Ondansetron attenuates CCK induced satiety and c-fos labeling in the dorsal medulla.

Serotonin 5-HT(3) antagonists have been suggested for treatment of several disorders involving altered gastrointestinal (GI) function. CCK also has well documented GI actions on both food intake and vago-vagal reflexes. To evaluate potential interactions, the effect of a 5-HT(3) antagonist, ondansetron, on exogenous CCK induced satiety and c-fos activation was determined. Ondansetron reduced both actions of CCK by approximately 50%. The reduction in c-fos was localized to a specific subregion of the dorsal medulla, suggesting that a distinct subpopulation of CCK receptive fibers are modulated by 5-HT(3) ligands. Treatments using 5-HT(3) antagonists also may affect endogenous CCK functions.

Effect of decreasing afferent vagal activity with ondansetron on symptoms of bulimia nervosa: a randomised, double-blind trial.

BACKGROUND: Several lines of evidence have led us to postulate that afferent vagal hyperactivity could be an important factor in the pathophysiology of the eating disorder bulimia nervosa. Ondansetron is a peripherally active antagonist of the serotonin receptor 5-HT3, and is marketed for prevention of vagally-mediated emesis caused by cancer chemotherapeutic agents. We investigated the effects of ondansetron on bulimic behaviours in patients with severe and chronic bulimia nervosa in a randomised, double-blind, placebo-controlled study. METHODS: We enrolled patients with severe bulimia nervosa (at least seven coupled binge/vomit episodes per week). The patients were otherwise healthy, their weight was normal, and they were not receiving medical or psychiatric treatment. During the first week of the study, patients recorded all eating-behaviour events to establish a baseline. In the second week, all patients received placebo, but were told that they were receiving either placebo or active drug. At the end of this single-blind phase, patients were randomly assigned placebo or ondansetron (24 mg daily) for a further 4 weeks. The primary outcome measure was the number of binge/vomit episodes per week. Data were analysed by intention to treat. FINDINGS: 29 patients met the inclusion criteria, of whom 28 completed the baseline study, and 26 completed the single-blind placebo week. 12 patients were assigned placebo, and 14 ondansetron; one patient in the ondansetron group dropped out owing to accidental injury. During the 4th week of double-blind treatment, mean binge/vomit frequencies were 13.2 per week (SD 11.6) in the placebo group, versus 6.5 per week (3.9) in the ondansetron group (estimated difference 6.8 [95% CI 4.0-9.5]; p<0.0001). The ondansetron group also showed significant improvement, compared with the placebo group, in two secondary indicators of disease severity. The amount of time spent engaging in bulimic behaviours was decreased on average by 7.6 h per week in the ondansetron group, compared with 2.3 h in the placebo group (estimated difference 5.1 [0.6-9.7]). Similarly, the number of normal meals and snacks increased on average by 4.3 normal eating episodes without vomiting per week in the ondansetron group, compared with 0.2 in the placebo group (estimated difference 4.1 [1.0-7.2]). INTERPRETATION: The decrease in binge-eating and vomiting under ondansetron treatment was not achieved by compensatory changes in eating behaviour such as by a smaller number of binges of longer duration, or by not eating, or by binge-eating without vomiting. Instead, our findings indicate a normalisation of the physiological mechanism(s) controlling meal termination and satiation. Since meal termination and satiety are mainly vagally mediated functions, since binge-eating and vomiting produce intense stimulation of vagal afferent fibres, and since ondansetron and other 5-HT3 antagonists decrease afferent vagal activity, the symptom improvement may result from a pharmacological correction of abnormal vagal neurotransmission.

Characterization of the extent of pontomesencephalic cholinergic neurons' projections to the thalamus: comparison with projections to midbrain dopaminergic groups.

We sought to determine whether pontomesencephalic cholinergic neurons which we have been shown previously to project to the substantia nigra and ventral tegmental area also contribute to the thalamic activation projection from the pedunculopontine and laterodorsal tegmental nuclei. Retrograde tracing, immunohistochemical localization of choline acetyltransferase and statistical methods were used to determine the full extent of the cholinergic projection from the pedunculopontine and laterodorsal tegmental nuclei to the thalamus. Progressively larger Fluoro-Gold injections in to the thalamus proportionally labeled increasing numbers of pontomesencephalic cholinergic cells both ipsi- and contralaterally in the pedunculopontine and laterodorsal tegmental nuclei. Multiple large thalamic injections left only a small fraction of the ipsilateral pontomesencephalic cholinergic group unlabeled. This small remainder did not correspond to the populations which project to the substantia nigra and ventral tegmental area, thereby indicating that substantia nigra- and ventral tegmental area-projecting cholinergic neurons must also project to the thalamus. We examined whether there existed any set of cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei which did not innervate a thalamic target. The distribution of descending projections of the pedunculopontine and laterodorsal tegmental nuclei demonstrated that the unlabeled remainder cannot correspond to a purely descending group. We also show that substance P-positive cholinergic cells in the laterodorsal tegmental nucleus project to the thalamus. Further studies demonstrated that the small population of cholinergic cells left unlabeled from the thalamus were the smallest sized cholinergic cells, and included two groups of small, light-staining cholinergic cells located in the parabrachial area and central gray, adjacent to the main pedunculopontine and laterodorsal tegmental nuclei cholinergic groups. These small cells, in contrast to thalamic-projecting cholinergic cells, did not stain positively for reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Taken together, these results indicated that all of the reduced nicotinamide adenine dinucleotide phosphate diaphorase-positive/choline acetyltransferase-positive neurons of the pedunculopontine/laterodorsal tegmental nuclei ascend to innervate some portion of the thalamus, in addition to the other targets they innervate. These findings indicate that the diverse physiological and behavioral effects attributed to the activity of pontomesencephalic cholinergic neurons should not be dissociated from their activating effects in the thalamus.

A preliminary report on pain thresholds in bulimia nervosa during a bulimic episode.

Subjects with bulimia nervosa (BN) have been shown to exhibit abnormal satiety responses. Short-term satiety is largely mediated by afferent vagal activity. Activation of afferent vagal fibers has also been found to stimulate a descending pain inhibitory pathway that leads to elevation in somatosensory pain thresholds. Therefore, the study of pain thresholds in BN subjects may lead to a better understanding of afferent vagal function in this disorder. In this preliminary study, pressure pain thresholds were assessed in nine subjects with BN on 3 consecutive days during a binge-eating and vomiting (B/V) episode, during a normal meal, and after an overnight fast. A significant time versus condition effect was found with a significant change in the pain threshold in BN subjects under the B/V condition only. These data are consistent with the hypothesis that vagal afferent activation by a B/V episode also activates the descending pain inhibitory pathway.

Phenomenological and pharmacological study of provoked obsessive/anxiety symptoms in obsessive-compulsive disorder: a preliminary study.

BACKGROUND: Inclusion of obsessive-compulsive disorder (OCD) as an anxiety disorder in DSM-i.v. assumes that anxiety is the primary symptom of OCD; however, persuasive empirical evidence in support of this view has not been presented yet. In the present study we hypothesized that provoked anxiety symptoms respond better to intravenous diazepam than would provoked obsessions. We, therefore, reasoned that anxiety symptoms are secondary symptoms of OCD. METHODS: To test the hypothesis we designed a double-blind, randomized, placebo-controlled crossover study. Patients underwent four experimental conditions in which the sequence of symptom provocation and i.v. injection of (placebo or diazepam) were alternated. Baseline and i.v. injection-induced symptom changes were assessed using visual analogs. RESULTS: Obsessions and anxiety correlated strongly for all four experimental conditions in which the sequence of the symptom provocation and diazepam i.v. injections was alternated. i.v. diazepam injection before and after symptom provocation failed to preferentially modulate anxiety symptoms over obsessions. Unexpectedly, in the group in which i.v. diazepam injection preceded the symptom provocation, reduction of mean obsessions was even more pronounced. CONCLUSIONS: Strong correlations between anxiety and obsessions at baseline, during symptom provocation, and after i.v. diazepam infusion suggest that anxiety and obsessions are tightly coupled phenomena in OCD.

Transplantation of fetal neocortex ameliorates sensorimotor and locomotor deficits following neonatal ischemic-hypoxic brain injury in rats.

Ischemic brain injury in neonates can result in the degeneration of cortical and subcortical areas of brain and is associated with neurologic deficits. One approach to restoring function in conditions of ischemic brain injury is the use of neural transplants to repair damaged connections. This approach has been shown to reestablish neural circuitry and to ameliorate associated motor deficits in models of neonatal sensorimotor cortex damage. In this study, we utilized the Rice et al. rodent model of neonatal ischemic-hypoxic (IH) brain injury to assess whether transplantation of fetal neocortical tissue can promote functional recovery in tests of sensorimotor and locomotor ability throughout development and as adults. We show that animals that received neocortical grafts 3 days following the IH injury performed significantly better as adults on two measures of motor ability, the Rota-Rod treadmill and apomorphine-induced rotations, than did control animals that received sham transplants after the IH injury. Transplants were identifiable in 72% of the animals 10-12 weeks after implantation. Histochemical studies revealed that while the transplanted tissue did not establish normal cortical cytoarchitecture, cells and fibers within the grafts stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), choline acetyl transferase (ChAT), cholecystokinin (CCK), and glial fibrillary acidic protein (GFAP). These results suggest that transplantation of fetal neocortical tissue following IH injury in the neonatal period is associated with amelioration of motor deficits and that the grafted tissue demonstrated a neurochemical phenotype that resembled normal neocortex. This approach warrants continued investigation in light of potential therapeutic uses.

Nerve growth factor-independent reduction in choline acetyltransferase activity in PC12 cells expressing mutant presenilin-1.

Mutations in the presenilin genes (PS-1 and PS-2) are linked to early onset familial Alzheimer's disease (AD), but the mechanisms by which these mutations cause the cognitive impairment characteristic of AD are unknown. Basal forebrain cholinergic neurons are involved in learning and memory processes, and reductions in choline acetyl-transferase (ChAT) activity are a characteristic feature of AD brain. We therefore hypothesized that presenilin mutations suppress expression of the cholinergic phenotype. In rat PC12 cells stably transfected with the human PS-1 gene containing the Leu --> Val mutation at codon 286 (L286V), we observed a drastic reduction (>90%) in basal ChAT activity compared with cells transfected with vector alone. By immunocytochemistry, a similar decrease in ChAT protein levels was found in the mutant transfectants. In cells differentiated with nerve growth factor, ChAT activity was again markedly lower in L286V-expressing cells than in control cells. We also observed reductions in ChAT activity in PC12 cells expressing the wild-type human PS-1 gene but to a lesser extent than in L286V-expressing cells. The viability of cells transfected with either the wild-type or the mutant PS-1 gene was not compromised. Our results suggest that PS-1 mutations may contribute to the cognitive impairment in AD by causing a nontoxic suppression of the cholinergic phenotype.

Glutamate-enriched cholinergic synaptic terminals in the entopeduncular nucleus and subthalamic nucleus of the rat.

Several lines of evidence suggest that the cholinergic neurons of the mesopontine tegmentum contain elevated levels of glutamate and are the source of cholinergic terminals in the subthalamic nucleus and entopeduncular nucleus. The object of this study was to test whether cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, also express relatively high levels of glutamate. To address this, double immunocytochemistry was performed at the electron microscopic level. Perfuse-fixed sections of rat brain were immunolabelled to reveal choline acetyltransferase by the pre-embedding avidin-biotin-peroxidase method. Serial ultrathin sections of cholinergic terminals in both the entoped uncular nucleus and subthalamic nucleus were then subjected to post-embedding immunocytochemistry to reveal glutamate and GABA. Quantification of the immunogold labelling showed that choline acetyltransferase-immunopositive terminals and boutons in both regions were significantly enriched in glutamate immunoreactivity and had significantly lower levels of GABA immunoreactivity in comparison to identified GABAergic terminals. Furthermore, the presumed transmitter pool of glutamate i.e. that associated with synaptic vesicles, was significantly greater in the choline acetyltransferase-positive terminals than identified GABA terminals, albeit significantly lower than in established glutamatergic terminals. In the entopeduncular nucleus, a small proportion of cholinergic terminals displayed high levels of GABA immunoreactivity. Taken together with other immunocytochemical and tracing data, the elevated levels of glutamate in cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, is further evidence adding weight to the suggestion that acetylcholine and glutamate may be co-localized in both the perikarya and terminals of at least a proportion of neurons of the mesopontine tegmentum.

Specificity of fiber innervation by cholinergic neurons transplanted into the retrosplenial cortex of adult rats as revealed by choline acetyltransferase immunocytochemistry.

The retrosplenial cortex (RSC) is a target of the forebrain cholinergic projecting system. It receives extensive cholinergic innervation from the medial septal nucleus and the diagonal band of Broca. These cholinergic afferents travel along the paths of cingulate bundle and fornix. In the present study we investigated the ability of cholinergic fetal septal grafts to reinnervate the deafferented RSC. Four groups of rats were used: (1) normal control rats (NC); (2) rats with bilateral transections of the cingulate bundle (CgX); (3) rats with simultaneous lesions of both the cingulate bundle and the fornix (FX), and (4) rats with intra-retrosplenial fetal septal grafts and lesions in both cingulate bundle and the fornix (RSCsep-TPL). We found that lesions in the cingulate bundle alone produced a modest reduction of cholinergic innervation whereas lesions in both the fornix and cingulate bundle resulted in a complete loss of cholinergic inputs in this area, indicating that both the cingulate bundle and the fornix are involved in mediating cholinergic projections from the septal-diagonal area to the RSC. Transplantation of cholinergic fetal septal neurons into the RSC of animals with simultaneous lesions in both the fornix and cingulate bundle restored the cholinergic innervation pattern to that which is typical of the normal septo-retrosplenial inputs. These results provide the neuroanatomical basis for behavioral studies which have documented graft-mediated recovery of spatial memory function in rats with lesions of the cholinergic septo-retrosplenial pathways.

PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy.

Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.

Effects of immunological sympathectomy on postnatal peptide expression in the rat adrenal medulla.

Administration of monoclonal antibodies against acetylcholinesterase (AChE-mabs) to adult rats leads to a selective degeneration of the acetylcholine esterase-(AChE), choline acetyltransferase-(ChAT) and enkephalin-(ENK) positive preganglionic fibres of the splanchnic nerve innervating the adrenal gland. Here we used this approach of immunological sympathectomy, performed at postnatal day 2 (P2), in an attempt to study the development role of the preganglionic fibres in the adrenal medulla in more detail. Analysis was performed at P16 and revealed that the effect of this treatment varied considerably between animals, as judged by the number of remaining AChE-, ChAT- and ENK-positive fibres. The number and intensity especially of ENK fibres in the adrenal medulla correlated negatively with the number and staining intensity of ENK-immunoreactive chromaffin cells, suggesting a 'dose-response' relationship. Thus, the high early postnatal levels of ENK-like immunoreactivity generally persisted in chromaffin cells of adrenals with a successful immunosympathectomy, i.e. in those adrenals that lacked AChE-, ChAT- and ENK-positive nerves. In contrast, calcitonin gene-related peptide-like immunoreactivity in nerves and chromaffin cells was not affected. Large and strongly AChE-positive intra-adrenal ganglion neurones, recently termed type I ganglion neurones, were present also after AChE-mab treatment and had an apparently normal morphology. These results indicate a role for preganglionic fibres in the developmental regulation of ENK in the chromaffin cells. However, these fibres appear less important for the postnatal development of the type I ganglion neurones.

The cholinergic innervation of the adrenal gland and its relation to enkephalin and nitric oxide synthase.

Using a monoclonal antibody against rat brain choline acetyltransferase (ChAT) the cholinergic innervation of the adult rat adrenal gland was visualized. Almost all ChAT-positive fibres contained nitric oxide synthase (NOS), whereas enkephalin (ENK) was exclusively found in ChAT fibres among adrenaline chromaffin cells. The ChAT/NOS/ENK fibres disappeared after immunological sympathectomy, indicating a preganglionic origin. ChAT was not found in the superficial peptide- and NOS containing fibre plexus in the adrenal cortex or in small or large intra-adrenal ganglion neurones under control conditions. Even after colchicine treatment only one single ChAT-positive small ganglion neurone was found. It is possible, therefore that some small intra-adrenal ganglion neurones, which express NOS- and VIP-like immunoreactivities, are noncholinergic, nonadrenergic neurones.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Distribution of pontomesencephalic cholinergic neurons projecting to substantia nigra differs significantly from those projecting to ventral tegmental area.

Locations of pontomesencephalic cholinergic projection neurons from the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei to midbrain dopaminergic nuclei were mapped. Stereotaxic microinjections of Fluoro-Gold- or rhodamine-labeled microspheres were made either to substantia nigra (SN) or ventral tegmental area (VTA) in rat. Choline acetyltransferase was visualized immunohistochemically. Labeled cells were digitally mapped at multiple levels of the nuclei using an interactive computer/microscope system. SN-projecting neurons were distributed predominantly ipsilaterally in distinct regions of the PPTg: either at its rostral pole or caudally in an area ventromedial to the superior cerebellar peduncle. Few SN-projecting neurons were found in LDTg. VTA-projecting neurons were distributed bilaterally throughout the cholinergic group, primarily in the densest regions of the LDTg and caudal PPTg. Neurons were not strictly segregated into these patterns. Scattered cells belonging to either projection could be found throughout the cholinergic group on either side. Hierarchical log-linear analysis showed these differences in topographic distribution to be statistically significant. Subtraction of cell density images demonstrated well delineated regions of the cholinergic group where the projections were predominately either to SN or VTA. These data indicate a high degree of internal organization within the pontomesencephalic cholinergic group based on the location of efferent projections to SN or VTA. These findings support the concept that this cholinergic group is functionally organized in a manner which selectively innervates motor (SN) and limbic (VTA) dopaminergic nuclei.

Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections.

This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 micron in diameter, 1.6 microns 2 in surface, and 0.12 micron 3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II-III, 11% in IV, 21% in V, 14% in VI), for a I-VI average of 14%. These synaptic junctions were usually single, symmetrical (> 99%), and occupied a small portion of the surface of varicosities (< 3%). A majority were found on dendritic branches (76%), some on spines (24%), and none on cell bodies. On the whole, the ACh junctional varicosities were significantly larger than their nonjunctional counterparts, and both synaptic and nonsynaptic varicosities could be observed on the same fiber. A subsample of randomized single thin sections from these whole varicosities yielded similar values for size and synaptic frequency as the result of a stereological extrapolation. Also analyzed in single sections, the microenvironment of the ChAT-immunostained varicosities appeared markedly different from that of unlabeled varicosity profiles randomly selected from their vicinity, mainly due to a lower incidence of synaptically targeted dendritic spines. Thus, the normal ACh innervation of adult rat parietal cortex is predominantly nonjunctional (> 85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity.

Distinct choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) bipolar neurons project to local blood vessels in the rat cerebral cortex.

Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is not known if the cells of origin are intra- or extracortical nor if perivascular fibers colocalize ACh and VIP. Cortical cholinergic innervation arises primarily from the basal forebrain and to a small extent from intrinsic bipolar ACh neurons thought to be the sole source of cortical VIP. In order to evaluate if intracortical perivascular ACh terminals could be distinguished from those of the basal forebrain by their colocalization with VIP, we performed a double immunofluorescence study and determined the percentage of colocalization of choline acetyltransferase (ChAT) and VIP in cortical neurons, as well as in terminal fields associated with intracortical blood vessels. From a total of 2103 cells examined in all cortical areas, VIP neurons accounted for the largest population (58.3%) followed by ChAT-positive cells (28.2%) with only 13.5% of cells being double-labelled for VIP and ChAT. Of the cortical ChAT-immunostained cells (n = 878), 32.3% colocalized VIP whereas only 18.8% of VIP neurons (n = 1509) also contained ChAT. In various cortical areas, ChAT cell bodies were seen to be contacted by VIP terminals which surrounded closely their cell soma and proximal dendrites. Perivascular fibers studied by double immunofluorescence and confocal microscopy were of three categories including cholinergic, VIPergic with a smaller population of fibers which costained for both ChAT and VIP. These results show that cortical VIP neurons are much more numerous than those containing ChAT, and that a majority of VIP neurons do not colocalize with ChAT. This observation indicates that ACh and VIP are primarily located in distinct neuronal populations and that VIP cannot be used as a marker of intracortical ACh neurons and terminals. Our results further suggest that intracortical blood vessels are primarily under the influence of distinct ChAT and VIP perivascular fibers. Also, the presence of a subset of VIP and ChAT/VIP fibers in association with intracortical blood vessels strongly suggests a role for cortical bipolar neurons in local cerebrovascular regulation. The origin of the perivascular ChAT fibers which do not colocalize VIP, however, remains unknown.

Light and electron microscopic immunocytochemical analysis of the neurovascular relationships of choline acetyltransferase and vasoactive intestinal polypeptide nerve terminals in the rat cerebral cortex.

Acetylcholine or vasoactive intestinal peptide (VIP) nerve terminals closely related to intracortical blood vessels have previously been reported. Recent physiological evidence indicates that these central neuronal systems are involved in the fine control of local cerebral blood flow. In the present study, the intimate associations between choline acetyltransferase (ChAT) and VIP axon terminals and intracortical microvessels were characterized by light (LM) and electron microscopic (EM) immunocytochemistry. In semithin sections, LM analysis of the distribution of ChAT- and VIP-immunostained puncta juxtaposed to small intraparenchymal blood vessels demonstrated that neither type of terminal was enriched or impoverished around microvessels within the cerebral cortex. At the EM level, most ChAT- or VIP-immunolabelled elements located within a 3 microns perimeter around vessel walls were axon terminals. These perivascular terminals were associated primarily with capillaries but also, to a lesser extent, with microarterioles. Even though ChAT and VIP terminals were frequently found in the immediate vicinity (< or = 0.25 microns) of microvessels, they almost never contacted the outer basal lamina, usually abutting onto perivascular astroglial leaflets. There were no membrane specializations at the site of contact between ChAT or VIP terminals and perivascular astroglia. In all cortical areas examined, the average size of VIP-immunolabelled varicosities (0.56 +/- 0.04 microns 2) was significantly larger than that of their ChAT counterparts (0.32 +/- 0.02 microns 2; P < 0.001). Perivascular VIP terminals were more frequently engaged in synaptic contact than those immunostained for ChAT, which rarely exhibited a synaptic junction even in serial thin sections. Neither VIP nor ChAT immunostaining was ever observed in endothelial cells. These results suggest that both acetylcholine and VIP exert their effects on intracortical microvessels through indirect, paracrine mechanisms. The marked difference in synaptic incidence and average size between both types of perivascular terminals indicates that these two vasoactive agents are primarily located in distinct neuronal populations. Further, our results show that the astrocytic glia is the major direct target for both ChAT and VIP perivascular terminals and suggest that neuronal/glial/vascular interactions are a key element in the neurogenic control of the intracortical microcirculation.

Immunohistochemical localization of chicken gonadotropin-releasing hormones I and II (cGnRH I and II) in turkey hen brain.

The distribution of cells and fibers immunoreactive (ir) for either chicken gonadotropin-releasing hormone I (cGnRH I; [Gln8]GnRH) or II ([His5,Trp7,Tyr8]GnRH) was determined in brains of turkey hens to reveal whether these peptides occur in separate neuronal systems. ir-cGnRH I cells were located: along the medial aspect of the ventriculus lateralis, nucleus accumbens, and bed nucleus of the stria terminalis; ventral to the tractus septomesencephalicus and extending medially to the third ventricle, and caudally into the lateral hypothalamic area; and in a diffuse band extending from the nucleus preopticus medialis to the nucleus dorsomedialis anterior thalami. cGnRH I fibers were evident in these areas in addition to the hippocampus, nucleus subhabenularis medialis, nucleus ventromedialis hypothalami, and median eminence. Two groups of ir-cGnRH II cells were observed: a magnocellular group lying between the substantia grisea centralis and the nucleus ruber; and a parvicellular group lying medial to the nucleus of the basal optic root and extending into the lateral hypothalamic area. ir-cGnRH II fibers were prominent in limbic structures (cortex piriformis, lateral to nucleus taeniae, hippocampus); olfactory areas (tuberculum olfactorium, nucleus subhabenularis lateralis, nucleus septalis lateralis); areas that in other avian species have steroid-concentrating cells or receptors (medial edge of lobus parolfactorius, nucleus septalis medialis, nucleus periventricularis magnocellularis, nucleus dorsomedialis posterior thalami); and areas containing ir-GnRH I cells or fibers but not in median eminence. These results suggest that cGnRH I and II occur in separate neuronal systems and that cGnRH II does not directly promote pituitary gonadotropin secretion.

Localization of NADPH diaphorase in neurons of the rostral ventral medulla: possible role of nitric oxide in central autonomic regulation and oxygen chemoreception.

We studied whether neurons containing nitric oxide synthase (NOS) are localized to the rostral ventrolateral medulla (RVM) and, if so, whether they are distinct from the adrenergic neurons of the C1 group. NOS-containing neurons and/or C1 neurons were visualized using NADPH diaphorase histochemistry and phenylethanolamine N-methyltransferase (PNMT) immunohistochemistry, respectively. A column of NADPH diaphorase-positive neurons, extending 2 mm in the rostrocaudal plane, was observed lateral to the inferior olive and medial to the C1 neurons. Double labelling studies showed that NADPH diaphorase-positive neurons were not immunoreactive for PNMT, indicating that the two enzymes were localized in the different cells. Furthermore, only a small fraction of NADPH diaphorase neurons were retrogradely labelled after injections of fluorogold into the thoracic cord. We conclude that the RVM contains a well-defined group of neurons endowed with NOS that are distinct from the adrenergic neurons of the C1 group and have only limited monosynaptic projections to the spinal cord. Since the RVM is involved in the control of arterial pressure and in oxygen-conserving reflexes, the findings raise the possibility that nitric oxide participates in central autonomic regulation and oxygen chemoreception.