Nitric Oxide Synthase-containing Neurons Research Articles

Localization of Nitric Oxide Synthase-containing Neurons in the Bat Visual Cortex and Co-localization with Calcium-binding Proteins.

Microchiroptera (microbats) is a suborder of bats thought to have degenerated vision. However, many recent studies have shown that they have visual ability. In this study, we labeled neuronal nitric oxide synthase (nNOS)—the synthesizing enzyme of the gaseous non-synaptic neurotransmitter nitric oxide—and co-localized it with calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the visual cortex of the greater horseshoe bat (Rhinolophus ferrumequinum, a species of microbats). nNOS-immunoreactive (IR) neurons were found in all layers of the visual cortex. Intensely labeled neurons were most common in layer IV, and weakly labeled neurons were most common in layer VI. Majority of the nNOS-IR neurons were round- or oval-type neurons; no pyramidal-type neurons were found. None of these neurons co-localized with CB, CR, or PV. However, the synthesis of nitric oxide in the bat visual cortex by nNOS does not depend on CB, CR, or PV.

Sexual experience influences mating-induced activity in nitric oxide synthase-containing neurons in the medial preoptic area

Nitric oxide (NO) acts in the medial preoptic area (mPOA) of the hypothalamus to facilitate the expression of male sexual behavior and has also been widely implicated in mechanisms of experience, learning, and memory. Using immunohistochemistry for Fos, as a marker for neural activity, and nitric oxide synthase (NOS), the enzyme that catalyzes the production of nitric oxide (NO), we examined whether sexual activity and sexual experience influence Fos co-expression in NOS-containing neurons in the mPOA of male rats. Consistent with previous findings, results indicate that mating increased activity in the mPOA, and that sexual experience facilitated the expression of sexual behaviors, together with increased mating-induced Fos and NOS in the mPOA. Results also indicate that mating increased co-expression of Fos in NOS-containing neurons, and that this increase was highest in animals undergoing their first sexual encounter, indicating that initial sexual experience increases NO production in the mPOA of male rats.

Developmental exposure to chlorpyrifos and diazinon differentially affect passive avoidance performance and nitric oxide synthase-containing neurons in the basolateral complex of the amygdala

Chronic exposure to low doses of organophosphates during brain development can induce persistent neurochemical and behavioral effects. This study sought to determine the long-lasting effects of developmental exposure to chlorpyrifos (CPF) and diazinon (DZN) on passive avoidance (PA) performance and neuronal nitric oxide synthase (nNOS)-containing neurons in the subnuclei within basolateral complex of amygdala (BLC). Developing rats were exposed to daily dose (1mg/kg) of CPF or DZN during gestational days 15–18 and postnatal days (PND) 1–4. PA performance was assessed in young adulthood (PND 60). Brain sections were also processed by NADPH-diaphorase (NADPH-d) and nNOS immunohistochemistry. Gestational exposure to CPF increased NADPH-d+/nNOS-immunoreactive (IR) neurons within the basolateral nucleus (BL) and medial paracapsular intercalated cluster, which was along with PA retention impairment in both male and female rats. Prenatal exposure to DZN did not significantly change the number of NADPH-d+/nNOS-IR neurons in the BLC while impaired PA retention in females. Postnatal exposure to CPF decreased NADPH-d+/NOS-IR neurons in the BL without affecting PA performance. Exposure to DZN during early postnatal period impaired PA retention in both sexes, albeit to a lesser extent in females, and was along with a considerable sex independent reduction of NADPH-d+/NOS-IR neurons in all BLC subnuclei. Our data suggest that developmental exposure to apparently subtoxic dose of CPF and DZN elicit long-lasting impairment in PA retention that are associated, but not necessarily correlated with effects on NADPH-d+/NOS-IR neurons in BLC of the amygdala.

Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs

Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum. Inhibitory neuromuscular transmission was investigated using in vitro ileal longitudinal muscle myenteric plexus (LMMP) preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 weeks postnatal) guinea pigs. Amperometric measurements of nicotine-induced nitric oxide (NO) release (measured as an oxidation current) from myenteric ganglia revealed larger currents in neonatal (379 +/- 24 pA) vs adult (119 +/- 39 pA, P < 0.05) tissues. Nicotine-induced oxidation currents were blocked by the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (NLA, 100 micromol L(-1)). Nicotine-induced, NLA-sensitive oxidation currents could be detected in the tertiary plexus of neonatal but not adult tissues. Immunohistochemistry demonstrated stronger NOS immunoreactivity in neonatal compared with adult myenteric ganglia. Western blot studies revealed higher levels of NOS in neonatal compared with adult LMMP. Cell counts revealed that the total number of myenteric neurons in the small intestine was greater in adults than in neonatal guinea pigs, however, the ratio of NOS : Calbindin neurons was significantly higher in neonatal compared with adult tissues. Nitric oxide signaling to the longitudinal muscle is stronger in neonatal compared with adult guinea pig ileum. Nitric oxide synthase-containing neurons are diluted postnatally by cholinergic and other, as yet unidentified neuronal subtypes.

Choline acetyltransferase-and nitric oxide synthase-containing neurons of the major pelvic ganglion projecting to the external urethral sphincter in rats

Objective To observe the choline acetyltransferase (ChAT)-and nitric oxide synthase ( NOS) -containing neurons of the major pelvic ganglion ( MPG) projecting to the external urethral sphincter (EUS). Methods Twenty-four Sprague-Dawley rats were divided into two groups: group A (n = 12) and group B (n = 12). In group A, the pelvic ganglia neurons innervating the EUS were labeled by retrograde axonal tracing methods with the use of horseradish peroxidase (HRP) injected into the EUS and identified with TMB-HRP reaction. Then immunohistochemical (ChAT) and histochemical methods ( NADPH-diaphorase activity and nitric oxide synthase immunoreactivity) were used to identify the neurotransmitters of HRP-labeled neurons projecting to the EUS. In group B, Malt agglutinator binding horseradish peroxidase (WGA-HRP) was injected into the MPG for antegrade tracing. Results In group A, HRP-labeled neurons with spherical or oval shape mainly distributed in the MPG. The HRP-labeled neurons were positve for ChAT (155/358, 43.3%) and NOS (49/339, 14.5%), respectively. In group B, HRP positive axon terminals were observed in the EUS after WGA-HRP was injectec into MPG. Conclusion ChAT and NOS-containing neurons of the major pelvic ganglion project to the ELS. Acetylcholine and nitric oxide may be as neurotransmitters or modulators to participate the procedure of urinary storage and voiding. Key words: Pelvic ganglion; External urethral sphincter; Choline acetyltransferase; Nitric oxide svnthase

Role of nitric oxide synthase-containing hypothalamic paraventricular-spinal neurons in the CRF-induced activation of the central sympatho-adrenal outflow in rats

Role of nitric oxide synthase-containing hypothalamic paraventricular-spinal neurons in the CRF-induced activation of the central sympatho-adrenal outflow in rats

Heterogeneity of nitric oxide synthase-containing neurons in the mouse main olfactory bulb

The distribution and structural features of nitric acid synthase (NOS) containing intrinsic neurons were studied in the mouse main olfactory bulb (MOB). NOS positive neurons were heterogeneous, including some subpopulations of periglomerular cells, granule cells, interneurons in the external plexiform layer, superficial and deep short-axon cells and stellate cells. NOS positive periglomerular cells were frequently calretinin immunoreactive and, although rarely, calbindin positive. Importantly, some middle and external tufted cells were also confirmed to be NOS positive, some of which were also cholecystokinin (CCK) positive. Retrograde tracer experiments showed that some NOS positive tufted cells, which were also CCK positive, constitute the intrabulbar association system and the projection system to the olfactory tubercle. In addition, another particular subpopulation of NOS positive neurons with no or little CCK immunoreactivity appeared to project to areas covering the dorsal endopiriform nucleus, claustrum and insular cortex. Furthermore, diverse types of neurons other than mitral/tufted cells were also suggested to be projection neurons of the MOB. The present study revealed the diversity of NOS positive neurons in the mouse MOB and further revealed that they were different from those reported previously in the rat MOB in structural and chemical properties.

Nitric oxide synthase-containing neurons in the amygdaloid nuclear complex of the rat

The nitric oxide-producing neurons in the rat amygdala (Am) were studied, using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. Almost all nuclei of the Am contained NADPHd-positive neurons and fibers, but the somatodendritic morphology and the intensity of staining of different subpopulations varied. The strongly stained neurons displayed labeling of the perikaryon and the dendritic tree with Golgi impregnation-like quality, whilst the dendrites of the lightly stained neurons were less successfully followed. Many strongly positive neurons were located in the external capsule and within the intraamygdaloid fiber bundles. A large number of small, strongly stained cells was present in the amygdalostriatal transition area. In the Am proper, a condensation of deeply stained cells occurred in the lateral amygdaloid nucleus. In the basolateral nucleus, the strongly NADPHd-positive neurons were few, and were located mainly along the lateral border of the nucleus. These cells clearly differed from the large, pyramidal, and efferent cells. The basomedial nucleus contained numerous positive cells but most of them were only lightly labeled. A moderate number of strongly stained neurons appeared in the medial division of the central nucleus, and a larger accumulation of strongly positive cells was present in the lateral and the capsular divisions. The medial amygdaloid nucleus contained numerous moderately stained neurons and displayed the strongest diffuse neuropil staining in Am. In the nucleus of the lateral olfactory tract, the first layer contained only NADPHd-stained axons, in the second layer, there were numerous moderately stained cells, and in the third layer, a few but deeply stained neurons. From the cortical nuclei, the most appreciable number of stained neurons was seen in the anterior cortical nucleus. The anterior amygdaloid area contained numerous NADPHd-positive neurons; in its dorsal part the majority of cells were only moderately stained, whereas in the ventral part the neurons were very strongly stained. The intercalated amygdaloid nucleus lacked NADPHd-positive neurons but an appreciable plexus of fine, tortuous axons was present. In the intra-amygdaloid part of the bed nucleus of the stria terminalis (st) some lightly stained cells were seen but along the entire course of st strongly stained neurons were observed. Some Am nuclei, and especially the central lateral nucleus and the intercalated nucleus, display considerable species differences when compared with the primate Am. The age-related changes of the nitrergic Am neurons, as well as their involvement in neurodegenerative diseases is discussed.

Neuropeptide Y Protects against Methamphetamine-Induced Neuronal Apoptosis in the Mouse Striatum

Methamphetamine (METH) is an illicit drug that causes neuronal apoptosis in the mouse striatum, in a manner similar to the neuronal loss observed in neurodegenerative diseases. In the present study, injections of METH to mice were found to cause the death of enkephalin-positive projection neurons but not the death of neuropeptide Y (NPY)/nitric oxide synthase-positive striatal interneurons. In addition, these METH injections were associated with increased expression of neuropeptide Y mRNA and changes in the expression of the NPY receptors Y1 and Y2. Administration of NPY in the cerebral ventricles blocked METH-induced apoptosis, an effect that was mediated mainly by stimulation of NPY Y2 receptors and, to a lesser extent, of NPY Y1 receptors. Finally, we also found that neuropeptide Y knock-out mice were more sensitive than wild-type mice to METH-induced neuronal apoptosis of both enkephalin- and nitric oxide synthase-containing neurons, suggesting that NPY plays a general neuroprotective role within the striatum. Together, our results demonstrate that neuropeptide Y belongs to the class of factors that maintain neuronal integrity during cellular stresses. Given the similarity between the cell death patterns induced by METH and by disorders such as Huntington's disease, our results suggest that NPY analogs might be useful therapeutic agents against some neurodegenerative processes.

Immunocytochemical Localization of Nitric Oxide Synthase-containing Neurons in Mouse and Rabbit Visual Cortex and Co-Localization with Calcium-binding Proteins

Immunocytochemical Localization of Nitric Oxide Synthase-containing Neurons in Mouse and Rabbit Visual Cortex and Co-Localization with Calcium-binding Proteins

Fetal alcohol syndrome and pyloric stenosis: alcohol induced or an association?

We report a newborn with fetal alcohol syndrome with severe feeding intolerance and failure to thrive due to pyloric stenosis. This illustrates the importance of early recognition of pyloric stenosis in fetal alcohol syndrome to improve nutrition and growth. We speculate that pyloric stenosis in neonates results from the absence or immaturity of intrinsic nitric oxide synthase-containing neurons in the pyloric muscle in children of alcohol-addicted mothers.

Detection of nitric oxide synthase (NOS) immunoreactive neurons in the human septal area: a matter of method?

There is a remarkable discrepancy between biochemical and cell morphological findings with regard to the presence of NADPH diaphorase/neuronal nitric oxide synthase (NOS) in the primate septal area. Whereas considerable concentrations of neuronal nitric oxide synthase and high enzyme activities have been measured in postmortem human septal nuclei, histochemical studies were either unable to detect any nitric oxide synthase immunoreactivity in primate septal neurons, or found only a very few nitrergic neurons in this region. This study aimed to investigate the possible presence of nitrergic neurons in human the septal region in greater detail. After having studied a total of 16 postmortem human brains we conclude that the immunohistochemical demonstration of nitric oxide synthase in human septal neurons is largely dependent on the mode of tissue handling: in brain specimens which were fixed en-bloc with paraffin and embedded in paraplast, nitric oxide synthase immunoreactivity is barely detectable, whereas a satisfying immunostaining is obtained on free-floating frozen sections after an immersion–fixation with 4% paraformaldehyde and 0.5% glutaraldehyde, followed by sucrose protection of the specimens. We show herein that there are indeed nitric oxide synthase-containing neurons in the human septum, thus supporting results from previous biochemical studies.

Premotor nitric oxide synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog

In this study we investigate the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway. As the first step, nitric oxide synthase immunohistochemistry was used to distinguish nitric oxide synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10–11, axotomy-induced retrograde changes consisting in a strong upregulation of nitric oxide synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of nitric oxide synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for nitric oxide synthase immunocytochemistry revealed the presence of Fluorogold-labeled and nitric oxide synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and nitric oxide synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII–IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

Two phases of nitrergic neuropathy in streptozotocin-induced diabetic rats.

The distinction between metabolic and structural changes occurring in autonomic neurons during diabetes has not been fully clarified. Here we demonstrate that nitric oxide synthase-containing (nitrergic) neurons innervating the penis and gastric pylorus of streptozotocin-induced diabetic rats undergo a selective degenerative process in two phases. In the first phase, nitrergic nerve fibers lose some of their neuronal nitric oxide synthase content and function. In the second phase, nitrergic degeneration takes place in the cell bodies in the ganglia, leading to complete loss of nitrergic function. The changes in the first phase are reversible with insulin replacement; however, the neurodegeneration in the second phase is irreversible. Neurodegeneration is due to apoptotic cell death in the ganglia, which is selective for the nitrergic neurones.

Distribution of carbon monoxide–producing neurons in human colon and in Hirschsprung's disease patients

Hirschsprung's disease (HSCR) is characterized by the absence of ganglion cells and impaired relaxation of the gut. Nitric oxide (NO) and, more recently, carbon monoxide (CO) have been identified as inhibitory neurotransmitters causing relaxation. A deficiency in NO has been reported in aganglionic gut; we hypothesized that CO could also be involved in impaired gut motility in HSCR. The aim of the study was to determine the distribution of CO-and NO-producing enzymes in the normal and aganglionic gut. We performed laser capture microdissection, reverse transcription-polymerase chain reaction, and immunohistochemistry on colon biopsies of normal controls (n = 9) and patients with HSCR (n = 10). The mRNA expression of heme oxygenase-2 (HO-2), immunoreactivities of HO-2 and NO synthase, was determined and compared. Results show a high level of expression of HO-2 mRNA localized in the myenteric plexus. Expression of HO-2 mRNA was also detected in the mucosa, submucosa, and muscular layer. Down-regulation of HO-2 mRNA expression was detected in the aganglionic colon. Immunoreactivities of HO-2 and NO synthase were localized mainly to the ganglion plexus and to nerve fibers within the muscle in the control colons and normoganglionic colons. HO-2–containing neurons were more abundant than NO synthase-containing neurons in the myenteric plexus. Nearly all of the NO synthase-containing neurons also contained HO-2. HO-2 and NO synthase were selectively absent in the myenteric and submucosal regions and in the muscle of the aganglionic colon. Our findings suggest involvement of both CO and NO in the pathophysiology of HSCR. HUM PATHOL 33:1030-1036. Copyright 2002, Elsevier Science (USA). All rights reserved.

The Case for the Bulbospinal Respiratory Nitric Oxide Synthase-Immunoreactive Pathway in the Dog

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3–C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 μm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2–C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide attenuate glutamate-induced nNOS activation and cytotoxicity.

Both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in the central and peripheral nervous systems. Attention has been focused on these neuropeptides because among their numerous biological activities, they have been confirmed to show neuroprotective effects against ischemia and glutamate-induced cytotoxicity. It is well established that glutamate has excitatory effects on neuronal cells, and that excessive glutamate shows potent neurotoxicity, especially in neuronal nitric oxide synthase-containing neurons. Glutamate stimulates the production of nitric oxide (NO) in neurons, and the NO generated is tightly associated with the delayed death of neurons. We examined the effects of these neuropeptides on the glutamate-induced neural actions using PC12 cells, and we confirmed the important activities of PACAP/VIP on the production of NO as well as the delayed cell death stimulated by glutamate.

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

GABA neurons provide a rich input to microvessels but not nitric oxide neurons in the rat cerebral cortex: A means for direct regulation of local cerebral blood flow

Basal forebrain neurons project to microvessels and the somata of nitric oxide (NO) synthase-containing neurons in the cerebral cortex, and their stimulation results in increases in cortical perfusion. gamma-Aminobutyric acid (GABA) is the second major neurotransmitter synthesized by these neurons and it has also been reported to modify cerebromicrovascular tone. We thus investigated by light and electron microscopy the association of GABA neurons (labeled for glutamic acid decarboxylase [GAD]) with cortical microvessels and/or NO neurons (identified by nicotinamide adenine dinucleotide [NADPH-D] histochemistry) within the frontoparietal and perirhinal cerebral cortex in the rat. On thick and semithin sections, a high density of GAD puncta was observed, several surrounded intracortical blood vessels and neuronal perikarya. In contrast, NADPH-D cell somata and proximal dendrites were only occasionally contacted by GAD nerve terminals. Perivascular and perisomatic GAD appositions were identified at the ultrastructural level as large (0.44-0.50 microm(2)) neuronal varicosities located in the immediate vicinity of, or being directly apposed to, vessels or unstained neuronal cell bodies. In both cortical areas, perivascular GAD terminals were located at about 1 microm from the vessels and were seen to frequently establish junctional contacts (synaptic frequency of 25-40% in single thin sections) with adjacent neuronal but not vascular elements. Ibotenic or quisqualic acid lesion of the substantia innominata did not significantly affect the density of cortical and perivascular GAD terminals, suggesting that they mostly originated locally in the cortex. These results suggest that GABA terminals can interact directly with the microvascular bed and that the somata and proximal dendrites of NO neurons are not a major target for cortical GABA neurotransmission. However, based on the colocalization of GABA and NADPH-D in a subset of cortical neurons, we suggest that these interneurons could be implicated in the cortical vascular response elicited by stimulation of basal forebrain neurons.