Search

Five approaches for the preparation of adrenal chromaffin cell lines have been developed. Initially, continuous chromaffin lines were derived from spontaneous pheochromocytoma tumors of the medulla, either from murine or human sources, such as the rat PC12 cell line and the human KNA and KAT45 cell lines. Over the last few decades, more sophisticated molecular methods have allowed for induced tumorigenesis and targeted oncogenesis in vivo, where isolation of specific populations of mouse cell lines of endocrine origin have resulted in model cells to examine a variety of regulatory pathways in the chromaffin phenotype. As well, conditional immortalization with retroviral infection of chromaffin precursors has provided homogeneous and expandable chromaffin cells for transplant studies in animal models of pain. This same strategy of immortalization with conditionally expressed oncogenes has been expanded recently to create the first disimmortalizable chromaffin cells, with an excisable oncogenic cassette, as might be envisioned for the creation of human chromaffin cell lines. Eventually, as we increase our understanding of regulating the phenotypic fate of chromaffin cells in vitro, stem or progenitor adrenal medullary cell lines will be derived as an alternative source for expansion and clinical use.

Phencyclidine (PCP) is a non-competitive inhibitor of the nicotinic acetylcholine receptor (nAChR) with biphasic characteristics. At low and high micromolar concentrations, PCP inhibits nAChR from fetal mouse muscle, whereas at intermediate concentrations PCP does not inhibit the receptor. The present study was performed to determine whether the high and low concentration effects of PCP on mouse nAChR were due to interactions of this blocker with channel lining amino acids. In order to test this hypothesis, we examined the ability of PCP to inhibit acetylcholine-induced currents from wild-type nAChR and nAChR in which amino acid substitutions were made in the 6′, 8′ and 10′ positions of the M2 transmembrane segments of the receptor. Fetal mouse nAChR from BC3H-1 cells were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp technique. The results of this study reveal that in native fetal muscle receptor, PCP potency is not affected by membrane potential between −80 mV and −30 mV. The potency of PCP is increased by mutations in M2 6′, 8′, and 10′ positions. This increase in potency cannot be explained merely by either changes in hydrophobicity/hydrophilicity of amino acids at these positions or by side-chain size. A model proposing extra-luminal inhibitory and regulatory sites for PCP explains the lack of voltage-dependency, the biphasic effect of PCP, and the fact that all M2 mutations increased PCP potency (by disrupting the link with the regulatory sites). J. Neurosci. Res. 61:44–51, 2000. © 2000 Wiley-Liss, Inc.

Intraperitoneal injection of rimcazole, a sigma ligand, into male rats increased plasma concentrations of corticosterone in a dose- and time-related fashion. Concurrently, rimcazole increased concentrations of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the paraventricular nucleus (PVN) of the hypothalamus, suggesting that the drug activates noradrenergic neurons terminating in this nucleus. This latter suggestion was confirmed by the finding that rimcazole also increased concentrations of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in the PVN. Pentazocine, a sigma ligand was without effect per se, but blocked the ability of rimcazole to increase concentrations of MHPG in the PVN and corticosterone in plasma. Taken together, these results suggest that rimcazole activates noradrenergic neurons projecting to the PVN via a mechanism involving sigma binding sites, and this action may be responsible for the ability of this drug to increase secretion of corticosterone.

Potential efferent projections of A13 dopaminergic (DA) neurons were identified in the present study by examining the distribution of labelled fibers following iontophoretic injection of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into the medial zona incerta (MZI), the region of the diencephalon containing A13 DA neuronal perikarya. One week after injection, PHA-L labelled fibers were found throughout the brain with the heaviest labelling occurring ipsilateral to the injection site in the anterior hypothalamic area, lateral hypothalamus, lateral preoptic area, horizontal diagonal band of Broca, and parvocellular region of the paraventricular nucleus. Moderate labelling was observed in the ipsilateral median preoptic nucleus, lateral septum, lateral aspect of the bed nucleus of the stria terminalis, and central nucleus of the amygdala. Moderate labelling was also found in the contralateral MZI and parvocellular region of the paraventricular nucleus. Light labelling was detected in the ipsilateral medial preoptic area, supraoptic nucleus, ventromedial nucleus, arcuate nucleus, vertical limb of the diagonal band of Broca, and in the contralateral lateral hypothalamus. Few immunopositive fibers were present in the dorsomedial nucleus of the hypothalamus or the magnocellular region of the paraventricular nucleus. These results reveal that neurons located in the MZI (possibly A13 DA neurons) have ipsilateral efferent axonal projections to a variety of brain regions including the lateral hypothalamus, lateral preoptic area, and the limbic structures at the diencephalic-telencephalic juncture.

The medial zona incerta (MZI) and dorsomedial nucleus of the hypothalamus (DMN), which contain cell bodies and terminals of incertohypothalamic dopaminergic (DA) neurons, are densely innervated by both noradrenergic (NE) and 5-hydroxytryptaminergic (5-HT) neurons. In view of emerging anatomical and pharmacological evidence suggesting possible interactions between 5-HT and catecholaminergic neurons, the effects of experimental procedures that inhibit or disrupt 5-HT neurons on the activities of catecholaminergic neurons terminating in these regions were examined in the present study. Catecholaminergic neuronal activity was estimated by measuring catecholamine synthesis (accumulation of 3,4-dihydroxyphenylalanine [DOPA] after administration of a decar☐ylase inhibitor) and metabolism (concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the norepinephrine metabolite 3-methoxy-4-hydroxyphenyleneglycol (MHPG) in the MZI and DMN of both male and female rats. Inhibition of 5-HT neurons following administration of the 5-HT1A autoreceptor agonist8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) increasedthe accumulation of DOPA in the DMN and the concentrations of DOPAC in the MZI and DMN, indicating an activation of catecholaminergic neurons in these regions. Concentrations of MHPG were increased in the MZI and DMN by 8-OH-DPAT or 5,7-dihydroxytryptamine-induced lesions of 5-HT neurons, revealing that NE neurons terminating in these regions were activated following procedures that decrease 5-HT neuronal function. Following destruction of NE neurons projecting to the MZI and DMN, 8-OH-DPAT no longer increased DOPAC concentrations in these brain regions. Taken together, these results reveal that 5-HT neurons tonically inhibit the activity of NE neurons terminating in the MZI and DMN, but do not influence the activity of incertohypothalamic DA neurons.

Current understanding of chronic pain points a decrease in level of the inhibitory neurotransmitter GABA, in the spinal dorsal horn, leading to an imbalance between excitatory and inhibitory pathways. A subcloned derivative of the human NT2 cell line (hNT2.17) which, after neuronal differentiation, secretes different inhibitory neurotransmitters such as GABA and glycine has been recently isolated. In this study, we have investigated the effect of this new cell line on peripheral nerve injury induced by chronic constriction (CCI) and notably the effect on the cellular GABAergic pathway. Our data show that the decrease in GABA expression in the spinal dorsal horn of injured animals is concomitant with a decline of its synthetic enzyme GAD67-Ir and mRNA but not GAD65. Interestingly, in transplanted animals we observed a strong induction of GAD67 mRNA with one week after graft, which is followed by a recovery of GAD67 and GABA Ir. This effect paralleled a reduction of hindpaw hypersensitivity and thermal hyperalgesia induced by CCI. These results suggest that hNT2.17 GABA cells can modulate neuropathic pain after CCI certainly by minimizing the imbalance and restoring the cellular GABAergic pathway.

The effects of two dopaminergic (DA) antagonists, raclopride (S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy- 6-methoxybenzamide(+)-tartrate) and remoxipride (S(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide hydrochloride monohydrate), were compared on the DA receptor-mediated regulation of incertohypothalamic and nigrostriatal DA neurons. Both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, which contains the terminals of the nigrostriatal DA neurons. On the other hand, raclopride but not remoxipride increased concentrations of DOPAC in the medial zona incerta and dorsomedial hypothalamic nucleus, regions that contains cell bodies and terminals, respectively, of incertohypothalamic DA neurons. These results suggest that raclopride blocks a population of DA receptors that regulates the activity of incertohypothalamic DA neurons, whereas remoxipride does not.

The purpose of the present study was to characterize opioid receptor-mediated regulation of incertohypothalamic dopaminergic (DA) neurons in the rat brain by examining the acute effects of selective mu or kappa opioid receptor agonists and antagonists on concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the medial zona incerta (MZI) and the dorsomedial hypothalamic nucleus (DMN) which contain cell bodies and terminals, respectively, of these neurons. Morphine caused a dose- and time-related increase in concentrations of DOPAC in MZI and DMN; this stimulatory effect was blocked by the mu opioid receptor antagonist naltrexone. In contrast, activation or blockade of kappa opioid receptors following administration of U-50,488 or nor-binaltorphimine, respectively, had no effect on DOPAC concentrations in either the MZI or DMN. The basal activity of incertohypothalamic DA neurons and their response to morphine was similar in male and female rats. Morphine also increased the concentrations of 5-hydroxyindoleacetic acid in MZI and DMN, indicating that morphine increases the activity of 5-hydroxytryptamine (5HT) neurons projecting to these regions. This might suggest that morphine-induced activation of incertohypothalamic DA neurons is mediated by 5HT neurons; but 5,7-dihydroxytryptamine-induced lesions of 5HT neurons did not alter the ability of morphine to increase DOPAC concentrations in MZI and DMN. These results indicate that the stimulatory effects of mu opioid receptor activation on incertohypothalamic DA neurons is not dependent upon the presence of 5HT neurons.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

The present study examined the effects of intraperitoneal administration of selective D1 (SKF 38393) and D2 (quinelorane) dopaminergic receptor agonists on Fos-like immunoreactivity (Fos-LI) and levels of corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (cAMY). Ninety minutes after administration of the D1 agonist SKF 38393, Fos-LI was increased in both the PVN and cAMY. Administration of SCH 39166, a selective D1 antagonist, blocked and attenuated the SKF 38393-induced increase in Fos-LI in the PVN and cAMY, respectively. Similarly, 90 minutes after intraperitoneal injection of the D2 agonist quinelorane, Fos-LI was increased in both PVN and cAMY. Administration of the selective D2 antagonist raclopride prevented the ability of quinelorane to increase Fos-LI in the PVN and cAMY. Both SKF 38393 and quinelorane stimulated the expression of CRH and mRNA in the PVN, but failed to alter its expression in the cAMY. Taken together, these results indicate that stimulation of either D1 and D2 dopaminergic receptors activates CRH neurons in the PVN. Stimulation of either D1 or D2 receptors activates neurons in the cAMY, but these changes do not appear to be occurring in CRH neurons.

The role of tuberoinfundibular and periventricular-hypophysial dopaminergic neurons in mediating rimcazole-induced decreases in plasma concentrations of prolactin and alpha-melanocyte stimulating hormone was assessed. Dopaminergic neuronal activity was estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence and intermediate lobe of the pituitary which contain terminals of tuberoinfundibular and periventricular-hypophysial dopaminergic neurons, respectively. Rimcazole decreased plasma concentrations of both prolactin and alpha-melanocyte stimulating hormone, increased the concentration of DOPAC in median eminence, but did not alter DOPAC concentrations in the intermediate lobe of the pituitary. Pretreatment with a 'putative' sigma receptor agonist, pentazocine, prevented the rimcazole-induced increase of the concentration of DOPAC in the median eminence, but did not block the ability of rimcazole to decrease plasma concentrations of prolactin. The results of this study reveal that the ability of rimcazole to decrease alpha-melanocyte stimulating hormone secretion is not mediated by a dopaminergic mechanism, whereas the ability of rimcazole to decrease prolactin secretion appears to be mediated by both dopaminergic and non-dopaminergic mechanisms.

Studies utilizing fluorescent histochemical techniques first revealed that A13 dopaminergic (DA) perikarya located in medial zona incerta (MZI) project to various regions within the hypothalamus; accordingly, these DA neurons were designated the 'incertohypothalamic' DA neuronal system. More recently, it has been shown that the anterograde neuronal tract tracer Phaseolus vulgaris leucoagglutinin, after injection into MZI, is identified in nerve terminals outside of the hypothalamus; for example, in horizontal limb of the diagonal band of Broca (HDB) and central nucleus of the amygdala (cAMY). The purpose of the present study was to determine, using neurochemical techniques, if A13 DA neurons project to the HDB and cAMY. Concentrations of dopamine and one of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in HDB and cAMY following: (1) electrical stimulation of MZI, (2) electrolytic lesion or knife ablation of MZI, and (3) administration of gamma-hydroxybutyric acid (GHBA) into MZI. For comparison, similar measurements were made in nucleus accumbens (N. Acc.), a terminal region of A10 DA neurons in the ventral tegmental area (VTA). Electrical stimulation of MZI increased DOPAC concentrations in HDB and cAMY, whereas electrolytic or ablative lesions of MZI decreased dopamine concentrations in both of these regions. By contrast, neither stimulation nor lesion of MZI had any effect on DOPAC or dopamine concentrations in N. Acc. Intracerebral injection of GHBA into MZI increased dopamine concentrations in MZI and HDB, but not in cAMY or N. Acc. Intracerebral administration of GHBA into VTA increased dopamine concentrations in HDB and N. Acc., but not in MZI or cAMY.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to examine the effects of quinelorane (LY163502), a potent and selective D 2 dopaminergic (DA) receptor agonist, on the activity of tuberoinfundibular DA neurons in male and female rats as estimated by determining the concentration of the primary metabolite of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), in terminals of these neurons in the median eminence (ME). In males, quinelorane produced dose- and time-related increases in the concentration of DOPAC in the ME which was blocked by the D 2 receptor antagonist raclopride. The activity of tuberoinfundibular neurons in female rats is higher than it is in males because circulating levels of prolactin tonically stimulate these neurons in the female. In female rats, quinelorane markedly lowered plasma concentrations of prolactin but failed to alter DOPAC concentrations in the ME. Pretreatment of female rats with prolactin antiserum induced hypoprolactinemia and reduced DOPAC concentrations in the ME; in these animals quinelorane increased ME DOPAC concentrations. These results indicate that by acting on D 2 receptors quinelorane is able to stimulate tuberoinfundibular DA neurons in both male and female rats, but in female rats the ability of quinelorane to reduce circulating levels of prolactin indirectly reduces the activity of tuberoinfundibular DA neurons and thereby masks the stimulatory action of this drug on these neurons.

The purpose of the present study was to determine the effects of quinelorane, a selective D2 receptor agonist, on concentrations of prolactin and alpha-melanocyte-stimulating hormone (alpha-MSH) in plasma of male rats. Quinelorane decreased plasma concentrations of prolactin but not of alpha-MSH, whereas, the D2 receptor antagonist raclopride increased plasma concentrations of both hormones. Quinelorane reversed the effects of raclopride on circulating levels of prolactin, but not alpha-MSH. The results of this study suggest that secretion of hormones from melanotrophs and lactotrophs is regulated by different subtypes of D2 receptors.

The activities of periventricular-hypophysial dopaminergic (DA) neurons were compared in male and female rats by measuring dopamine synthesis (accumulation of 3,4-dihydroxyphenylalanine [DOPA] after inhibition of L-aromatic amino acid decarboxylase) and metabolism (concentrations of 3,4-dihydroxyphenylacetic acid [DOPAC]) in terminals of these neurons in the intermediate lobe of the pituitary. For comparison, the synthesis and metabolism of dopamine in the neural lobe of the pituitary and median eminence were also determined. The concentrations of DOPAC and accumulation of DOPA were higher in females than in males in both the intermediate lobe and median eminence, revealing a sexual difference in the basal activity of periventricular-hypophysial and tuberoinfundibular DA neurons. In contrast, there were no differences between male and female rats in activity of DA neurons terminating in the neural lobe. One week following gonadectomy, DOPA accumulation in the median eminence was decreased in females and increased in males, but remained unchanged in the intermediate lobe. These results indicate that sexual differences in the activity of periventricular-hypophysial DA neurons terminating in the intermediate lobe are not dependent upon the presence of circulating gonadal steroids, and in this respect, these neurons differ from tuberoinfundibular DA neurons.

Persistent absence from school is often associated with anxiety and with difficulty in relating to other people. These and other factors were examined to determine their contribution to the identification of persistent absenteeism in the upper junior and lower secondary age groups. Questionnaires were administered to 190 children, including persistent absentees, to measure: (a) their relationship with parents, teachers and peers and (b) their anxiety level. Other information was obtained from teachers and pupil records. A multiple regression analysis was used to determine the predictive efficiency of the variables at both age levels. The strength of peer and teacher relationships, together with ability, suggested that thecauses of persistent absenteeism were likely to be school related. The uncertainty about the causal importance of the home was underlined by the weakness of the home related factors. Anxiety seemed to be of value in differentiating between truants and other persistent absentees.

To obtain an effective perimeter intrusion detection system requires careful sensor selection, procurement, and installation. The selection process involves a thorough understanding of the unique site features and how these features affect the performance of each type of sensor. It is necessary to develop procurement specifications to establish acceptable sensor performance limits. Careful explanation and inspection of critical installation dimensions is required during on-site construction. The implementation of these activities at a particular site is discussed.