Distribution of neuronal cells which contain dopamine ß-hydroxylase, tyrosine hydroxylase, neuropeptide Y and vasoactive intestinal polypeptide in the human internal carotid nerve.

The human internal carotid nerve (ICN) occasionally has a swelling beneath the external opening of the carotid canal. In this study, the presence and distribution of neuronal cells were investigated in the bilateral ICNs of nine human cadavers. Among 44.4% of the cadavers, swellings were detected in the ICN. Their diameters ranged from 1.7 to 3.6 mm (average ± SD = 2.6 ± 0.7 mm). Thirty-eight percent of these swellings were large (diameter > 3 mm) and showed an oval shape. The large swelling contained many neuronal cells. However, the ICNs with or without a swelling <3 mm diameter were mostly free from neuronal cells (93.3%). Only in one human cadaver, the right ICN without a swelling had a small number of neuronal cells. By the present immunohistochemical method, ICN neurons contained catecholamine-synthesizing enzymes and neuropeptides. Dopamine-beta hydroxylase- and tyrosine hydroxylase-immunoreactivity were mostly expressed by ICN neurons. More than half of them also contained neuropeptide Y-immunoreactivity. However, vasoactive intestinal polypeptide-immunoreactive ICN neurons were relatively infrequent. Substance P- and calcitonin gene-related peptide-immunoreactive ICN neurons could not be detected. By the cell size analysis, neuropeptide Y-immunoreactive neurons were significantly smaller than neuropeptide Y-immunonegative neurons in the ICN. The present study suggests that ICN neurons have a sympathetic function in the human.

Determination of Dopamine-ß-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection.

Dopamine-ß-hydroxylase (DBH, EC 1.14.17.1) is an enzyme with implications in various neuropsychiatric and cardiovascular diseases and is a known drug target. There is a dearth of cost effective and fast method for estimation of activity of this enzyme. A sensitive UHPLC based method for the estimation of DBH activity in human sera samples based on separation of substrate tyramine from the product octopamine in 3 min is described here. In this newly developed protocol, a Solid Phase Extraction (SPE) sample purification step prior to LC separation, selectively removes interferences from the reaction cocktail with almost no additional burden on analyte recovery. The response was found to be linear with an r2 = 0.999. The coefficient of variation for assay precision was < 10% and recovery > 90%. As a proof of concept, DBH activity in sera from healthy human volunteers (n = 60) and schizophrenia subjects (n = 60) were successfully determined using this method. There was a significant decrease in sera DBH activity in subjects affected by schizophrenia (p < 0.05) as compared to healthy volunteers. This novel assay employing SPE to separate octopamine and tyramine from the cocktail matrix may have implications for categorising subjects into various risk groups for Schizophrenia, Parkinson's disease as well as in high throughput screening of inhibitors.

Catecholamine-Synthesizing Enzymes in Pheochromocytoma and Extraadrenal Paraganglioma.

In chromaffin cells, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine ß-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are mainly involved in catecholamine synthesis. In this study, we evaluated the association between the status of catecholamine-synthesizing enzymes and histopathological features of pheochromocytoma and extraadrenal paraganglioma with special emphasis upon their postoperative clinical behavior. Immunohistochemical evaluation of TH, DBH, AADC, PNMT, Ki 67, and S-100 was performed in 29 pheochromocytoma and 10 extraadrenal paraganglioma and one lymph node harboring metastatic pheochromocytoma. Among these cases, metastasis was subsequently developed in three cases. Urinary normetanephrine (U-NM) levels were significantly higher in clinical metastatic cases than non-metastatic ones. Ki 67 labeling index was significantly higher in both clinical metastatic cases and the Adrenal Gland Scaled Score (PASS) score of ≧ 4 cases than PASS < 4 cases, although this score was originally used in pheochromocytoma. H-score of AADC and DBH were significantly lower in PASS ≧ 4 cases than those with < 4 cases, and in the cases associated with intratumoral necrosis (n = 4), the presence of spindle shaped tumor cells (n = 4), and large nests of cells or diffuse growth (n = 5). Lower status of intratumoral AADC could be related to poor differentiation of tumor cells in both catecholamine production and morphology and could be related to aggressive biological behavior of both pheochromocytoma and extraadrenal paraganglioma.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Role of the area postrema in the hypophagic effects of oleoylethanolamide.

The satiety-promoting action of oleoylethanolamide (OEA) has been associated to the indirect activation of selected brain areas, such as the nucleus of the solitary tract (NST) in the brainstem and the tuberomammillary (TMN) and paraventricular (PVN) nuclei in the hypothalamus, where noradrenergic, histaminergic and oxytocinergic neurons play a necessary role. Visceral ascending fibers were hypothesized to mediate such effects. However, our previous findings demonstrated that the hypophagic action of peripherally administered OEA does not require intact vagal afferents and is associated to a strong activation of the area postrema (AP). Therefore, we hypothesized that OEA may exert its central effects through the direct activation of this circumventricular organ. To test this hypothesis, we subjected rats to the surgical ablation of the AP (APX rats) and evaluated the effects of OEA (10mgkg-1 i.p.) on food intake, Fos expression, hypothalamic oxytocin (OXY) immunoreactivity and on the expression of dopamine beta hydroxylase (DBH) in the brainstem and hypothalamus. We found that the AP lesion completely prevented OEA's behavioral and neurochemical effects in the brainstem and the hypothalamus. Moreover OEA increased DBH expression in AP and NST neurons of SHAM rats while the effect in the NST was absent in APX rats, thus suggesting the possible involvement of noradrenergic AP neurons. These results support the hypothesis of a necessary role of the AP in mediating OEA's central effects that sustain its pro-satiety action.

The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping.

Amylin reduces meal size by activating noradrenergic neurons in the area postrema (AP). Neurons in the AP also mediate the eating-inhibitory effects of salmon calcitonin (sCT), a potent amylin agonist, but the phenotypes of the neurons mediating its effect are unknown. Here we investigated whether sCT activates similar neuronal populations to amylin, and if its anorectic properties also depend on AP function. Male rats underwent AP lesion (APX) or sham surgery. Meal patterns were analysed under ad libitum and post-deprivation conditions. The importance of the AP in mediating the anorectic action of sCT was examined in feeding experiments of dose-response effects of sCT in APX vs. sham rats. The effect of sCT to induce Fos expression was compared between surgery groups, and relative to amylin. The phenotype of Fos-expressing neurons in the brainstem was examined by testing for the co-expression of dopamine beta hydroxylase (DBH) or tryptophan hydroxylase (TPH). By measuring the apposition of vesicular glutamate transporter-2 (VGLUT2)-positive boutons, potential glutamatergic input to amylin- and sCT-activated AP neurons was compared. Similar to amylin, an intact AP was necessary for sCT to reduce eating. Further, co-expression between Fos activation and DBH after amylin or sCT did not differ markedly, while co-localization of Fos and TPH was minor. Approximately 95% of neurons expressing Fos and DBH after amylin or sCT treatment were closely apposed to VGLUT2-positive boutons. Our study suggests that the hindbrain pathways engaged by amylin and sCT share many similarities, including the mediation by AP neurons.

Developmental origins of central norepinephrine neuron diversity.

Central norepinephrine-producing neurons comprise a diverse population of cells differing in anatomical location, connectivity, function and response to disease and environmental insult. The mechanisms that generate this diversity are unknown. Here we elucidate the lineal relationship between molecularly distinct progenitor populations in the developing mouse hindbrain and mature norepinephrine neuron subtype identity. We have identified four genetically separable subpopulations of mature norepinephrine neurons differing in their anatomical location, axon morphology and efferent projection pattern. One of the subpopulations showed an unexpected projection to the prefrontal cortex, challenging the long-held belief that the locus coeruleus is the sole source of norepinephrine projections to the cortex. These findings reveal the embryonic origins of central norepinephrine neurons and provide multiple molecular points of entry for future study of individual norepinephrine circuits in complex behavioral and physiological processes including arousal, attention, mood, memory, appetite and homeostasis.

Age-related changes of dopamine, noradrenaline and adrenaline in adrenal glands of mice.

AIM: Catecholamines, which are physiologically important neurotransmitters and hormones, apparently decrease in the brain and plasma as some species age. Because this observation has engendered controversy, we used mice to investigate whether age-related changes occur in adrenal catecholamine levels and in the expression of catecholamine synthetic enzymes. METHODS: Adrenal glands were collected from male C57BL/6NCr mice at the ages of 6, 12 and 24 months. Catecholamines, such as dopamine (DA), noradrenaline (NA) and adrenaline (AD) from those glands, were measured by using a highly sensitive liquid chromatographic method with peroxyoxalate chemiluminescence reaction detection. Tyrosine hydroxylase (TH), dopa decarboxylase, dopamine beta hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression levels were measured by quantitative real-time polymerase chain reaction. RESULTS: Although DA levels in the adrenals of 24-month-old mice were higher than in 6- and 12-month-old mice, the AD content decreased with age. In such mice, the ratio of DA to NA at 24 months was lower than at 12 months, and the ratio of NA to AD content at 24 months was significantly lower than at 6 months. The mRNA expression ratios in TH, DBH and PNMT in 24-month-old mice were all lower than in 12-month-old mice. CONCLUSIONS: These results strongly suggest that catecholamine synthesis, in general, declines with aging in the adrenal glands of mice and that AD, in particular, undergoes a significant decrease with advancing age.

The accessory posterolateral nerve: an immunohistological analysis.

BACKGROUND: Recent endoscopic dissection studies have redefined the postganglionic pterygopalatine autonomic pathways suggesting that neurovascular rami, termed "accessory posterolateral nerves," project directly through the palatine bone to innervate the posterolateral nasal mucosa rather than traveling with trigeminal arborizations. The goal of this study was to characterize these accessory posterolateral nerves by immunohistochemistry to determine their morphology and composition. METHODS: This is an Institutional Review Board approved study in seven patients in whom the presence of accessory posterolateral nerves were surgically identified exiting the perpendicular plate of the palatine bone and sampled. The presence of neural tissue was confirmed by hematoxylin and eosin and S-100 protein staining. Nerves were then stained with anti-human choline acetyl-transferase (ChAT; 1:100) and anti-human dopamine beta-hydroxylase (DBH; 1:100) followed by a fluorescein isothiocyanate-labeled secondary antibody to test for the presence of peripheral parasympathetic and sympathetic fibers, respectively. Human cadaveric sensory nerves were used as a negative control. RESULTS: All seven samples contained neural elements. Two specimens were also associated with arteries. All nerves were comprised of a single fascicle containing an approximately equal distribution of ChAT(+) and DBH(+) fibers. CONCLUSION: This histological study supports prior descriptions defining a newly recognized neural pathway for innervation of the nasal mucosa. Our data confirm that these accessory posterolateral nerves project directly through the perpendicular plate of the palatine bone and are composed of autonomic fibers. Recognition of this pathway may be exploited in the treatment of sinonasal disease resulting from autonomic dysregulation.

Analysis of the chemical coding of neurons in the intermediate thoracic ganglion of the pig.

The pig has been widely used as a model in cardiovascular research. A unique feature of the porcine extrinsic sympathetic cardiac nerves is that they arise from intermediate ganglia in the thoracic cavity. The localization and pattern of distribution of nerve cell bodies and fibers containing tyrosine hydroxylase (TH), dopamine B-hydroxylase (DBH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET) as well as calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was studied with immunohistochemistry. Almost all the neurons showed immunoreactivity to TH. Immunoreactivity to NPY, VIP, SOM, GAL, MET and PACAP was displayed by nerve cell bodies while nerve fibers exhibited immunoreactivity to all the neuropeptides studied. Therefore, it seems that the chemical coding of neurons and especially nerve fibers in the porcine intermediate ganglion share general similarities (with certain neurochemical variability), with porcine prevertebral ganglia (e.g., celiacomesenteric and caudal mesenteric ganglia).

Chronic individual housing-induced stress decreased expression of catecholamine biosynthetic enzyme genes and proteins in spleen of adult rats.

OBJECTIVE: Social isolation is regarded as one of the most relevant causes of diseases in mammalian species. The activation of the sympathoneural system represents one of the key components of the stress response. The sympathetic nervous system is one of the major pathways involved in immune-neuroendocrine interactions. The aim of the present study was to determine plasma epinephrine and norepinephrine in individually housed rats, as well as to find out whether splenic gene expression of catecholamine synthesizing enzymes and their protein levels are affected by chronic psychosocial stress. METHODS: Tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels were quantified by quantitative real-time RT-PCR. The TH, DBH and PNMT immunoproteins were assayed by Western blot. RESULTS: Chronic social isolation of adult male rats produced a significant increase in plasma catecholamine levels and a decrease in splenic TH mRNA, DBH mRNA and PNMT mRNA. Protein levels of TH, DBH and PNMT were also reduced. CONCLUSION: These results suggest that increased plasma catecholamines and decreased gene expression and protein levels of catecholamine biosynthetic enzymes in the spleen of chronically individually housed animals might reduce catecholamine synthesis, thus leaving the immunocompetent tissues depleted of catecholamines and consequently leading to an impairment of immune response.

Immunohistochemical characterization of neurones in the hypoglossal nucleus of the pig.

In this study, the presence of several neurotransmitters and transmitter synthesizing enzymes was studied in hypoglossal nucleus (HN) of the juvenile (4 months old) female pigs (n = 3). Double-labeling immunofluorescence revealed neurones expressing cholinacetyltranspherase (ChAT), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), and somatostatin (SOM). Nerve fibers within HN were ChAT, CGRP, NOS, SOM, substance P (SP), Leu-5-enkephalin (Leu-5-Enk), ss-dopamine hydroxylase (DssH), neuropeptide Y (NPY) positive. Virtually all the perikarya contained ChAT, whereas CGRP was present in 47% of the neurones. Nerve cell bodies containing NOS or SOM were only occasionally observed. Immunoreactive nerve fibers were found in a close vicinity of the perikarya, often forming baskets around nerve cell bodies. The results obtained were compared with similar data obtained in other species. The presence of immunoreactive structures, origin of the nerve fibers, and functional significance of the findings are discussed.

Copper deficiency in rodents alters dopamine beta-mono-oxygenase activity, mRNA and protein level.

Cu is an essential cofactor for at least twelve mammalian enzymes including dopamine beta-mono-oxygenase (DBM), which converts dopamine (DA) to noradrenaline (NA). Previous studies reported that certain Cu-deficient (Cu-) rat tissues have lower NA and higher DA than Cu-adequate (Cu+) tissues, suggesting that DBM function was impaired. However, in vitro studies suggested that DBM activity is higher in Cu- tissue. Experiments were conducted on adrenal glands (AG), medulla oblongata/pons (MO), vas deferens (VD) and heart (HT) from a single rat experiment to provide data to help clarify this puzzling contradiction. In vitro DBM activity assays showed Cu- samples had significantly higher activity than Cu+ samples in both AG and MO, but not VD. Activity data were confirmed by Western immunoblots. Quantitative real-time PCR demonstrated higher DBM mRNA in Cu- tissues but unaltered levels of several other cuproenzymes and Cu-binding proteins. Previous pharmacological data implied that high DBM was associated with low NA. HPLC analyses confirmed that NA and DA levels in Cu- MO, VD and HT were significantly lower and higher, respectively, than in Cu+ tissues. However, the NA content of AG was not statistically lower. Furthermore there was no correlation between higher DBM mRNA and lower NA in four Cu-tissues. Adequate dietary Cu is essential to support DBM function in vivo but additional studies are needed to determine the mechanism for increased DBM transcription associated with Cu deficiency.

Anatomical evidence for ileal Peyer's patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Ultrastructural evidence for selective noradrenergic innervation of CNS vagal projections to the fundus of the rat.

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

Structure and chemical organization of the accessory olfactory bulb in the goat.

The structure and chemical composition of the accessory olfactory bulb (AOB) were examined in male and female goats. Sections were subjected to either Nissl staining, Klüver-Barrera staining, lectin histochemistry, or immunohistochemistry for nitric oxide synthase (NOS), neuropeptide Y (NPY), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and glutamic acid decarboxylase (GAD). The goat AOB was divided into four layers: the vomeronasal nerve layer (VNL), glomerular layer (GL), mitral/tufted (M/T) cell layer (MTL), and granule cell layer (GRL). Quantitative and morphometric analyses indicated that a single AOB contained 5,000-8,000 putative M/T cells with no sex differences, whereas the AOB was slightly larger in males. Of the 21 lectins examined, 7 specifically bound to the VNL and GL, and 1 bound not only to the VNL, but also to the MTL and GRL. In either of these cases, no heterogeneity of lectin staining was observed in the rostrocaudal direction. NOS-, TH-, DBH-, and GAD-immunoreactivity (ir) were observed in the MTL and GRL, whereas NPY-ir was present only in the GRL. In the GL, periglomerular cells with GAD-ir were found in abundance, and a subset of periglomerular cells containing TH-ir was also found. Double-labeling immunohistochemistry revealed that virtually all periglomerular cells containing TH-ir were colocalized with GAD-ir.

A co-localization study on the ovine pancreas innervation.

The expression of DbetaH and several neuropeptides was investigated in neuronal elements of the ovine pancreas using double immunocytochemical stainings. Immunoreactivities to DbetaH, NPY, VIP and SP were seen to various extents in nerve terminals associated with the acini, islets, ducts, blood vessels, interlobular connective tissue as well as in the neurons of intrapancreatic ganglia. The expression of CGRP was limited to nerve fibers lying in the connective tissue septa, amongst the acini and in close vicinity to the pancreatic blood vessels. Single GRP-positive nerve endings were located around the acini, ducts and in the interlobular connective tissue. With the exception of the ductal system in a co-localization of NPY with DbetaH was frequently found in all regions of the pancreas. Moderately numerous blood vessel-associated VIP-positive nerve fibers as well as the vast majority of VIP-containing intrapancreatic neurons were found to co-express DbetaH. Single SP-immunoreactive (IR) nerve fibers of the exocrine pancreas and interlobular connective tissue as well as SP-positive intrapancreatic neurons additionally showed the presence of DbetaH. The co-localization of VIP and NPY was found in nerve terminals located around the blood vessels and acini, in the connective tissue septa as well as in numerous pancreatic neuronal perikarya. Rare nerve terminals located between the acini and around small blood vessels as well as several neurons of intrapancreatic ganglia were VIP-IR/ SP-IR. Simultaneous expression of SP and CGRP was found in nerve fibers supplying large pancreatic arteries and veins, interlobular connective tissue and, occasionally, around the acini. Throughout the pancreas the population of CGRP-positive nerve endings showed lack of VIP and NPY. In a moderate number of GRP-containing nerve fibers, a co-expression of NPY was noted. Nerve terminals containing both GRP and VIP were detected sporadically, whereas none of the GRP-positive nerve terminals showed expression of SP. We conclude that the presented noradrenergic as well as peptidergic innervation patterns of the ovine pancreas are species-dependent. On the basis of the occurrence of DbetaH, NPY, VIP and SP (alone or in combination) in pancreatic neuronal elements we can suggest that these substances presumably act as regulators of the endocrine and/or exocrine pancreas. Involvement of CGRP and GRP in the ovine pancreas physiology seems to be of minor importance. The co-localization study indicated that the pancreas of the sheep is innervated from several sources including intrinsic as well as extrinsic ganglia.

Possible role of insulin-like growth factor-II C-peptide on catecholamine release and ultrastructural aspects of chromaffin cells in the adrenal gland of the frog.

The present study was undertaken to demonstrate that insulin-like growth factor-II C-peptide (IGF-II C-peptide) affects the function of the adrenal gland of Rana ridibunda (Anura, Amphibia) by stimulating chromaffin cells. Previous studies have shown that insulin-like growth factors affect adrenal gland function in mammals. On the basis of these findings, frogs were injected with IGF-II C-peptide (2.5 microg/0.2 ml), whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 12 and 48 h after injection and fixed, embedded in paraffin wax and Epon, and examined by immunohistochemistry and transmission electron microscopy to investigate whether there were structural changes and activation of chromaffin cells in the frog adrenal gland. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase was shown by the peroxidase-antiperoxidase-3, 3'-diaminobenzidine tetrachloride method. After injection of IGF-II C-peptide, chromaffin cells released serotonin and synthesized dopamine beta-hydroxylase. The most pronounced effect of IGF-II C-peptide on the chromaffin cells was observed at 12h after injection. Our results indicate that there is a possible role of IGF-II C-peptide on chromaffin cell activity enhancing catecholamine release in the adrenal gland of the frog.

Dopamine effects on identified rat vagal motoneurons.

Catecholaminergic neurons of the A2 area play a prominent role in brain stem vagal circuits. It is not clear, however, whether these neurons are noradrenergic or adrenergic, i.e., display tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) immunoreactivity (-IR) or dopaminergic (i.e., TH- but not DbetaH-IR). Our aims were to investigate whether a subpopulation of neurons in the A2 area was dopaminergic and, if so, to investigate the effects of dopamine (DA) on the membrane of gastric-projecting vagal motoneurons. We observed that although the majority of A2 neurons were both TH- and DbetaH-IR, a small percentage of nucleus tractus solitarius neurons were TH-IR only, suggesting that DA itself may play role in these circuits. Whole cell recordings from thin brain stem slices showed that 71% of identified gastric-projecting motoneurons responded to DA (1-300 microM) with either an excitation (28%) or an inhibition (43%) of the membrane; the remaining 29% of the neurons were unresponsive. The DA-induced depolarization was mimicked by SK 38393 and prevented by pretreatment with SCH 23390. Conversely, the DA-induced inhibition was mimicked by bromoergocryptine and prevented by pretreatment with L741626. When tested on the same neuron, the effects of DA and NE were not always similar. In fact, in neurons in which DA induced a membrane depolarization, 77% were inhibited by NE, whereas 75% of neurons unresponsive to DA were inhibited by NE. Our data suggest that DA modulates the membrane properties of gastric-projecting motoneurons via D1- and D2-like receptors, and DA may play different roles than norepinephrine in brain stem vagal circuits.