Connectivity of GABAergic calretinin-immunoreactive neurons in rat primary visual cortex

In rat visual cortex neurons that are immunoreactive for the calcium- binding protein calretinin (CR⁺) constitute a distinct family which accounts for 17% of γ-aminobutyric acid (GABA)-expressing cells. It is not clear, however, (i) whether CR is expressed exclusively in GABAergic neurons and (ii) how CR⁺ neurons are incorporated into neuronal circuits of rat visual cortex. To address these questions we studied synaptic relationships of CR⁺ neurons with GABA⁺ and GABA^- elements in the neuropil of rat primary visual cortex (area 17). All CR⁺ neurons are nonpyramidal cells with smooth or sparsely spiny and often beaded dendrites. Of all CR⁺ neurons, 56% are located in layers 1 and 2/3. In layer 2/3, most CR⁺ neurons are bipolar- shaped and have vertically oriented dendrites. Many ascending dendritic branches reach layer 1 where they run parallel to pial surface. CR⁺ axons are thin, highly branched near the cell body and often send descending collaterals to layers 5 and 6. Double immunofluorescence labeling revealed GABA in 94% of CR⁺ cell bodies in layer 2/3. Electron microscopic analysis shows that all CR⁺ axon terminals contain elongated vesicles and form symmetric synapses. Postembedding staining shows that 98% of CR⁺ terminals are GABA⁺. GABA-immunoreactivity is also present in somata and thick dendrites of CR⁺ neurons but many thin dendrites are GABA^-. CR⁺ somata, dendrites and axon terminals are enriched in mitochondria. Somata and thick CR⁺ dendrites are densely innervated. At least 68% of the targets of CR⁺ terminals in layer 2/3 are GABA⁺ and ≥50% of these are other CR⁺ neurons. The remainder (32%) of targets of CR⁺ terminals are thin dendrites of GABA^- cells. In contrast, in layers 5 and 6, 60% of CR⁺ terminals form synapses with GABA^- somatic profiles. The preferential interactions of layer 2/3 CR⁺ neurons with GABAergic neurons, and with CR⁺ neurons in particular, suggests that these cells play a role in the inhibition of inhibitory neurons of the same layer. Through these interactions CR⁺ cells may reduce inhibition of pyramidal cells in layers 2/3, 5 and 6 and thus disinhibit a column of neurons.