Calcium imaging method to visualize the spatial patterns of neural responses in the pygmy squid, Idiosepius paradoxus, central nervous system.

BACKGROUND: Cephalopods exhibit unique behaviors such as camouflage and tactile learning. The brain functions correlated to these behaviors have long been analyzed through behavioral observations of animals subject to surgical manipulation or electrical stimulation of brain lobes. However, physiological methods have rarely been introduced to investigate the functions of each individual lobe, though physiological work on giant axons and slices of the vertical lobe system of the cephalopods have provided deep insights into ion conductance of nerves and long-term synaptic plasticity. The lack of in vivo physiological work is partly due to difficulties in immobilizing the brain which is contained within the soft body and applying calcium indicators to the cephalopod central nervous system. NEW METHOD: We here present a calcium imaging method to visualize neural responses in the central nervous system of the smallest squid, Idiosepius paradoxus. RESULTS: We injected calcium indicator Cal-520 into the brachial lobes and revealed a spatiotemporal pattern of neural responses to the electrical stimulations of the axial nerve cord in the first arm. COMPARISON WITH EXISTING METHODS: We established a method to immobilize the central nervous system which is contained within the soft body and record the calcium responses from the intact central nervous system. CONCLUSIONS: Our method provides a novel approach to investigate the mechanisms of how the characteristic organization of the cephalopod brain functions to induce their unique behaviors.

Three-dimensional brain atlas of pygmy squid, Idiosepius paradoxus, revealing the largest relative vertical lobe system volume among the cephalopods.

Cephalopods have the largest and most complex nervous system of all invertebrates, and the brain-to-body weight ratio exceeds those of most fish and reptiles. The brain is composed of lobe units, the functions of which have been studied through surgical manipulation and electrical stimulation. However, how information is processed in each lobe for the animal to make a behavioral decision has rarely been investigated. To perform such functional analyses, it is necessary to precisely describe how brain lobes are spatially organized and mutually interconnected as a whole. We thus made three-dimensional digital brain atlases of both hatchling and juvenile pygmy squid, Idiosepius paradoxus. I. paradoxus is the smallest squid and has a brain small enough to scan as a whole region in the field-of-view of a low-magnification laser scan microscope objective. Precise analyses of the confocal images of the brains revealed one newly identified lobe and also that the relative volume of the vertical lobe system, the higher association center, in the pygmy squid represents the largest portion compared with the cephalopod species reported previously. In addition, principal component analyses of relative volumes of lobe complexes revealed that the organization of I. paradoxus brain is comparable to those of Decapodiformes species commonly used to analyze complex behaviors such as Sepia officinalis and Sepioteuthis sepioidea. These results suggest that the pygmy squid can be a good model to investigate the brain functions of coleoids utilizing physiological methods. J. Comp. Neurol. 524:2142-2157, 2016. © 2016 Wiley Periodicals, Inc.