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. 2014 May;4(3):317-36.
doi: 10.1002/brb3.218. Epub 2014 Feb 12.

Characterization of NO-producing neurons in the rat corpus callosum

Paolo Barbaresi  1 , Mara Fabri  1 , Emanuela Mensà  1
Affiliations

Characterization of NO-producing neurons in the rat corpus callosum

Paolo Barbaresi et al. Brain Behav. 2014 May.

Abstract

Introduction: The aim of this study was to determine the presence and distribution of nitric oxide (NO)-producing neurons in the rat corpus callosum (cc).

Material and methods: To investigate this aspect of cc organization we used nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunocytochemistry.

Results: Intense NADPH-d-positive (NADPH-d+) neurons were found along the rostrocaudal extension of the cc (sagittal sections). They were more numerous in the lateral cc and gradually decreased in the more medial regions, where they were very few or absent. The Golgi-like appearance of NADPH-d+ intracallosal neurons allowed dividing them into five morphological types: (1) bipolar; (2) fusiform; (3) round; (4) polygonal; and (5) pyramidal. The number of NADPH-d+ neurons (both hemispheres) was counted in two brains using 50-μm thick sections. In the first brain, counts involved 145 sections and neurons were 2959; in the second, 2227 neurons were counted in 130 sections. The distribution and morphology of nNOS-immunopositive (nNOSIP) neurons was identical to that of NADPH-d+neurons. Some of these neurons were observed in the cc ependymal region, where they might be in contact with cerebrospinal fluid (CSF), monitoring its composition, pH, and osmolality changes, or playing a role in regulating the synthesis and release of several peptides. The somatic, dendritic, and axonal processes of many NADPH-d+/nNOSIP neurons were closely associated with intracallosal blood vessels.

Conclusions: Such close relationship raises the possibility that these neurons are a major source of NO during neural activity. As NO is a potent vasodilator, these findings strongly suggest that NO-positive neurons transduce neuronal signals into vascular responses in selected cc regions, thus giving rise to hemodynamic changes detectable by neuroimaging.

Keywords: Colocalization; GFAP; NADPH-d; immunocytochemistry; nNOS; nitric oxide.

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Figures

Figure 1
Figure 1
Comparison of cc borders defined by CO activity and those defined by neutral red counterstaining. (A–C) Anterior and posterior part of the rat cc (same mediolateral level). Neutral red counterstaining. (B–D) CO reaction, section adjacent to the one shown in (A–C). (E–F) Central part of the rat-cc. (E) Neutral red; (F), CO staining. Adjacent sections. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. VI, sixth layer of the cerebral cortex; wm, white matter. Calibration bars: 500 μm.
Figure 2
Figure 2
Distribution of NADPH-d+ neurons from lateral to medial (from A to D; right hemisphere) and from medial to lateral (from E to H; left hemisphere) in the rat corpus callosum. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. Calibration bar: 1 mm
Figure 3
Figure 3
Distribution of nNOS-immunopositive (nNOSIP) neurons in the rat corpus callosum from lateral to medial. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. Calibration bar: 1 mm
Figure 4
Figure 4
Low-power photomicrographs showing the distribution of NADPH-d+ neurons at different mediolateral levels of the rat corpus callosum. (A) Low-power photomicrograph showing many NADPH-d+ neurons in the lateral rat cc. (B) Absence of NADPH-d+ neurons at the medial level. The framed region in B is enlarged in B′. At this mediolateral level NADPH-d+ neurons are located around the genu. (C) Photomicrograph showing some positive neurons; one of them has a dendrite crossing the white matter and reaching layer VI. (D) Splenium of the rat cc showing a positive neuron (arrow). Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. wm: white matter. VI, sixth layer of the cerebral cortex Calibration bars: 250 μm in A, in B′ and C; 500 μm in D.
Figure 5
Figure 5
Photomicrographs of nNOSIP neurons in the rat corpus callosum. (A) Low-power photomicrograph showing the distribution of nNOSIP neurons. (B) A bipolar neuron close to an intracallosal blood vessel. Framed area enlarged in C. (C) Enlarged area showing a dendritic spine (arrowhead). (D) Triangular and ovoid nNOSIP intracallosal neurons. (E) nNOSIP neurons in the ependymal region. (F) A round nNOSIP neuron near the lateral ventricle. (G) Bipolar nNOSIP intracallosal neuron. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982) on bottom left side. Calibration bars: 250 μm in A; 25 μm in B–G.
Figure 6
Figure 6
Morphology of NADPH-d+ neurons in the rat corpus callosum. (A) A bipolar NADPH-d+ intracallosal neuron with long dendrites extending along the rostrocaudal axis of the corpus callosum. (B) A pyriform NADPH-d+ neuron in the ependymal region. (C) Three NADPH-d+ neurons close to an intracallosal blood vessel. (D) NADPH-d+ neurons with vertically oriented dendrites. (E) A cluster of NADPH-d+ intracallosal neurons in the ependymal region. (F) A bipolar NADPH-d+ intracallosal neuron with dendrites close to a blood vessel. (G) An inverted pyriform NADPH-d+ intracallosal neuron with vertically oriented dendrites. bv, blood vessel. Calibration bars: 25 μm for B and G; 50 μm for A, D, F; 100 μm for C, E.
Figure 7
Figure 7
Camera lucida drawings of three bipolar (fusiform) NADPH-d+ neurons in the rat corpus callosum. Neurons in A and B are oriented vertically, neuron in C is oriented horizontally. Ax, axon. Calibration bars: 25 μm.
Figure 8
Figure 8
Camera lucida drawings of two rectangular NADPH-d+ neurons (A) in the middle and (B) splenium of the corpus callosum. A dendrite from the neuron in A reaches the alveus of hippocampus. Ax, axon. Calibration bar: 50 μm.
Figure 9
Figure 9
Camera lucida drawings of three NADPH-d+ round neurons. (A) Two of them (A-1 and A-2) lie in the forceps major of the corpus callosum; note the wide dendritic field. (B) Round neuron in the middle corpus callosum showing a narrow, elliptical dendritic field. Calibration bars: 50 μm.
Figure 10
Figure 10
Camera lucida drawings of two polygonal NADPH-d+ neurons found (A) in the middle and (B) splenium of the corpus callosum wide dendritic fields taken from two different anteroposterior and mediolateral levels. Calibration bar: 50 μm.
Figure 11
Figure 11
Camera lucida drawings of two pyriform (A1) and (A2) and two pyramidal (B1) and (B2) neurons from different callosal regions. Dendrites from neurons in A2 and B1 reach the overlying white matter (wm). Calibration bar: 50 μm.
Figure 12
Figure 12
Photomicrographs showing NADPH-h+ neurons lying close to blood vessels. (A), (D) NADPH-d+ neurons in the splenium of the corpus callosum. Cell bodies and their processes are closely apposed to the wall of a longitudinal blood vessel. Photomicrographs are rotated 90° counterclockwise. (B) A NADPH-d+ neuron apposed to a callosal blood vessel encircles the wall with one of its processes. (C) A callosal vessel with stained fibers containing many varicosities and puncta. (E) Large blood vessel with stained fibers containing numerous varicosities and a spray-like distribution of NADPH-d+ puncta. (F) An intensely labeled callosal neuron wrapped around a blood vessel. Calibration bars: 25 μm in A, B, C, D, E, and G; 50 μm in F.
Figure 13
Figure 13
Confocal laser scanning photomicrographs showing the lack of colocalization (C) of GFAP+ (A, red fluorescence) and nNOSIP neurons (B, green fluorescence) in the rat corpus callosum. Calibration bar: in C for A–C 25 μm.
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