Differential distribution of nicotinamide adenine dinucleotide phosphate-diaphorase and neural nitric oxide synthase in the rat choroid plexus. A histochemical and immunocytochemical study

This study used NADPH diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase immunocytochemistry to examine the localization of nitric oxide synthase in the choroid plexus of the lateral ventricles and the fourth ventricle of rat brain. That the NADPHd reaction product in choroid plexus was specific to nitric oxide synthase was evaluated: (i) by comparison to immunocytochemical labelling for nitric oxide synthase; and (ii) by comparing NADPHd histochemical staining in choroid plexus and brain (rich in nitric oxide synthase-positive and NADPHd-positive neurons) in the presence or absence of iodonium diphenyl or dichlorophenolindophenol, two potent albeit non-selective inhibitors of nitric oxide synthase activity. In brain, NADPHd histochemistry homogeneously stained neuronal cell bodies, axons and dendrites, while it produced particulate cytoplasmic staining of all epithelial cells in the choroid plexuses of the lateral and fourth ventricles. Within the choroid plexus of the lateral ventricles, NADPHd-positive nerve fibres were also observed around blood vessels and coursing among the epithelial cells. The distribution of immunoreactivity for nitric oxide synthase in brain and in nerve fibres in the choroid plexuses of the lateral ventricles resembled the distribution of histochemical labelling for NADPHd. Choroid plexus epithelial cells were, however, devoid of nitric oxide synthase immunoreactivity. Consistent with this, iodonium diphenyl and dichlorophenolindophenol (0.1 mM) inhibited NADPHd histochemical staining in brain neurons and in choroid plexus nerve fibres, but not in choroid plexus epithelial cells. These results demonstrate that the choroid plexus of the lateral ventricles in rat brain is innervated by nitric oxide synthase-positive nerve fibres. These nitric oxide synthase-positive nerve fibres may have an important role in the regulation of cerebrospinal fluid balance. Although choroid plexus epithelial cells contain an enzyme with NADPHd activity, this enzyme is not nitric oxide synthase.     

Projections of the estrogen receptor-immunoreactive ventrolateral hypothalamus to other estrogen receptor-immunoreactive sites in female guinea pig brain

The ventrolateral hypothalamus in female guinea pigs includes an estrogen receptor dense region adjacent to the ventromedial hypothalamus. This region is reciprocally connected with other estrogen receptor-containing areas suggesting that steroid hormone receptor-containing cells may be directly linked. Phaseolus vulgaris leucoagglutinin, an anterograde tract tracer, was specifically placed in this region with the aim of labeling some projections from estrogen receptor-containing neurons. These projections were colocalized immunocytochemically with the distribution of estrogen receptor-containing cells. Dense ventrolateral hypothalamic innervation was observed in some regions also containing a high concentration of estrogen receptor-containing cells. These regions included the medial preoptic area, the bed nucleus of the stria terminalis, the ventrolateral hypothalamus anterior and posterior to the injection site, and the midbrain central gray. A low density of ventrolateral hypothalamic fibers and terminals was observed in two regions rich in estrogen receptors, the amygdala and the arcuate nucleus. In general, ventrolateral hypothalamic fibers and terminals were present in all regions where estrogen receptors were found except the medial thalamus and habenular region. Labeled terminal boutons and perineuronal baskets were found around estrogen receptor-containing cells in most regions which contained estrogen receptor-containing cells. These close appositions were suggestive of synaptic contacts, suggesting that the ventrolateral hypothalamus may influence steroid-dependent behaviors via the modulation of estrogen receptor-containing cells. Furthermore, ventrolateral hypothalamic projections may include direct connections with estrogen receptor-containing cells, suggesting the presence of a network of interconnected estradiol-sensitive neurons involved in the regulation of estradiol-dependent functions.     

Nitric oxide-producing neurons remain intact after small bowel transplantation

This study documents the fate of nitric oxide neurons following small bowel transplantation. Heterotopic, syngeneic small bowel transplants were performed in five male Lewis rats. The grafts were harvested 7, 36, 55, 60, and 65 days postoperatively, together with the native bowel for comparison. NADPH diaphorase staining of cryostat sections was used to identify the nitric oxide-producing neurons. NADPH diaphorase activity was concentrated in the myenteric plexus, deep muscular, and submucous plexus. No differences were detected between the transplanted and native bowel at any time after transplantation. This suggests that nitric-oxide-producing neurons are well preserved after transplantation and that posttransplant dysmotility is probably not related to interrupted nitric oxide innervation.     

Angiotensin II interacts with nitric oxide-cyclic GMP pathway in the central control of drinking behaviour: mapping with c-fos and NADPH-diaphorase

Recognition of the role of nitric oxide in cell-to-cell communication has changed the concept of traditional neurotransmission. We have shown previously that N-methyl-D-aspartate receptors mediate dipsogenic responses and c-Fos expression induced by intracerebroventricular infusion of angiotensin II. Since these receptors are known to be linked to the nitric oxide-cyclic GMP pathway, the present study explores the contribution of this path to the behavioural and cellular effects of intracerebroventricular angiotensin II by using behavioural testing, NADPH-diaphorase histochemistry and immunocytochemical staining for the immediate-early gene, c-fos. N(G)-nitro-L-arginine methyl ester (125 and 250 microg, intracerebroventricular), an inhibitor of nitric oxide synthase, and Methylene Blue (100 microg), an inhibitor of guanylate cyclase activation, antagonized water intake induced by intracerebroventricular injection of 25 pmol angiotensin II. The effects of N(G)-nitro-L-arginine methyl ester were reversed by co-injection of L-arginine, the substrate for nitric oxide synthase. However, N(G)-nitro-L-arginine methyl ester did not alter the pattern of angiotensin II-induced c-fos expression in the organum vasculosum of the lamina terminalis, median preoptic nucleus, hypothalamic paraventricular nucleus and supraoptic nucleus. Double staining with NADPH-diaphorase histochemistry and c-Fos immunocytochemistry showed that neurons staining for both were localized to the anterior third ventricle. However, only 19-25% of the c-Fos-positive neurons expressed NADPH. There were also substantial numbers of neurons in which angiotensin II induced c-Fos that were NADPH-negative. Extensive co-distribution of NADPH-diaphorase-stained cells and those expressing c-fos in response to intracerebroventricular injection of angiotensin II, especially in the median preoptic nucleus, imply that nitric oxide might participate in the mechanism of angiotensin II-induced drinking behaviour. However, a low rate of co-localization of the two markers to individual cells suggests that angiotensin II stimulated the production of nitric oxide and c-Fos in different populations of neurons. Since our previous results showed that glutamate blockade, but not nitric oxide synthase inhibition, suppressed angiotensin II-induced c-Fos, the experiments reported here further suggest that nitric oxide release is not an essential requirement for the expression of c-fos elicited by angiotensin II. They also provide evidence that the dipsogenic and c-Fos responses to angiotensin II are dissociated at a cellular level.     

Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.     

The nitric oxide-cyclic GMP signalling pathway in rat brain

Nitric oxide is a novel signalling molecule in the brain and a potent activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. To determine if stimulation of cyclic GMP formation is a widespread mechanism of nitric oxide signal transduction, we have compared the distribution of the nitric oxide-generating enzyme (nitric oxide synthase) with that of nitric oxide-stimulated cyclic GMP accumulation, throughout the rat brain. The former was done using NADPH diaphorase histochemistry and the latter by cyclic GMP immunohistochemistry following perfusion of the nitric oxide donor, nitroprusside, in vivo. At a gross level, there was generally a good match when the two were compared in adjacent sections. Although the relative staining intensity varied from area to area, in no grey matter region did we observe cyclic GMP accumulation in the absence of nitric oxide synthase staining. In detail, the locations were complementary rather than identical. In some areas, nitric oxide synthase was found in postsynaptic structures and cyclic GMP accumulation in presynaptic elements and fibres; in others, the locations were reversed. Glial cells and their processes also accumulated cyclic GMP in the cerebellum. The results suggest that soluble guanylate cyclase is a major nitric oxide "receptor" throughout the brain. They also support the hypothesis that nitric oxide generated therein primarily functions as a mediator of cell-cell signaling rather than as a conventional second messenger acting within the cells in which it is produced. The types of communication subserved by nitric oxide appear to be extraordinarily diverse.     

NADPH diaphorase-positive neurons in the intracardiac plexus of human, monkey and canine right atria

Distribution of nitric oxide synthase in intracardiac ganglion cells located in human, monkey and canine right atria was histologically investigated using the reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase method and acetylcholinesterase histochemistry. In the intracardiac ganglion, many large neurons exhibited both positive reactions, whereas some of the NADPH diaphorase-positive small neuronal cells were shown with negative acetylcholinesterase reaction.     

Application of a 3D volume 19F MR imaging protocol for mapping oxygen tension (pO2) in perfluorocarbons at low field

Achalasia is a motility disorder of the esophagus characterized by the loss of inhibitory neurons in the distal esophagus. Although idiopathic in nature, autoimmune mechanisms have been proposed, and we set out to determine the presence of myenteric neuronal antibodies. We prospectively studied 18 patients with well-characterized achalasia (by clinical, x-ray, and manometric evidence), nine with gastroesophageal reflux disease, and analyzed the sera from 22 disease-free controls. Using double-label, indirect immunofluorescence techniques, rat esophageal and intestinal sections were double-labeled with sera (dilutions of 1:50 to 1:400) from the three groups and with neurofilament antibody to localize neurons. Seven of 18 achalasia patients had sera that stained the majority of neurons within plexi in the esophageal and intestinal sections, including both NADPH diaphorase (nitric oxide synthase) -positive and -negative neurons. None of the gastroesophageal reflux patients or the controls showed staining. Neuronal antibodies in achalasia provide an attractive hypothesis to explain this diffuse, possibly immune-based disorder.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.     

Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii

The anatomical relationship between vagal afferents and brain nitric oxide synthase containing terminals in the nucleus tractus solitarii was studied by means of anterograde tracing combined with immunocytochemistry and immuno-electron microscopy. Biotinylated dextran amine was injected into the nodose ganglion with a glass micropipette. Four to eight days following the injection, regions of the nucleus tractus solitarii containing biotinylated dextran amine-labelled vagal afferents and those containing nitric oxide synthase-immunopositive terminals were congruent. Many neurons exhibiting nitric oxide synthase immunoreactivity were found within the biotinylated dextran amine-containing terminal field. However dense labeling of terminals with biotinylated dextran amine precluded determination if the terminals were nitric oxide synthase-immunoreactive. Therefore, we combined degeneration of vagal afferents after removal of one nodose ganglion with nitric oxide synthase immuno-electron microscopy. Axon terminals that possessed characteristic vesicle clusters and were partially or completely engulfed by glial processes were identified as degenerating vagal afferents. Degenerating axon terminals comprised 38% of the total axon terminals in the nucleus tractus solitarii in a sample of sections; and of the degenerating axon terminals, 67% were nitric oxide synthase-immunoreactive. Nitric oxide synthase immunoreactivity was present in 41% of the non-degenerating axon terminals. Prominent staining of dendrites for nitric oxide synthase immunoreactivity indicated that much of the nitric oxide synthase in the nucleus tractus solitarii is not derived from peripheral afferents. Of the total number of dendritic profiles sampled, half were nitric oxide synthase-immunoreactive. Our data support the hypothesis that nitric oxide or nitric oxide donors may be present in primary vagal afferents that terminate in the nucleus tractus solitarii. While this study confirms that vagal afferents contain brain nitric oxide synthase, it demonstrates for the first time that the majority of nitric oxide synthase immunoreactivity in the nucleus tractus solitarii is found in intrinsic structures in the nucleus. In addition, our data show that second or higher order neurons in the nucleus tractus solitarii may be nitroxidergic and receive both nitroxidergic and non-nitroxidergic vagal input.     

Gadolinium chloride removes pulmonary intravascular macrophages and curtails the degree of ovine lentivirus-induced lymphoid interstitial pneumonia

Estrogens are produced by the aromatization of androgens. These steroids exert their actions after binding to their receptors. Past studies have shown that estrogen receptors (ER) and aromatase enzyme (AROM) reside in many of the same brain regions. Few studies, however, have examined the neural co-localization of these important components involved in estrogen-activated behaviors. In the present study we examined the co-localization of ER and AROM immunoreactive (ir) neurons in musk shrew (Suncus murinus) brains. Data were collected from a representative section from three neural regions, the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN). Here we report a sex difference in the number of ER-ir neurons from the analyzed section of the mPOA and BNST. Females have more ER-ir neurons in the mPOA and males have more in the BNST. In the sections we examined, males tended to have more aromatase containing neurons than females. Although there were no significant differences in the numbers of double-labeled cells, the VMN contains the greatest percentage of these cells in both males and females; followed by the mPOA and the BNST. In addition, in the mPOA of both sexes, a distinct nucleus of aromatase containing neurons which was devoid of ER immunoreactivity was noted. Area measurements of the AROM-ir nucleus showed that it was significantly larger in males than in females. Taken together, these data suggest that there is not extensive cellular co-localization of estrogen receptors and aromatase enzyme in the musk shrew brain. However, the presence of other genomic forms of ER (membrane and/or ERbeta) in AROM containing neurons has not been ruled out by this study. Thus, we hypothesize that estrogens produced in brain affect behavior by binding to ER in neurons other than those that contain aromatase enzyme.     

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.     

Sex differences in the activity of gamma-aminobutyric acidergic neurons in the rat hypothalamus

The rate of GABA turnover was determined in nine microdissected brain regions in adult male and female rats. In the medial preoptic nucleus (central aspect) and ventromedial nucleus (ventrolateral aspect) of the hypothalamus, areas involved in the regulation of gonadotropin secretion and sex behavior, GABAergic neuronal activity was about 2-fold greater in males than females. These results demonstrate a striking sexual dimorphism in the activity of specific populations of hypothalamic GABAergic neurons.     

Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus

The adipose tissue-derived hormone leptin regulates body weight homeostasis by decreasing food intake and increasing energy expenditure. The weight-reducing action of leptin is thought to be mediated primarily by signal transduction through the leptin receptor (LR) in the hypothalamus. We have used immunohistochemistry to localize LR-immunoreactive (LR-IR) cells in the rat brain using an antiserum against a portion of the intracellular domain of LR that is common to all LR isoforms. The antiserum recognized the short and long isoforms of LR in transfected hematopoietic BaF3 cells. To examine the chemical nature of target cells for leptin, direct double-labeling immunofluorescence histochemistry was applied. The results show extensive distribution of LR-like immunoreactivity (LR-LI) in the brain with positively stained cells present, e.g., in the choroid plexus, cerebral cortex, hippocampus, thalamus, and hypothalamus. In the hypothalamus, strongly LR-IR neurons were present in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), periventricular nucleus, arcuate nucleus, and lateral hypothalamus. Weaker LR-IR neurons were also demonstrated in the lateral and medial preoptic nuclei, suprachiasmatic nucleus, ventromedial and dorsomedial nuclei, and tuberomammillary nucleus. Confocal laser scanning microscopy showed LR-LI in the periphery of individual cells. In magnocellular neurons of the SON and PVN, LR-LI was demonstrated in vasopressin- and oxytocin-containing neurons. In parvocellular neurons of the PVN, LR-LI was demonstrated in many corticotropin-releasing hormone-containing neurons. LR-IR neurons were mainly seen in the ventromedial aspect of the arcuate nucleus, where LR-LI co-localized with neuropeptide Y. In the ventrolateral part of the arcuate nucleus, LR-LI was present in many large adrenocorticotropic hormone-IR proopiomelanocortin-containing neurons and in a few galanin-, neurotensin-, and growth hormone-releasing hormone-containing neurons. In the dorsomedial arcuate nucleus, few tyrosine hydroxylase (dopamine)-containing neurons were seen to have LR-LI. Melanin-concentrating hormone-containing neurons in the lateral hypothalamus had LR-LI. Based on the immunohistochemical results, possible interactions of leptin with brain mechanisms are discussed.     

Distribution and co-localization of nitric oxide synthase and argininosuccinate synthetase in the cat hypothalamus

Argininosuccinate synthetase (ASS) and nitric oxide synthase (NOS) comprise part of the cyclic metabolic pathway to produce nitric oxide (NO). ASS is one of the arginine synthesis enzymes which synthesizes argininosuccinate from aspartate and citrulline, and NOS forms NO and citrulline from arginine. This study examines the localization of ASS and NOS in the cat hypothalamus using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and immunohistochemistry against ASS and NOS. NADPH-d positive and/or ASS-immunoreactive neurons were localized in the following areas: the anterior hypothalamic area, the anterior hypothalamic nucleus, the supraoptic nucleus, the suprachiasmatic nucleus, the periventricular complex, the paraventricular nucleus, the parvocellular nucleus, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the dorsal hypothalamic area, the posterior hypothalamic area, and the supramammillary nucleus. NOS and ASS double-labeled neurons were found in the anterior hypothalamic area, the supraoptic nucleus, the central part of the paraventricular nucleus of the hypothalamus, the lateral hypothalamic area, ventral part of the parvocellular hypothalamic nucleus, the posterior hypothalamic area, and the supramammillary nucleus. Double-labeled neurons in the hypothalamus comprised 20.7-32.0% of ASS-immunoreactive neurons and 10.2-26.3% of NOS-immunoreactive neurons. The results suggest the existence of the 'NO cycle' in situ and the physiological importance of NO and argininosuccinate in several regions of the cat hypothalamus.     

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.     

Nitric oxide synthase-, N-methyl-D-aspartate receptor-, glutamate- and aspartate-immunoreactive neurons in the mouse arcuate nucleus: effects of neonatal treatment with monosodium glutamate

Glutamate-, aspartate-, N-methyl-D-aspartate receptor (NMDAR1 and 2 subunits)-, and nitric oxide synthase (NOS)-immunoreactive neurons were studied in the arcuate nucleus (AN) of mice treated neonatally with monosodium glutamate (MSG) which is known to cause extensive neuronal loss in this hypothalamic nucleus. It was found that intensely stained glutamate- and aspartate-immunoreactive neurons present in the AN of control mice were completely absent in the MSG-lesioned AN as well as the ventromedial nucleus lateral to the AN. Similarly, NMDAR1-immunoreactive neurons were mostly absent in the MSG-lesioned AN but remained intact in the ventromedial nucleus. There was also a substantial loss of NMDAR2 immunoreactivity within the AN. In contrast, NOS-immunoreactive neurons in the AN survived the neonatal glutamate treatment, although they appeared to be less intensely stained.     

A microfabricated electrostatic haptic display for persons with visual impairments

The distribution of the NADPH diaphorase activity was studied in mouse Leydig cells by means of light and electron microscopy. When observed by the light microscope, most Leydig cells appeared intensely stained; a few cells (about 10%) showed a slightly positive or apparently negative reaction. The inhibitory effects of N(G)-nitro-L-arginine and iodonium diphenyl on frozen sections suggest the colocalisation of NADPH diaphorase reaction with nitric oxide synthase. The ultrastructural study revealed that all the Leydig cells were positively stained for NADPH diaphorase; however, a small number of cells displayed weak enzymatic activity. The reaction product was located in the mitochondria, smooth endoplasmic reticulum and lipidic vacuoles, and the nuclear envelope was also stained. The possible meaning of the NADPH diaphorase activity in the Leydig cells of mice was discussed.     

Enteric neuronal plasticity and a reduced number of interstitial cells of Cajal in hypertrophic rat ileum

BACKGROUND: Partial obstruction of the ileum causes a notable hypertrophy of smooth muscle cells and enteric neurones in the proximally located intestine. AIMS: To study the expression of neuromessengers in the hypertrophic ileum of rat as little is known about neuromessenger plasticity under these conditions. To investigate the presence of interstitial cells of Cajal (ICC) in hypertrophic ileum. METHODS: Ileal hypertrophy was induced by circumferential application of a strip of plastic film for 18-24 days. Immunocytochemistry, in situ hybridisation, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, and ethidium bromide staining were used to investigate the number of enteric neurones expressing neuropeptides and nitric oxide synthase, and the frequency of ICC. RESULTS: In the hypertrophic ileum several neuronal populations showed changes in their expression of neuromessengers. Myenteric neurones expressing vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide, and galanin were notably increased in number. In submucous ganglia the number of VIP immunoreactive neurones decreased while those expressing VIP mRNA increased. NADPH diaphorase positive submucous neurones increased dramatically while the number of neuronal type nitric oxide synthase expressing ones was unchanged. The number of ICC decreased notably in hypertrophic ileum. CONCLUSION: Enteric neurones change their levels of expression of neuromessengers in hypertrophic ileum. ICC are also affected. The changes are presumably part of an adaptive response to the increased work load.