Abstract
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.
Highlights
The neuronal nitric oxide (NO) synthase isoform is expressed by distinct populations of neocortical neurons (Karagiannis et al, 2009; Kubota et al, 2011)
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)
Alternate sections were incubated for 48–72 h at 4 ̊C with one of the following antibodies diluted in phosphate buffered saline (PBS) containing triton (0.2%; PBST): rabbit anti-nitric oxide (NO) synthase (nNOS) antibody (1:500; Santa-Cruz sc-648), rabbit anti-PV (1:800; Swant PV28), rat anti-SOM (1:500; Millipore MAB354), or rabbit anti-vasoactive intestinal peptide (VIP; 1:500, ImmunoStar 20077)
Summary
The neuronal nitric oxide (NO) synthase (nNOS) isoform is expressed by distinct populations of neocortical neurons (Karagiannis et al, 2009; Kubota et al, 2011). Type I cells usually exhibit large somata, are intensely stained and represent about 2% of total GABAergic interneurons in the rat primary sensorimotor cortex (Kubota et al, 1994) In rodents, they are found in all cortical layers (Oermann et al, 1999), they are more frequently observed in infragranular layers (Vercelli et al, 2000; Wiencken and Casagrande, 2000; Garbossa et al, 2001; Lee and Jeon, 2005). Immunohistochemical studies indicate that type I cells frequently co-express neuropeptide Y (NPY) and somatostatin (SOM; Dawson et al, 1991; Kubota et al, 1994; Gonchar and Burkhalter, 1997; Estrada and Defelipe, 1998; Smiley et al, 2000)
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