Proceedings of The Physiological Society

University of Cambridge (2004) J Physiol 555P, PC72


NMDA receptor subunits immunoreactivity in the CNS of the cephalopods

Carlo Di Cristo and Anna Di Cosmo

Department of Biological and Environmental Sciences, University of Sannio, Via Port'Arsa 11, 82100, Benevento, Italy

Excitatory amino acids, such as glutamate and aspartate, are present in many synapses of the vertebrate and invertebrates central nervous system (CNS). Glutamate has a depolarizing effect on neurons and this effect is rapid and can be mimicked by other amino acids. The speed of this effect suggested that glutamate and its agonist evoke depolarization by acting directly on receptor channel.It can be distinguish two main groups of receptor channel activated by glutamate: this distinction is based on the affinity of the receptor for glutamate selective structural analogs, notably for N-methyl-D-aspartate (NMDA). Thus, ionotropic glutamate receptors have been subdivided into N-methyl-D-aspartate (NMDA), and AMPA/kainate classes.

NMDA receptor subunits 2A&B (NMDAR2A&B) immunoreactivity is shown to be present in specific regions of the central nervous system (CNS) of the cephalopod molluscs Sepia officinalis and Octopus vulgaris (n of experiments = 10; all the animals were humanely killed). An antibody which recognizes both NMDAR2A and NMDAR2B subunits equally (Chemicon) was used. SDS-PAGE/Western blot analysis revealed for both animal an immunoreactive band at 170 kDa.

Figure 1. NMDAR 2A&B immunopositive staining in the vertical-superior frontal system of Sepia officinalis and Octopus vulgaris. A Immunopositive staining of cell bodies (arrows) and neuropil in the vertical lobe of Sepia officinalis. Note the intense immunostaining in the fibers. B Subvertical lobe of Sepia officinalis. Cluster of immunopositive neurons in the central region of the suvbertical lobe under the vertical lobe. C. Strong immunopositive neurons in the precommissural lobe of Sepia officinalis. D Median superior frontal lobe of Octopus vulgaris. Staining in small cell bodies (arrows) and scattered immunopositive staining in the neuropil (arrowheads). E Immunopositive neurons in the lateral superior frontal lobe of Octopus vulgaris. The immunopositive axons reach the neuropil of the lobe. F Vertical lobe of Octopus vulgaris. Positive staining of cell bodies (arrows) and in fibers running to the neuropil of the lobule. Note few immunopositive amacrine cells. Magnification: A, B, C, E x900; D, F x500.

This same antibody was then used for immunohistochemical staining of serial sections of the CNSs to reveal localized specific staining of cell bodies and fibers in several lobes of the brain. Staining was found in lower motor centers, mainly posterior pedal and palliovisceral lobes; in some higher motor centers (anterior basal and peduncle lobes); in learning centers (vertical lobe system (Fig.1) and, in Octopus inferior frontal system); and in the optic lobes. Immunopositivity was also found in the areas of brain which control the activity of the optic gland, a gonadotropic endocrine gland. These findings suggest that glutamate via NMDA receptors may be involved as a signalling molecule in motor, learning, visual and olfactory systems in the cephalopod brain.

Where applicable, experiments conform with Society ethical requirements