GABAergic interneurons in the mouse neocortex have been classified into three supgroups based on gene expression: (1) parvalbumin-containing (PV+) neurons ; (2) somatostatin-immunoreactive (SOM+) neurons; (3) 5HT3a receptor-expressing neurons, including vasoactive intestinal polypeptide-expressing (VIP+) neurons and the others. Several recent reports demonstrated that VIP+ neurons had a disinhibitory effect to principal cells via other types of interneurons. In the present study, we conducted morphological examination on the excitatory and inhibitory inputs to VIP+ neurons in layer 2/3 of the primary somatosensory cortex by using VIP-Cre knock-in mice and adeno-associated viral vectors (AAV2/1).
We first developed AAV2/1 SynTet DIO-FGL, which made somata and dendrites of VIP+ neurons completely labeled with GFP under Cre exposure. The injected brain sections were then triple-immunostained for GFP, presynaptic markers and postsynaptic markers. The apposed presynaptic punctae on GFP-positive membrane were considered as putative input sites only when the postsynaptic markers existed on the apposed points.
After acquiring 3D z-stack images of somata and dendrites of VIP+ neurons in layer 2/3 under the confocal laser-scanning microscope, we reconstructed the dendrites and cell bodies by using NeuroLucida computer-assisted neuron-tracing system. VIP+ neurons had many branches in the distal portions; their tangential spread of dendrites was limited within 250 μm, and they extended in the vertical direction from layer 1 to 4.
Subsequently, the excitatory and inhibitory inputs to VIP+ neurons were counted. Both cortico- and thalamo-cortical excitatory inputs were frequently observed on the distal portions of dendrites of VIP+ neurons. There was no significant difference in total inhibitory inputs between proximal and distal dendrites, but each input of subgroups in GABAergic interneurons varied from proximal to distal; the inputs from PV+ neurons were frequently observed on the cell bodies and proximal dendrites of VIP neurons, whereas those of SOM+ neurons were mainly found on the distal portions of them.