Francisco Javier Maza

The interactions between memory processes and emotions are complex. Our previous investigations in the crab Neohelice led to an adaptation of the affective extension of sometimes opponent processes (AESOP) model. The model proposes that emotions generate separate emotive memory traces, and that the unfolding of emotional responses is a crucial component of the behavioral expression of reactivated memories. Here, we show that an aversive conditioning, that used changes in an innate escape response to an aversive visual stimulus, induced an emotional behavior that endured beyond the stimuli: the aversive memory training built an anxiety-like state evaluated in a dark/light plus-maze. We found that, after the training session, crabs displayed aversion to maze light areas, and an increased time immobilized in the dark zones of the maze, an anxiety-like behavior induced by stressors or physiological conditions in other crustaceans. The training-dependent anxiety-like behavior was blocked by pretraining administration of fluoxetine, suggesting an underlying serotonin-dependent phenomenon. We hypothesize that this training-induced anxiety-like state generates a separate emotive memory trace that is reinstated and crucial for the modulation of memory expression once the memory is reactivated.

High-order brain centers play key roles in sensory integration and cognition. In arthropods, much is known about the insect high-order centers that support associative memory processes, the mushroom bodies. The hypothesis that crustaceans possess structures equivalent to the mushroom bodies -traditionally called hemiellipsoid body- has been receiving neuroanatomical endorsement. The recent functional support is limited to the short term: in a structure of the true crab Neohelice granulata that has many insect-like mushroom bodies traits, the plastic learning changes express the context attribute of an associative memory trace. Here, we used in vivo calcium imaging to test whether neuronal activity in this structure is associated with memory reactivation in the long-term (i.e., 24 h after training). Long-term training effects were tested by presenting the training-context alone, a reminder known to trigger memory reconsolidation. We found similar spontaneous activity between trained and naïve animals. However, after training-context presentation, trained animals showed increased calcium events rate, suggesting that memory reactivation induced a change in the underlying physiological state of this center. Reflecting the change in the escape response observed in the paradigm, animals trained with a visual danger stimulus showed significantly lower calcium-evoked transients in the insect-like mushroom body. Protein synthesis inhibitor cycloheximide administered during consolidation prevented calcium mediated changes. Moreover, we found the presence of distinct calcium activity spatial patterns. Results suggest that intrinsic neurons of this crustacean mushroom body-like center are involved in contextual associative long-term memory processes.

The hypothesis of a common origin for high‐order memory centers in bilateral animals presents the question of how different brain structures, such as the vertebrate hippocampus and the arthropod mushroom bodies, are both structurally and functionally comparable. Obtaining evidence to support the hypothesis that crustaceans possess structures equivalent to the mushroom bodies that play a role in associative memories has proved challenging. Structural evidence supports that the hemiellipsoid bodies of hermit crabs, crayfish and lobsters, spiny lobsters, and shrimps are homologous to insect mushroom bodies. Although a preliminary description and functional evidence supporting such homology in true crabs (Brachyura) has recently been shown, other authors consider the identification of a possible mushroom body homolog in Brachyura as problematic. Here we present morphological and immunohistochemical data in Neohelice granulata supporting that crabs possess well‐developed hemiellipsoid bodies that are resolved as mushroom bodies‐like structures. Neohelice exhibits a peduncle‐like tract, from which processes project into proximal and distal domains with different neuronal specializations. The proximal domains exhibit spines and en passant‐like processes and are proposed here as regions mainly receiving inputs. The distal domains exhibit a “trauben”‐like compartmentalized structure with bulky terminal specializations and are proposed here as output regions. In addition, we found microglomeruli‐like complexes, adult neurogenesis, aminergic innervation, and elevated expression of proteins necessary for memory processes. Finally, in vivo calcium imaging suggests that, as in insect mushroom bodies, the output regions exhibit stimulus‐specific activity. Our results support the shared organization of memory centers across crustaceans and insects.

Significance: Mushroom bodies (MBs) are higher brain structures of several invertebrate groups, vastly studied in insects as a key structure involved in memory processes. Moreover, MBs and the vertebrate pallium have been proposed to share an ancestral common origin. In crustaceans, the hemiellipsoid bodies (HBs) are proposed to be homologues of the insect MBs. However, functional evidence for the involvement of HBs in memory processes is lacking. Here, in the crab Neohelice , we show memory traces in the HBs that, as for MBs, reflect the context attribute of memory. These results extend the homology based on anatomy and gene expression to the functional level. Consequently, present data support the hypothesis of a common origin for the arthropods’ high-order memory centers.