Morphological analysis of the mormyrid cerebellum using immunohistochemistry, with emphasis on the unusual neuronal organization of the valvula

Abstract

This study used immunohistochemistry, Golgi impregnation, and electron microscopy to examine the circuitry of the cerebellum of mormyrid fish. We used antibodies against the following antigens: the neurotransmitters glutamate and gamma-aminobutyric acid (GABA); the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N-methyl-D-aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1alpha and mGluR2/3; the intracellular signaling molecules calcineurin and calcium calmodulin kinase IIalpha (CAMKIIalpha); and the receptor for inositol triphosphate (IP3RIalpha). Purkinje cells are immunoreactive to anti-IP3R1alpha, anticalcineurin, and anti-mGluR1alpha. Cerebellar efferent cells (eurydendroid cells) are anticalretinin and anti-NR1 positive in the valvula but not in the corpus and caudal lobe. In contrast, climbing fibers are anticalretinin and anti-NR1 immunopositive in the corpus and caudal lobe but not in the valvula. Purkinje cells, Golgi cells, and stellate cells are GABA positive, whereas efferent cells are glutamate positive. Unipolar brush cells are immunoreactive to anti-mGluR2/3, anticalretinin, and anticalbindin. We describe a "new" cell type in the mormyrid valvula, the deep stellate cell. These cells are GABA, calretinin, and calbindin positive. They are different from superficial stellate cells in having myelinated axons that terminate massively with GAD- and gephyrin-positive terminals on the cell bodies and proximal dendrites of efferent cells. We discuss how the valvula specializations described here may act in concert with the palisade pattern of Purkinje cell dendrites for analyzing spatiotemporal patterns of parallel fiber activity.