of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus

The isoforms of cAMP-dependent protein kinase (PKA) show distinct biochemical properties and subcellular localization, suggesting different physiological functions, and conferring the fine-tuning between the activation of cAMP-PKA cascade and the cellular response. The critical role of PKA in memory and synaptic plasticity has been extensively demonstrated both in vertebrates and invertebrates, but the role of PKA isoforms is a matter of debate. Here we present experimental data showing differential PKA activation profiles after two different experiences: an instance of associative contextual learning (context-signal learning) and a single exposure to a novel context, both in the learning and memory model of the crab Chasmagnathus. Differences were found in the temporal course of activation and in the involvement of PKA isoforms. We found increased PKA activity immediately and 6 h after context-signal training correlating with the critical periods during which pharmacological inhibition of PKA disrupts memory formation. In contrast, PKA activity increased immediately but not 6 h after single exposure to a novel context. The amounts of PKA I and PKA II holoenzymes were analyzed to determine changes in holoenzyme levels and/or differential activation induced by both experiences. Results indicate that context-induced PKA activation is at least in part due to PKA II, and that PKA activation 6 h after context-signal learning coincides with an increase in the total level of PKA I. Considering the higher sensitivity of PKA I to cAMP, its increment can account for the PKA activation found 6 h after training and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.     

One-trial-a-day avoidance learning

A one-trial-a-day discrete-trial avoidance conditioning procedure run for 30 consecutive days was compared with a massed-trial procedure where Ss received 30 consecutive trials within 1 day. The Ss in both groups were equated for time each S spent in the nonshock compartment of the apparatus, number of times the transport box carrying Ss was lifted out of the nonshock compartment, and the number of times each S was handled. The main difference between groups was the intersession trial length of 24 h for the one-trial-a-day Ss. Learning was rapid for both groups. The groups did not differ reliably on six acquisition indices. The methodological advantages of the one-trial-a-day procedure and its theoretical importance were discussed.     

Signaled avoidance in the eye withdrawal reflex of the green crab

Learning in a signaled avoidance procedure was studied in the eye withdrawal reflex of the green crab, Carcinus maenas. A puff of air to the eye, which causes eye retraction, was used as the unconditioned stimulus (US). A mild vibration on the carapace, which has no effect on untrained animals, was used as a warning (conditioned) stimulus (CS). Eye withdrawal during the CS led to the omission of the otherwise scheduled US. Acquisition was rapid, reaching about 75% avoidance after 30 trials. Extinction occurred slowly over the course of 40 CS-only trials. Yoked controls did not perform as well. The behavior of experimental animals in the avoidance procedure was found to be essentially identical to the performance of animals subjected to a classical conditioning paradigm in which CS responses had no effect on US presentation. Additional groups of animals were subjected to experiments in which (a) avoidance conditioning (60 trials) was followed by classical conditioning (40 trials) or (b) classical conditioning was followed by avoidance. The behavior of these groups was, again, essentially identical. The results suggest that there may be an underlying Pavlovian mechanism for the learned response, although the contribution of an operant process is not excluded. The results expand the range of invertebrate animals in which fundamental conditioning phenomena can be demonstrated, and may provide a neuronal model for learning in a signaled avoidance procedure.     

Enhanced inhibitory avoidance learning prevents the long-term memory-impairing effects of cycloheximide,a protein synthesis inhibitor

Interference with activity of numerous cerebral structures produces memory deficiencies; in many instances, however, when animals are over-trained such interference becomes innocuous. Systemic administration of protein synthesis inhibitors impairs long-term retention; this effect has been interpreted to mean that protein synthesis is required for memory consolidation, though little is known about the effect of protein synthesis inhibitors on memory of enhanced learning in the rat. To further analyze the protective effect of enhanced learning against amnesic treatments, groups of Wistar rats were trained in a one-trial step-through inhibitory avoidance task, using different intensities of foot-shock during training. Cycloheximide (CXM; 2.8 mg/kg), an inhibitor of protein synthesis, was injected either 30 min before training or immediately after training. Twenty-four hours after training retention latencies were recorded. Our data showed that both pre- and post-training administration of CXM produced amnesia in those groups that had been trained with relatively low foot-shock intensities, but no impairment in retention was observed when relatively high intensities of foot-shock were administered. These and similar results lead us to conclude that protein synthesis inhibitors may interfere with memory consolidation, but their effect disappears when animals are submitted to an enhanced learning experience, calling into question the idea that protein synthesis is required for memory consolidation.     

Stimulus specificity in avoidance learning

The present experiments address the issue of stimulus specificity in fear and avoidance learning. First, it was established that light stimuli are effective warning signals (WS) in shuttle avoidance. Then light stimuli were shown to produce conditioned suppression in the conditioned emotional response situation comparable to that produced by noise conditioned stimuli. Finally, the effectiveness of noise onset and noise offset as feedback signals was tested. This was assessed under conditions of immediate and delayed termination of a light WS. Delayed termination of a light WS interfered with avoidance learning and the introduction of noise offset as a feedback signal enhanced it. The only demonstration of stimulus specificity was the failure of noise onset to function as a feedback signal.     

Posttraining inactivation of excitatory afferent input to the locus coeruleus impairs retention in an inhibitory avoidance learning task

These experiments examined whether the nucleus paragigantocellularis (PGi) contributes to memory storage processing via its ascending excitatory influence on locus coeruleus (LC) neuronal activity. Activation of the LC leads to memory enhancement and also results in a widespread release of norepinephrine in target structures, such as the amygdala and hippocampus. Infusion of norepinephrine into either structure also improves memory for several types of learned responses. Thus, the capacity for norepinephrine to modulate memory within limbic structures may be contingent upon the functional connections between PGi and the LC. To examine this hypothesis, male Sprague-Dawley rats were implanted with cannula aimed above PGi (Experiments 1 and 2) or 1.5 mm dorsal or medial to PGi (Experiment 3). Immediately following inhibitory avoidance training (0.45 mA, 0. 5 s), phosphate-buffered saline, lidocaine (Experiment 1), or 12.5 or 25 nmol/0.5 microl of the GABA agonist muscimol (Experiment 2) was infused into PGi. On a retention test given 48 h later, the latency to reenter the footshock compartment was significantly shorter for subjects given either lidocaine or 12.5 or 25.0 nmol of muscimol compared to controls. In Experiment 3, infusion of lidocaine or muscimol into areas 1.5 mm dorsal or medial to PGi did not significantly alter retention, indicating that the memory impairment observed in Experiments 1 and 2 was site specific and not due to the spread of drug to cell groups surrounding PGi. These findings suggest that PGi may serve a vital function in relaying biologically relevant information to forebrain structures involved in memory via its excitatory influence on the LC.     

Glycine(B) receptor antagonists and partial agonists prevent memory deficits in inhibitory avoidance learning

Activation of N-methyl-d-aspartate (NMDA) receptors has been hypothesized to mediate certain forms of learning and memory. This hypothesis is based on the ability of competitive and uncompetitive NMDA receptor antagonists to disrupt learning. We investigated the effects of glycine site antagonists and partial agonists on deficits of acquisition (learning) and consolidation (memory) in a single trial inhibitory avoidance learning paradigm. Posttraining administration of either hypoxia (exposure to 7% oxygen) or the convulsant drug pentylenetetrazole (PTZ) (45 mg/kg) to mice impaired consolidation without producing neuronal cell death. Pretreatment with the competitive glycine antagonist 7-chlorokynurenic acid (7KYN) and the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+)HA-966 prevented memory deficits induced by hypoxia and PTZ, but did not affect scopolamine-induced learning impairment. In addition, ACPC prevented consolidation deficits evoked by a nonexcitotoxic concentration of l-trans-pyrrolidine-2, 4-dicarboxylate, a competitive inhibitor of glutamate transport that increases extracellular levels of glutamate. Moreover, (+)HA-966, 7KYN, and ACPC facilitated both acquisition and consolidation of inhibitory avoidance training, an effect that was dose-dependent and reversed by glycine. These results indicate that memory deficits induced by both hypoxia and PTZ involve NMDA receptor activation. Furthermore, the present findings demonstrate that glycine site antagonists and partial agonists prevent memory deficits of inhibitory avoidance learning by affecting consolidation, but not acquisition processes.     

On how passive is inhibitory avoidance

Rats were trained in a step-down inhibitory avoidance task using a 25 X 25-cm platform and either a 0.3- or a 0.8-mA training footshock. Immediately after training retrieval was good in all animals; but at 24 h there was a decline in the group trained with 0.3-mA footshock. This decline was not observed in animals submitted to an immediate retrieval test and then tested again at 24 h. Thus, the immediate retrieval test apparently served the purpose of a rehearsal. A considerable degree of activity (rearing, ambulation, sticking the head out of the platform) was observed in test sessions. Activity scores were lower in the animals trained with the 0.8-mA footshock. The amount of activity, however, was unrelated to retrieval performance (i.e., to test session step-down latency).     

Long-lasting and context-specific freezing preference is acquired after spaced repeated presentations of a danger stimulus in the crab Chasmagnathus

A visual danger stimulus elicits an escape response in the crab Chasmagnathus that declines after repeated presentations. Previous results report that such waning may be retained as context-signal memory (CSM) or signal memory (SM): CSM is long lasting, associative, and produced by spaced training, while SM is an intermediate memory, nonassociative, and produced by massed training. The performances of both spaced and massed trained crabs are here examined, using video analysis to determine topographic changes in the behavioral response during and after training. During spaced training, escape vanishes and is mainly replaced by freezing, while during massed training, escape decreases over trials without being replaced by any defensive response. After 24 h, the marked proclivity to freezing persists in spaced trained crabs, while a high level of escaping is shown by massed trained crabs. The long-lasting freezing preference of spaced trained crabs proves to be context-specific and apparent from the very first presentation of the danger stimulus at testing, though freezing is not triggered by the sole exposure to the context. We conclude (a) that freezing preference is the acquired response of the CSM process; (b) that CSM can be properly categorized as an instance of contextual conditioning and SM of classical habituation; (c) that CSM and SM are not two phases of a memory processing but two distinctly types of memory; and (d) that therefore, the temporal distribution of training trials has a drastic effect on crab's memory, more dramatic than that previously described. The possibility that massed and spaced presentations of the same stimulus may represent two different stimulus types is discussed.