Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Because of the large electrophysiological, molecular, and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling, and singlecell reverse transcriptase-PCR designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons, we used and compared different unsupervised clustering algorithms based on laminar location and electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively GABAergic and consistently disclosed three main types of NPY-expressing interneurons.(1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent and superficial, and substantially expressed the neuronal isoform of nitric oxide synthase. (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I coexpressed somatostatin and were the most excitable type. (3) Among fast-spiking and parvalbumin-positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles.

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT3A promoter (5-HT3A:GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT3A:GFP mice, we identified 2 populations of 5-HT3A-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT3A mRNA detection showed that cortical 5-HT3A interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT3A receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT3A interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT3A subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

In the neocortex, neuronal nitric oxide (NO) synthase (nNOS) is essentially expressed in two classes of GABAergic neurons: type I neurons displaying high levels of expression and type II neurons displaying weaker expression. Using immunocytochemistry in mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we studied the distribution of type I and type II neurons in the barrel cortex and their expression of parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal peptide (VIP). We found that type I neurons were predominantly located in deeper layers and expressed SOM (91.5%) while type II neurons were concentrated in layer II/III and VI and expressed PV (17.7%), SOM (18.7%), and VIP (10.2%). We then characterized neurons expressing nNOS mRNA (n = 42 cells) ex vivo, using whole-cell recordings coupled to single-cell reverse transcription-PCR and biocytin labeling. Unsupervised cluster analysis of this sample disclosed four classes. One cluster (n = 7) corresponded to large, deep layer neurons, displaying a high expression of SOM (85.7%) and was thus very likely to correspond to type I neurons. The three other clusters were identified as putative type II cells and corresponded to neurogliaform-like interneurons (n = 19), deep layer neurons expressing PV or SOM (n = 9), and neurons expressing VIP (n = 7). Finally, we performed nNOS immunohistochemistry on mouse lines in which GFP labeling revealed the expression of two specific developmental genes (Lhx6 and 5-HT3A). We found that type I neurons expressed Lhx6 but never 5-HT3A, indicating that they originate in the medial ganglionic eminence (MGE). Type II neurons expressed Lhx6 (63%) and 5-HT3A (34.4%) supporting their derivation either from the MGE or from the caudal ganglionic eminence (CGE) and the entopeduncular and dorsal preoptic areas. Together, our results in the barrel cortex of mouse support the view that type I neurons form a specific class of SOM-expressing neurons while type II neurons comprise at least three classes.