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The postnatal development of nitric oxide (NO)-producing intracallosal neurons was studied in rats by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry from postnatal day 0 (P0) to P30. NADPH-d-positive neurons (NADPH-d+ Ns ) were detected already at P0, mainly in the rostral region of the corpus callosum (cc). Their location and the intensity of staining allowed them to be classified as type I NO-producing neurons. At P0, tufts of intensely labeled fibers, probably corresponding to the callosal septa described in the monkey and human cc, entered the ventral cc region and reached its dorsal portion. From P5, cell bodies and dendrites were often associated to blood vessels. The number of intracallosal NADPH-d+ Ns rose in the first postnatal days to peak at P5, it declined until P10, and then remained almost constant until P30. Their size increased from P0 to P30, dramatically so (>65%) from P0 to P15. From P10 onward their distribution was adult-like, i.e. NADPH-d+ Ns were more numerous in the lateral and intermediate portions of the cc and diminished close to the midline. In conjunction with previous data, these findings indicate that intracallosal NADPH-d+ Ns could have a role in callosal axon guidance, myelination, refinement processes, and callosal blood flow regulation., (Copyright © 2019. Published by Elsevier B.V.)

The corpus callosum (cc) contains nitric oxide (NO)-producing neurons. Because NO is a potent vasodilator, these neurons could translate neuronal signals into vascular responses that can be detected by functional brain imaging. Substance P (SP), one of the most widely expressed peptides in the CNS, also produces vasomotor responses by inducing calcium release from intracellular stores through its preferred neurokinin 1 (NK1) receptor, thus inducing NO production via activation of neuronal NO synthase (nNOS). Single- and double-labeling experiments were performed to establish whether NK1-immunopositive neurons (NK1IP -n) are found in the rat cc and the extent of NK1 colocalization with nNOS. NK1IP -n were seen to constitute a large neuronal population in the cc and had a distribution similar to that of nNOSIP neurons (nNOSIP -n). NK1IP -n were numerous in the lateral cc and gradually decreased in the more medial portions, where they were few or absent. Intracallosal NK1IP -n and their dendritic trees were intensely labeled, allowing classification into four morphological types: bipolar, round, polygonal, and pyramidal. Confocal microscopic examination demonstrated that nearly all NK1IP -n contained nNOS (96.43%) and that 84.59% of nNOSIP -n co-expressed NK1. These data suggest that the majority of intracallosal neurons can release NO as a result of the action of SP. A small proportion of nNOSIP -n does not contain NK1 and is not activated by SP; these neurons may release NO via alternative mechanisms. The possible mechanisms by which intracallosal neurons release NO are also reviewed., (© 2014 Wiley Periodicals, Inc.)

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.

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 μm(2) (PAG-dl) to 125.13 μm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 μm(2); neurons labeled only with NADPH-d or Calb measured 89.793 μm(2) and 113.48 μm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 μm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 μm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity., (Copyright © 2011 Wiley Periodicals, Inc.)

The development of intrinsic gamma-aminobutyric acid (GABA)-ergic neurons was studied in the first month of postnatal life in the rat periaqueductal gray matter (PAG) by light and electron microscopy using an anti-GABA serum. At birth (postnatal day 0: P0) GABA-immunopositive (GABA(IP)) neurons were detected only on the outer edge of dorsolateral PAG (PAG-DL) and were rare in the other PAG subdivisions. Their distribution did not change from P0 to P5, while they increased progressively from P5 to P10 in PAG-DL and began to be detected in ventrolateral PAG (PAG-VL). At the end of the second postnatal week the immunostaining pattern was nearly adult-like, and between P20 and P30 the adult pattern of GABA immunoreactivity was established. Quantitative light microscopic examination indicated that in the first postnatal month the cross-sectional area of GABA(IP) neurons gradually increased from 67.63 and 78.69 microm(2) at P0 to 122.15 and 119.16 microm(2) at P30 in PAG-DL and PAG-VL, respectively. Electron microscopic observations disclosed GABA labeling from P0 in cell bodies, dendrites, growth cones, and axon terminals. GABA(IP) terminals were few in neonatal rats and became more numerous and morphologically mature around the second week. Synapse development and maturation were examined by quantitative ultrastructural analysis. Synaptic vesicle number and size of GABA(IP) axon terminals progressively grew in the first postnatal month. In conclusion, the number and size of GABA(IP) cells progressively increase in postnatal PAG, with two populations of intrinsic neurons expressing their GABAergic nature in two different periods., (Copyright 2010 Wiley-Liss, Inc.)

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against gamma -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA(IP)) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35-36% of all terminals were GABA(IP); they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7-10% of the sample). Moreover, 49.15% of GABA(IP) axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA(IP) dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA(IP) and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.

Gamma-amino butyric acid (GABA) plasma membrane transporters (GATs) contribute to the modulation of GABA's actions and are implicated in neuropsychiatric diseases. In this study, the localization of GAT-3, the major glial GAT, was investigated in human cortex using immunocytochemical techniques. In prefrontal and temporal cortices, GAT-3 immunoreactivity (ir) was present throughout the depth of the cortex, both in puncta and in neurons. GAT-3-positive puncta were dispersed in the neuropil or closely related to cell bodies; neuronal staining was in perikarya, especially of pyramidal cells, and proximal dendrites. Electron microscopic studies showed that GAT-3 ir was in astrocytic processes as well as in neuronal elements. All GAT-3-positive neurons co-expressed heat shock protein 70. To test the possibility that the collection procedure of human samples induced the expression of GAT-3 in neurons which normally do not express it, we analyzed rat cortical tissue resected using the same procedure and found that numerous neurons are GAT-3-positive and that they co-express heat shock protein 70. Results show that in human cortex GAT-3 is expressed in astrocytic processes and in neurons and suggest that neuronal expression is related to the procedure used for collecting human samples.

SNAT1 mediates glutamine (Gln) influx into neurons and is believed to replenish the transmitters pools of glutamate (Glu) and gamma-aminobutyric acid (GABA). We investigated its distribution and cellular localization in the cerebral cortex and neighboring regions of rats and humans using light and electron microscopic immunocytochemical methods with specific antibodies. In the first somatic sensory cortex of rats and in areas 9, 10, 21 and 46 of the human cortex, numerous SNAT1-positive (+) cells were present in the cortical parenchyma and in the white matter; >95% of SNAT1+ cells were neurons, but some were astrocytes. Most SNAT1+ cells were pyramidal neurons, but numerous non-pyramidal neurons were also observed: SNAT1/GABA double-labeling studies showed that SNAT1 is expressed in all GABA+ neurons. SNAT1/synaptophysin studies showed that <0.1% of all synaptophysin+ puncta coexpressed SNAT1. SNAT1 immunoreactivity (ir) was also in leptomeninges, ependymal cells and choroid plexus. Electron microscopic studies showed that neuronal SNAT1 ir was almost exclusively observed in perikarya and dendritic profiles. SNAT1 ir was also in distal astrocytic processes, including end feet profiles, and in leptomeninges. These findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA.

The neuron-specific glutamate (Glu) transporter, excitatory amino acid carrier 1 (EAAC1), plays an important role in regulating Glu levels in the synaptic cleft. Using a specific EAAC1 monoclonal antibody, we investigated its regional distribution and ultrastructural localization in cat periaqueductal gray matter. In light microscopy EAAC1 immunoreactivity was randomly distributed to neurons and punctate structures. In electron microscopy, it was observed in the soma of many neurons, dendrites, in a discrete number of axon terminals, in ependymal cells and in few distal astrocytic processes. EAAC1 is thus not restricted to neurons, but could play an important role in glial cells. Moreover, EAAC1 protein could participate both in regulating the action of the Glu released at synaptic level and in other aspects of Glu metabolism.

Neuronal and glial high-affinity Na+/Cl(-)-dependent plasma membrane gamma-aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT-2 by immunocytochemistry with affinity-purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser-scanning light microscopic studies revealed intense GAT-2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT-2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double-labeled with GAT-2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT-2-positive profiles also were GFAP positive. Ultrastructural studies showed GAT-2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT-2-positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT-2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT-2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain.

N-Methyl-D-aspartate (NMDA) receptors play a critical role in many cortical functions and are implicated in several neuropsychiatric diseases. In this study, the cellular expression of the NMDAR1 (NR1) and NMDAR2A and B (NR2A and B) subunits was investigated in the human cerebral cortex by immunocytochemistry with antibodies that recognize the NR1 or the NR2A and B subunits of the NMDA receptor. In frontal (areas 10 and 46) and temporal (area 21) association cortices and the cingulofrontal transition cortex (area 32), NR1 and NR2A/B immunoreactivity (ir) were similar and were localized to numerous neurons in all cortical layers. NR1- and NR2A/B-positive neurons were mostly pyramidal cells, but some nonpyramidal neurons were also labeled. Electron-microscopic observations showed that NR1 and NR2A/B ir were similar. In all cases, labeling of dendrites and dendritic spines was intense. In addition, both NR1 and NR2A/B were consistently found in the axoplasm of some axon terminals and in distal astrocytic processes. This investigation revealed that numerous NMDA receptors are localized to dendritic spines, and that they are also localized to axon terminals and astrocytic processes. These findings suggest that the effects of cortical NMDA activation in the human cortex do not depend exclusively on the opening of NMDA channels located at postsynaptic sites, and that the localization of NMDA receptors is similar in a variety of mammalian species.

The morphology, distribution, proportion, size, and synaptic organization of periaqueductal gray matter neurons labeled with immunocytochemical techniques by an anti-glutamate (Glu) polyclonal serum were investigated in six adult cats (PAG-GLU 1-6). At the light microscopic level, numerous Glu-positive neurons were found throughout each subdivision of the periaqueductal gray matter. Their proportion and size, calculated in semi-thin sections (1-microm-thick), varied slightly among the subdivisions of the periaqueductal gray matter. The morphology of Glu-positive neurons was similar to that of the multipolar, triangular, and fusiform cells described in previous Golgi studies. Numerous puncta, interpreted as dendrites, axons, and axon terminals were also present in all subdivisions without preferential distribution. At the electron microscopic level, all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all presynaptic elements making asymmetric synapses were labeled. The vast majority of postsynaptic elements contacted by Glu-positive axon terminals were labeled and unlabeled dendrites. The present results describe for the first time the presence of both Glu-positive neurons and terminals in the feline periaqueductal gray matter and provide further evidence that Glu is the probable neurotransmitter of numerous excitatory neurons of this structure.

Experiments were carried out on the second somatic sensory area (SII) of cats to study 1) the laminar distribution of axon terminals from the ipsilateral first somatic sensory cortex (SI); and 2) the topographical relations between their terminal field and the callosal neurons projecting to the contralateral homotopic cortex. To label simultaneously in SII both ipsilateral cortical afferents and callosal cells, cats were given iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the forepaw zone of ipsilateral SI, and pressure injections of horseradish peroxidase (HRP) in the same zone of contralateral SII. The possibility that ipsilateral cortical axon terminals synapse callosal neurons was investigated with the electron microscope by combining lesion-induced degeneration with retrograde HRP labelling. Fibers and terminations immunolabelled with PHA-L from ipsilateral SI were distributed in SII in a typical patchy pattern and were mostly concentrated in supragranular layers. Labelled fibers formed a very dense plexus in layer III and ramified densely also in layers I and II. Labelled axon terminals were both en passant and single-stalked boutons. Counts of 8,303 PHA-L-labelled terminals of either type showed that 82.40% were in supragranular layers. The highest concentration was in layer III (43.99%), followed by layers II (30.32%) and I (8.09%). The remaining terminals were distributed among layers IV (6.96%), V (4.93%), and VI (5.68%). The same region of SII containing anterogradely labelled axons and terminals also contained numerous neurons retrogradely labelled with HRP from contralateral SII. Callosal projection neurons were pyramidal, dwelt mainly in layer III, and were distributed tangentially in periodic patches. Patches of anterograde and retrograde labelling either interdigitated or overlapped both areally and laminarly. In the zones of overlap, numerous PHA-L-labelled axon terminals were seen in close apposition to HRP-labelled pyramidal cell dendrites. Combined HRP-electron microscopic degeneration experiments showed that in SII axon terminals from ipsilateral SI form asymmetric synapses with HRP-labelled dendrites and dendritic spines pertaining to callosal projection neurons. These results are discussed in relation to the layering and function of the SI to SII projection, and to the evidence that SII neurons projecting to the homotopic area of the contralateral hemisphere have direct access to the sensory information transmitted from ipsilateral SI.

Experiments were carried out on cats to ascertain whether corticocortical neurones of somatosensory areas I (SI) and II (SII) could be labelled by retrograde axonal transport of D-[3H]aspartate (D-[3H]Asp). This tritiated enantiomer of the amino acid aspartate is (1) taken up selectively by axon terminals of neurones releasing aspartate and/or glutamate as excitatory neurotransmitter, (2) retrogradely transported and accumulated in perikarya, (3) not metabolized, and (4) visualized by autoradiography. A solution of D-[3H]Asp was injected in eight cats in the trunk and forelimb zones of SI (two cats) or in the forelimb zone of SII (six cats). In order to compare the labelling patterns obtained with D-[3H]Asp with those resulting after injection of a nonselective neuronal tracer, horseradish peroxidase (HRP) was delivered mixed with the radioactive tracer in seven of the eight cats. Furthermore, six additional animals received HRP injections in SI (three cats; trunk and forelimb zones) or SII (three cats; forelimb zone). D-[3H]Asp retrograde labelling of perikarya was absent from the ipsilateral thalamus of all cats injected with the radioactive tracer but a dense terminal plexus of anterogradely labelled corticothalamic fibres from SI and SII was observed, overlapping the distribution area of thalamocortical neurones retrogradely labelled with HRP from the same areas. D-[3H]Asp-labelled neurones were present in ipsilateral SII (SII-SI association neurones) in cats injected in SI. In these animals a bundle of radioactive fibres was observed in the rostral portion of the corpus callosum entering the contralateral hemisphere. There, neurones retrogradely labelled with silver grains were present in SI (SI-SI callosal neurones). Association and callosal neurones labelled from SI showed a topographical distribution similar to that of neurones retrogradely labelled with HRP. The laminar patterns of corticocortical neurones labelled with D-[3H]Asp or with HRP were also similar, with one exception. In the inner half of layer II, SII-SI association neurones and SI-SI callosal neurones labelled with the radioactive marker were much less numerous than those labelled with HRP. In cats injected in SII, D-[3H]Asp retrogradely labelled cells were present in ipsilateral SI (SI-SII association neurones). Their topographical and laminar distribution overlapped that of neurones labelled with HRP but, as in cats injected in SI, association neurones labelled with silver grains were unusually rare in the inner layer III.(ABSTRACT TRUNCATED AT 400 WORDS)