Postsynaptic structures of axon terminals of cortical double bouquet cells

Y. Kubota, Y. Kawaguchi,

Division of Cerebral Circuitry

National Institute for Physiological Sciences

Aichi Okazaki 444-8787, Japan

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GABAergic nonpyramidal cells regulate activities of cortical pyramidal cells. Several subtypes of cortical nonpyramidal cells have been found based on their intrinsic firing property, axonal innervation pattern and neurochemical content. Recently several dynamic properties of cortical circuitry have been revealed, but the precise intracortical wiring of each nonpyramidal cell subtype remains largely unknown. We investigated synaptic connections made by double bouquet cells, a subtype of nonpyramidal cells, in rat frontal cortex. In layers II/III, calretinin and corticotrophin releasing factor (CRF) were identified in double bouquet cells. Using the double fluorescent immunohistochemical method, calretinin and CRF were found in largely separate subgroups. Thus, calretinin-positive and CRF-positive double bouquet cells may be considered as different cell subtypes in layers II/III. We further studied postsynaptic structures of these two subtypes of double bouquet cells.

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Non-pyramidal cells were identified in isolated slices of frontal cortex from young rats (18-22 days postnatal) by whole cell, current-clamp recording, followed by intracellular injection of biocytin. After overnight fixation, cells were immunohistochemically double stained with antiserums against calretinin and CRF, then the slices were histochemically stained with DAB and embedded in Epon. The stained double bouquet cells had small soma and several descending axonal arbors.

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The postsynaptic structures were reconstructed three-dimensionally from serial ultra-thin sections of the intracellulary-stained axon terminals. This showed that double bouquet cells innervated dendritic shafts, spine heads, spine necks, and, rarely, somata. About half of the dendrites contacted by double bouquet cell axons received numerous asymmetrical synapses on their dendritic shafts, which identified these postsynaptic targets as nonpyramidal cells. The rest of the target dendrites of calretinin positive cells were proximal dendrites and distal dendrites of both pyramidal and nonpyramidal cells. For the CRF positive cells, the other part of the target dendrites may consist of proximal dendrites and distal dendrites of nonpyramidal cells. The target spines of the double bouquet cells usually had an asymmetrical input. This may indicate that the double bouquet cell specifically inhibits this excitatory input. The excitatory input was probably thalamic in origin, since vesicular glutamate transporter 2 (VGLUT2) was immunohistochemically identified in this terminal type. Another interesting finding was that the synaptic junction area of the axon terminals linearly increased with the circumference of postsynaptic dendritic shafts, and the junctional area on spine heads linearly increased with the volume of the spine heads.

These observations indicate that the double bouquet cells have several specific synaptic targets and may have a specific role in the regulation of cortical activity.

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