The Characterization Of Neuronal Nitric Oxide Synthase Expression In The Rat Dentate Gyrus

Background and ObjectivesInterneurons are cells that surround principal cells with their axons projecting to the same brain region and function to regulate the excitability of principal cells. Expression of special molecular markers can be used for classification of interneurons. Many molecular substances such as calcium-binding proteins (parvalbumin, calretinin, and calbidin etc.), neuropeptides (somatostatin, neuropeptides Y and vasoactive intestinal peptide etc.), enzymes and receptors have been shown to express in certain interneuron population(s) specifically. Even not all but certain interneuronal markers are closely related to the morphology, electrophysiological properties, axonal projection of a given interneuron population.Neuronal nitric oxide synthase (nNOS) is one of three nitric oxide synthase isoforms; it is responsible for nitric oxide production in neurons. The expression of nNOS is particularly strong in a fraction of interneurons. Previous studies have shown that the density of nNOS-expressing interneurons is different among brain regions. Up-to-date, data on the laminar distribution, anatomical features, electrophysiological properties, axonal projections of nNOS-expressing neurons are lack. The present study examined nNOS-expressing interneurons in the rat dentate gyrus of hippocampal formation by focusing on their laminar distribution, GABAergicity, and co-localization with other commonly known interneuronal markers.Materials and MethodsExperimental were conducted in15adult male Sprague-Dawley rats (450±50g,11-13weeks). After an individual rat was paralyzed with intraperitoneal injection of sodium pentobarbital, the rat was transcardially perfused with4%paraformaldehyde in0.1M phosphate-buffered saline. The brain was removed from the skull, and post-fixed in the same fixative at4℃overnight. Thereafter the brain tissue block was cryoprotected with gradually increased sucrose in0.1M PBS. After the tissue block was embedded with a medium that consisted of chicken egg albumin, gelatin and glutaradehyde, it was horizontally cut into60and μm in thickness. Hippocampal slices were collected every eight section. The collected sections were immunofluorescently stained with the free-floating procedure. The primary antibodies used were mouse and rabbit-anti-nNOSs, mouse anti GAD67, mouse anti parvalbumin (PV), rabbit anti-somatostatin (SOM), rabbit anti-neuropeptide Y (NPY) and rabbit anti vasoactive intestinal peptide (VIP). Stained sections were visualized with a fluorescence microscope and photographed. Some stained adjust sections scanned with a confocal microscopy. Immunoreactive positive cells were counted in different layers of dentate gyrus and the corresponding areas were measured with Image-Pro plus6. Densities of immunoreactive positive cells (cells/mm2) were calculated by dividing the number of cells with the area in each layer. Data are expressed as mean±standard deviation (SD) and statistically analyzed with one-way ANOVA followed by Newman-Keuls for post hoc comparisons.ResultsnNOS-expressing cells were present in all three layers of the dentate gyrus; the densities of nNOS-positive cells were23.1±1.9/mm2,24.1±10.7/mm2and143.1±13.8/mm2in the molecular layer (ML), granule cell layer (GCL) and the hilus of the dentate gyrus (n=6), respectively. The percentages of nNOS-expressing cells that co-expressed with GAD67were96.2±2.5%(n=6) and95.6±5.6%(n=6) in ML and GCL, respectively. However, the percentage of nNOS-expressing cells positive for GAD67in the hilus went down to82.3±4.7%. Co-localization of nNOS with other commonly known interneurons was infrequent. The percentages of nNOS-expressing cells positive for PV, SOM, NPY and VIP are8.6±2.6%,10.3±0.6%,13.8±3.0%and1.6±0.5%, respectively.Conclusions1. The distribution of nNOS-expressing neurons in the rat dentate gyrus is laminar dependent.2. Almost all nNOS-expressing cells in the molecular layer and granule cell layer are GABAergic but approximately1/5of nNOS-expressing cells in the hilus are not.3. The percentages of nNOS-expressing cells that co-express with other commonly known interneuronal markers range from near zero for VIP to14%for NPY with rest of them at about10%.4. Future studies are needed to determine if there are interneurons that express nNOS but not other commonly known interneuronal molecular markers and the specific functions that nNOS-expressing interneurons play.