Food odor, visual danger stimulus, and retrieval of an aversive memory trigger heat shock protein HSP70 expression in the olfactory lobe of the crab Chasmagnathus granulatus.

Although some of the neuronal substrates that support memory process have been shown in optic ganglia, the brain areas activated by memory process are still unknown in crustaceans. Heat shock proteins (HSPs) are synthesized in the CNS not only in response to traumas but also after changes in metabolic activity triggered by the processing of different types of sensory information. Indeed, the expression of citosolic/nuclear forms of HSP70 (HSC/HSP70) has been repeatedly used as a marker for increases in neural metabolic activity in several processes, including psychophysiological stress, fear conditioning, and spatial learning in vertebrates. Previously, we have shown that, in the crab Chasmagnathus, two different environmental challenges, water deprivation and heat shock, trigger a rise in the number of glomeruli of the olfactory lobes (OLs) expressing HSC/HSP70. In this study, we initially performed a morphometric analysis and identified a total of 154 glomeruli in each OL of Chasmagnathus. Here, we found that crabs exposed to food odor stimuli also showed a significant rise in the number of olfactory glomeruli expressing HSC/HSP70. In the crab Chasmagnathus, a powerful memory paradigm based on a change in its defensive strategy against a visual danger stimulus (VDS) has been extensively studied. Remarkably, the iterative presentation of a VDS caused an increase as well. This increase was triggered in animals visually stimulated using protocols that either build up a long-term memory or generate only short-term habituation. Besides, memory reactivation was sufficient to trigger the increase in HSC/HSP70 expression in the OL. Present and previous results strongly suggest that, directly or indirectly, an increase in arousal is a sufficient condition to bring about an increase in HSC/HSP70 expression in the OL of Chasmagnathus.

High environmental salinity induces memory enhancement and increases levels of brain angiotensin-like peptides in the crab Chasmagnathus granulatus.

Previous work on the brackish-water crab Chasmagnathus granulatus demonstrated that an endogenous peptide similar to angiotensin II plays a significant role in enhancing long-term memory that involves an association between context and an iterative danger stimulus (context-signal memory). The present results show that this memory enhancement could be produced by moving crabs from brackish water to sea water (33.0%) and keeping them there for at least 4 days. The possibility that such a facilitatory effect is due to osmotic stress is ruled out. Coincidentally, the level of angiotensin-II-like peptides in crab brain, measured by radioimmunoassay, increases with the length of exposure to sea water, reaching a significantly different level at the fourth day. The presence of angiotensin-II-like immunoreactive material in neural structures of the supraoesophageal and eyestalk ganglia was confirmed by immunohistochemical analysis. The results are interpreted as supporting the hypothesis that exposure to water of high salinity is an external cue triggering a process mediated by angiotensins that leads to enhanced memory in these crabs.

Inhibitors of protein and RNA synthesis block context memory and long-term habituation in the crab Chasmagnathus.

The crab Chasmagnathus granulatus reacts to a shadow passing overhead (a danger stimulus) with an escape response that habituates quickly and for at least 5 days. Recently, it has been reported that cycloheximide (CY) disrupts this long-term habituation and the corresponding context memory. In the present article, experiments with CY and an inhibitor of RNA synthesis, actinomycin-D (ACT), were parallelly conducted. An injection of CY (20 micrograms) or ACT (0.62 microgram) reduced the incorporation of [14C]-aminoacid into cerebral plus thoracic ganglia by 80% for 2 h and 59.7% for 1 h, respectively, but no inhibition was found at 24 h. Both ACT (0.62 microgram) and CY (20 micrograms) administered immediately after training (15 trials with the danger stimulus) impaired either long-term habituation or context memory when tested at 24 h. Because ACT and CY have in common only their direct or indirect inhibitory effect on protein synthesis, this finding is considered as an additional evidence that long-term memory in Chasmagnathus requires de novo protein synthesis. However, pretraining ACT or CY impaired context memory at 24 h but not long-term habituation. Such a disparity is explained by an unspecific attenuating effect upon the response, attributed to drug x training interaction. Neither ACT nor CY affected short-term habituation.

Cycloheximide inhibits context memory and long-term habituation in the crab Chasmagnathus.

A shadow moving over head elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. The effect of the protein synthesis inhibitor cycloheximide (CY) on this long-term memory was analyzed. Two hours after injection, 10 micrograms CY inhibited [14C]-amino amino acid incorporation into cerebral plus thoracic ganglia by 88% and 20 micrograms by 92%, but no inhibition was found at 24 h. A single injection of 10-20 micrograms CY given 30 min before training, failed to affect the short-term habituation. Similar doses impaired both context memory (CM) and long-term habituation (LTH) when tested at 72 and 120 h but only CM at 24 h. Such a disparity was explained by an unspecific depressing effect upon the response, attributed to an interaction between CY and training. The hypothesis was confirmed, because CY injected immediately after training disclosed amnestic effect at 24 h on both CM and LTH. A similar effect was proven when animals were injected at 2 h but not at 6 h after training. Results from experiments with pretraining and pretesting injections put aside a state-dependence or retrieval deficit effects of the drug. Taken together, findings of this article argue strongly for de novo protein synthesis as a mechanism of LTH and for the close relation between CM and LTH.

Passive avoidance learning in the crab Chasmagnathus granulatus.

Male crabs Chasmagnathus granulatus were trained by means of a method similar to the standard inhibitory avoidance technique widely used in vertebrates. Each crab was placed in the dark compartment (DC) of a double-chamber device, allowed to move towards the light compartment (LC) and latency to enter measured. Experimental crabs received a shock in LC, but controls were not punished. After 1 min, both experimental and control crabs were free to return to DC. On completion of 1, 2, 3 or 24 hr intertrial interval in DC a retention test was administered and latency to enter LC was measured. A single trial was proven enough to establish a LC-shock association that was detected up to 3 hr later, but no retention was proved after 24 hr. Memory was disrupted when crabs were removed from the apparatus during the 3 hr intertrial interval. Similarities and differences between the passive avoidance method used with crabs and that used with vertebrates are discussed.