Neuroanatomical distribution of angiotensin-II-like neuropeptide within the central nervous system of the crab Chasmagnathus; physiological changes triggered by water deprivation.

The angiotensins constitute a neuropeptidergic system that emerged early in evolution. Their classical osmoregulatory and dipsogenic functions and their mnemonic actions have been demonstrated both in vertebrates and in some invertebrates. Previously, we have shown that, in the euryhaline and semiterrestrial crab Chasmagnathus granulatus, water deprivation correlates with an increased level of brain angiotensin-II-like neuropeptide/s (ANGII-like) and improves memory processes through ANGII receptors. We have proposed that the release of brain angiotensins in response to water shortages is an ancient mechanism for coordinating various functions that, together, enable organisms to tolerate this environmental change. Here, we have evaluated the physiological changes in ANGII-like levels in diverse structures of the central nervous system of these animals during water deprivation. The neuroanatomical distribution of ANGII-like is described in the optic lobes and brain of Chasmagnathus granulatus and the physiological changes in ANGII-like distribution in various brain neuropils is evaluated after water deprivation. Our results indicate that ANGII-like is widely distributed, especially in the medial protocerebrum. After 2 h of water deprivation, ANGII-like immunoreactivity increases in the central body and decreases in the olfactory neuropil and, after 6 h of water deprivation, is markedly reduced in several brain areas. Although further experiments are needed to establish that the angiotensinergic system is involved in the balance of body fluids in this crab, our results suggest that ANGII regulates several functions during water shortages.

Both heat shock and water deprivation trigger Hsp70 expression in the olfactory lobe of the crab Chasmagnathus granulatus.

Heat-shock proteins (Hsp) are synthesized in the central nervous system in response to traumas but also after physical exercise and psychophysiological stress. Therefore, an increase in Hsp expression is a good marker of changes in metabolic activity. In the crab Chasmagnathus, a powerful memory paradigm has been established. Memory modulation is possible by water shortage. The brain areas activated by either training protocols and/or water-deprivation are still unknown. Hsp expression might be a marker to sensing the increase in metabolic activity in crab Chasmagnathus brain neuropils engaged in the physiological responses triggered by water deprivation and cognitive processing. Here, we observed an increase in brain Hsp of 70kDa (Hsp70) expression after a heat-shock treatment. Additionally, immunohistochemistry analysis revealed that, under basal conditions, some glomeruli of the olfactory lobes showed Hsp70 immunoreactivity in an on-off manner. Both a hot environment and water deprivation increased the number of glomeruli expressing Hsp70. This marker of neuropil's activity might turn out to be a powerful tool to test whether crustacean olfactory lobes not only process olfactory information but also integrate multimodal signals.

Phosphorylation of extra-nuclear ERK/MAPK is required for long-term memory consolidation in the crab Chasmagnathus.

It was previously demonstrated that mitogen-activated protein kinase (MAPK) signaling plays a pivotal role in neural plasticity and memory processes both in rodents and mollusks. Although the MAPK pathways are highly conserved, no evidence was found for its participation in memory models in other animal groups. Here we found ERK-like and JNK-like cross-immunoreactivity in the crab brain with phospho-specific antibodies and we estimated ERK and JNK activity during long-term memory consolidation in the context-signal learning paradigm of the crab Chasmagnathus. At 0, 1, 3 and 6h after training ERK and JNK activity was measured. ERK-like activation was found 1h after spaced training in cytosolic but not in nuclear fractions of brain homogenates, while JNK activity remained unchanged in both fractions. Passive (context exposure) and active (continuous stimulation) controls showed cytosolic ERK and JNK activation immediately after training, which decayed 1h later. In coincidence with this time course of activity, an ERK1/2 pathway inhibitor, PD098059, induced amnesia only when administered 45 min after training but not when administered immediately pre- or post-training. These data support that: (1) cytoplasmic but not nuclear ERK substrates must be differentially phosphorylated during memory consolidation, and (2) ERK phosphorylation and consequent activation 1h after training is necessary for long-term memory consolidation in this arthropod model.

CS-US delay does not impair appetitive conditioning in Chasmagnathus.

Habituation and appetitive conditioning have been already described in the crab Chasmagnathus. The purpose of this work is to study whether associative learning can be obtained despite a long conditioned stimulus-unconditioned stimulus interval. Results of the first experiment show that the weakening of temporal contiguity does not prevent appetitive conditioning to occur while after a long 4-h delay, conditioning wanes completely. A second experiment was conducted, after one and three days of training respectively, confirming the above results. Though initially neutral the context trace may be still available immediately after training and for the period of two but not after 4:00 h, demonstrating a forward limit for the conditioning window. After 3 days of training, a further decrease in the exploratory activity suggested that a longer training could increase the relative weight of habituation. Conditioning and habituation seem to work as opponent processes in the crab CHASMAGNATHUS GRANULATUS: if habituation training in the box is followed by the administration of reinforcement after a short period of time, appetitive conditioning will take place. However, as this interval is increased, habituation prevails. A persistent effect of the exposure to a given environment that may underlie trace conditioning in this crab is discussed in adaptive terms.