Corticotrophin-Releasing Hormone Stimulates Neurohypophyslal Hormone Release through an Interaction with the Intermediate Lobe of the Pituitary

Corticotrophin-releasing hormone is found co-localized with oxytocin in the magnocellular-neurohypophysical system but its function in this context is unknown. We tested its effects on neurohypophysical hormone secretion in vitro , in the presence and absence of the intermediate lobe of the pituitary. Corticotrophin-releasing hormone caused significant, calcium-dependent secretion of oxytocin and vasopressin from neural lobes in contact with intermediate lobes, i.e. neurointermediate lobes. This effect was inhibited by the dopamine agonist, bromocriptine. Corticotrophin-releasing hormone had no effect on isolated neural lobes in the absence of the intermediate lobe, but α- and γ-melanocyte-stimulating hormone produced an increase in secretion that was comparable in pattern and magnitude to the effect of corticotrophin-releasing hormone on neurointermediate lobes. These findings suggest that corticotrophin-releasing hormone released with oxytocin may act in a paracrine fashion to stimulate release of intermediate peptides which, in turn, can directly evoke release of oxytocin and vasopressin from neural lobe terminals.     

Glucocorticoid Receptor Immunoreactivity in the Rat Intermediate Lobe

Cells containing Type II glucocorticoid receptor (GR) immunoreactivity were identified in the rat pituitary gland by immunocytochemistry using a specific monoclonal antibody. At light microscopic level, GR immunoreactive cells were located in the intermediate lobe in addition to the well known GR-containing cell population in the anterior lobe. In both groups of cells GR appeared predominantly in the cell nuclei. Adrenalectomy resulted in a decrease in staining intensity of the anterior lobe and changed the pattern of fluorescence in a minority of cells where cytoplasmic staining became predominant. These changes appeared less marked in the intermediate lobe. Dexamethasone administration reversed the adrenalectomy-induced alterations of GR staining in both lobes. At the electron microscopic level, GR immunoreactive sites were revealed by the protein A-gold technique. In contrast to the distribution of fluorescence, GR was localized in cell nuclei as well as in the cytoplasm in both lobes. Quantitative estimates indicate that about 40% more immunoreactive sites are present in the anterior lobe than in the intermediate lobe. The presence of GR in the intermediate lobe suggests that this pituitary region, like the anterior lobe, is influenced by glucocorticoid hormones.     

Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.     

Cholinergic projections from the midbrain reticular formation and the parabigeminal nucleus to the lateral geniculate nucleus in the tree shrew

The distribution and sources of putative cholinergic fibers within the lateral geniculate nucleus (GL) of the tree shrew have been examined by using the immunocytochemical localization of choline acetyltransferase (ChAT). ChAT-immunoreactive fibers are found throughout the thalamus but are particularly abundant in the GL as compared to other principal sensory thalamic nuclei (medial geniculate nucleus, ventral posterior nucleus). Individual ChAT-immunoreactive fibers are extremely fine in caliber and display numerous small swellings along their lengths. Within the GL, ChAT-immunoreactive fibers are more numerous in the layers than in the interlaminar zones and, in most cases, the greatest density is found in layers 4 and 5. Two sources for the ChAT-immunoreactive fibers in the GL have been identified--the parabigeminal nucleus (Pbg) and the pedunculopontine tegmental nucleus (PPT)--and the contribution that each makes to the distribution of ChAT-immunoreactive fibers in GL was determined by combining immunocytochemical, axonal transport, and lesion methods. The projection from the Pbg is strictly contralateral, travels via the optic tract, and terminates in layers 1, 3, 5, and 6 as well as the interlaminar zones on either side of layer 5. The projection from PPT is bilateral (ipsilateral dominant) and terminates throughout the GL as well as in other thalamic nuclei. Lesions of the Pbg eliminate the ChAT-immunoreactive fibers normally found in the optic tract but have no obvious effect on the density of ChAT-immunoreactive fibers in the contralateral GL. In contrast, lesions of PPT produce a conspicuous decrease in the number of ChAT-immunoreactive fibers in the GL and in other thalamic nuclei on the side of the lesion but have no obvious effect on the number of ChAT-immunoreactive fibers in the optic tract. These results suggest that there are two sources of cholinergic projections to the GL in the tree shrew which are likely to play different roles in modulating the transmission of visual activity to the cortex. The Pbg is recognized as a part of the visual system by virtue of its reciprocal connections with the superficial layers of the superior colliculus, while the PPT is a part of the midbrain reticular formation and is thought to play a non-modality-specific role in modulating the activity of neurons throughout the thalamus and in other regions of the brainstem.     

Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high‐affinity choline transporter and choline acetyltransferase

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non‐neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high‐affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112‐1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112‐1 cells showed increased binding of the CHT1 inhibitor [³H]hemicholinium‐3 (HC‐3) and greater [³H]choline uptake and ACh synthesis than NIH3T3ChAT 103‐1 cells, a CHT1‐negative control cell line. HC‐3 significantly inhibited ACh synthesis in NIH3T3ChAT 112‐1 cells but did not affect synthesis in NIH3T3ChAT 103‐1 cells. ACh synthesis in NIH3T3ChAT 112‐1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low‐affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112‐1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes. © 2009 Wiley‐Liss, Inc.     

Ontogeny of cholinergic neurons in the mouse forebrain

The development of cholinergic neurons in the mouse forebrain was studied by immunocytochemistry with a monoclonal antibody to choline acetyltransferase (ChAT), the rate-limiting enzyme for acetylcholine synthesis. Since this antibody stained dividing cells in ventricular germinal zones as well as differentiating neurons, likely routes of migration could be inferred on the basis of the location of immunoreactive (IR) cells at different gestational ages. Germinal zones for cholinergic cells were observed in all ventricular zones of the forebrain with the ventral zones generating the earliest cells by gestational day 13.5 (GD13.5). On GD14, ChAT IR cells were visible in the germinal zones of the eye, olfactory ventricle, anterior horn, and dorsolateral aspect of the lateral ventricle, lateral ganglionic eminence, ventro- and dorsolateral third ventricle, and in the pineal anlage (epiphysis). ChAT IR neurons continued to develop in these and additional germinal zones on GD15, including the medial, dorsal, and dorsomedial walls of the lateral ventricle, and the medial and dorsal ganglionic eminence. On GD16, ChAT IR neurons were located in the prelimbic, pyriform, and parietal cortices and the lamina terminalis, and a cluster of IR cells was observed in the ventricular zone of the caudatopallial angle. On GD17-18, neurons in the anterior olfactory nucleus, olfactory tubercle, horizontal and vertical nucleus of the diagonal band, and medial septal nucleus stained more darkly and were multipolar, whereas immature bipolar neurons appeared to continue their migration into the hippocampus and along major fiber tracts, such as the corpus callosum, external capsule, fornix and anterior commissure. This study provides a comprehensive view of the zones of origin, probable routes of migration, and final destination of cholinergic neurons in the mouse forebrain.     

Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia

We have studied the expression of catecholaminergic and cholinergic phenotypes in sympathetic ganglia removed from 7- to 10-day-old quail embryos and grown in vitro under different conditions. Quantitative data were obtained by measuring the conversion of (³H) tyrosine and (³H) choline to catecholamines (CA) and acetylcholine (ACh), respectively. In explant cultures, large amounts of both neurotransmitters were synthesized from the onset, but CA generally predominated, the molar ratios of CA:ACh being, on average, of the order of 2:1. If the ganglia were dissociated before plating, there was a selective increase in ACh synthesis (three- to fivefold) such that the CA:ACh ratio fell strikingly. The early expression of the cholinergic phenotype appears to be species-specific in that, under identical conditions, dissociated cell cultures of newborn mouse superior cervical ganglia were overwhelmingly catecholaminergic (CA:ACh ratio of approximately 40:1) and ACh synthesis was only just detectable. Addition of veratridine (1.5 μM) either to explant or to dissociated cell cultures of embryonic quail sympathetic ganglia barely altered CA-synthesizing ability; in contrast, ACh synthesis and accumulation were stimulated about threefold. This effect, which we found to correspond to a quantitatively similar increase in the activity of choline acetyltransferase (ChAT), was completely blocked by tetrodotoxin, indicating that it was due to Na⁺-dependent depolarization. A preferential stimulation of ACh production was also observed when the concentration of K⁺ was raised to 20 mM. Veratridine treatment of cultures of presumptive sympathoblasts, in the form of sclerotome-associated neural crest cells, had identical effects. Our results reveal the quantitative importance of ACh-related properties in avian sympathetic ganglia from the earliest stages of their development and suggest that depolarization may be one of the factors selectively enhancing expression of the cholinergic phenotype during ontogeny. In these respects, the neurochemical differentiation of sympathetic neurons unfolds according to dissimilar scenarios in birds and mammals. © 1993 Wiley-Liss, Inc.     

Acetylcholine Metabolism in the Inflamed Rat Intestine

Acetylcholine (ACh) is a major neurotransmitter in the enteric nervous system. Since increasing evidence suggests that inflammation alters neural regulation of intestinal function, we examined the synthesis and breakdown of ACh in smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of the rat during inflammation caused by infection with the nematode parasiteTrichinella spiralis. Both total and neuron-specific uptake of the ACh precursor [³H]choline into SM/MP preparations was increased by over twofold on Day 6 postinfection. Further, a radiochemical assay of choline acetyltransferase activity showed significant increase by Day 1, with peak values reached by Day 3 and maintained without reversal thereafter. Despite the enhancement of these steps, measurement of the conversion of [³H]choline into [³H]ACh in SM/MP preparationsin vitroshowed a nearly fourfold decrease by Day 6, implying a large decrease in ACh production in the inflamed jejunum. Examination of acetylcholinesterase in the rat jejunum showed decreased histochemical staining intensity in the muscle wall, and quantitative evaluation showed significantly decreased (>50%) acetylcholinesterase activity in SM/MP preparations. These results show that cholinergic innervation of the intestine can undergo rapid and long-lasting alterations during inflammation. Upregulation of major steps in the synthetic pathway for ACh was not matched by increased ACh production, suggesting that defects in ACh packaging, storage, and granule exocytosis may also be present.     

Cytochemical identification of cholinergic amacrine cells in cat retina

Golgi studies of cat retina have revealed the presence of matching subpopulations of starburst-like amacrine and displaced amacrine cells that are morphologically similar to the cholinergic cells of rabbit retina. The displaced amacrines appear identical with the A14 cells described by Kolb et al. (Kolb, Nelson, and Mariani: Vision Res. 21:1081-1114, 1981). In order to determine whether these cells may be cholinergic, we carried out autoradiography to localize newly synthesized (3H)acetylcholine and immunocytochemistry to demonstrate the distribution of choline acetyltransferase. Autoradiographs showed labeling in somas of both amacrine and displaced amacrine cells. Choline acetyltransferase was found in amacrine cells that ramify in sublamina a of the inner plexiform layer and in displaced amacrine cells ramifying in sublamina b. The pattern of cholinergic neurons in the cat is similar to that in other vertebrates and suggests that acetylcholine may play an important and consistent role in retinal function.     

The effect of selective lesions on vestibular nuclear complex choline acetyltransferase activity in the rat

Biochemical, physiological and behavioral evidence suggests that acetylcholine (ACh) may play a neurotransmitter role in central vestibular function. However, the anatomic basis for a possible cholinergic influence on the vestibular nuclear complex (VNC) is unknown. To investigate vestibular cholinergic anatomy, we have made selective lesions of neurons intrinsic to the VNC, and of most known afferents to the VNC, and we have measured the activity of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, following such lesions. We found that a kainic acid lesion of the VNC, and lesions of vestibular afferents, including the VIIIth cranial nerve, cerebellum, spinal cord, vestibular commissure and the interstitial nucleus of Cajal, did not affect VNC ChAT activity. We conclude that there are no cholinergic neurons intrinsic to the VNC, and that these lesioned afferents are not cholinergic. It is likely, therefore, that a cholinergic projection to the VNC arises from a region other than those lesioned; possibilities include the nuclei of the reticular formation, the upper cervical cord and local pontomedullary nuclei.     

A population of oligodendrocytes derived from multipotent neural precursor cells expresses a cholinergic phenotype in culture and responds to ciliary neurotrophic factor

Because oligodendrocytes and their precursors possess receptors for classical transmitters, and neurotransmitters such as glutamate and noradrenaline can mediate oligodendroglial proliferation and differentiation, it is possible that other neurotransmitters can also exert regulatory roles in oligodendrocyte function. We used mitogen-proliferated multipotent neuroepithelial precursors (neurospheres) and identified oligodendroglia that expressed markers traditionally found in cholinergic neurons. Regardless of culture conditions, there existed a large population of cells that resembled oligodendrocytes morphologically and coexpressed the oligodendrocyte-specific marker galactocerebroside (GalC) and the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT). These cells did not express neuronal markers, and whole-cell recordings from cells with similar morphology displayed only outward currents in response to depolarizing voltage steps, further supporting their oligodendroglial identity. Another cholinergic marker, the vesicular ACh transporter, was also detected in GalC(+) oligodendrocytes. Furthermore, neurospheres cultured in the presence of the cholinergic receptor antagonist atropine showed a decrease in the number of GalC(+) spheres, implicating the muscarinic ACh receptor in oligodendrocyte development. The actions of neurotrophins and ciliary neurotrophic factor (CNTF) on these ChAT(+) oligodendrocytes were examined. Among these, CNTF treatment significantly increased oligodendrocytic process outgrowth. These results demonstrate classical cholinergic neuronal markers in oligodendrocytes as well as an effect of muscarinic receptor blockade on oligodendrocyte differentiation.     

Topographical localization of choline acetyltransferase within the human spinal cord and a comparison with some other species

Choline acetyltransferase (ChAT), which is known to be a specific marker of cholinergic structures, was assayed in small tissue samples punched out from cryosections of human, bovine, cat and rat spinal cords. The relative distribution patterns of spinal ChAT were similar between the different species. An area of high activity in the ventrolateral part of the ventral horn was found. This activity is probably located in the motor neurons, as it could be traced into the ventral root region. In addition, in the dorsal horn of the cord from man and cow another area with high ChAT activity was found. Subcellular studies suggest that this activity is mainly located at nerve terminals.     

Stimulation of choline acetyl transferase activity by l- and d-carnitine in brain areas of neonate rats

Acetyl-CoA supply to the cytosol and its regulatory influence on acetylcholine biosynthesis is still an unsolved question. Acetylcarnitine through the carnitine acetyl transferase (CarAT) system has been proposed to be the acetyl donor in this process. Carnitine isomers were injected into rat developing brains every day for 14 days after birth. Results showed that carnitine and its associated forms produced a choline acetyl transferase (ChAT) activity increase in the striatum and the hippocampus. Carnitine acetyl transferase activity was stimulated by the treatment of 1-carnitine in the hippocampus but it remained unchanged in the striatum and the cerebral cortex. These results suggest that ChAT and CarAT activities might be modulated by Acetyl-CoA derived preferentially from acetylcarnitine. It is suggested that ChAT activity enhancement depends on intrinsic and extrinsic cholinergic afferents to these brain areas. © 1995 Wiley-Liss, Inc.     

An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat

These experiments were directed at determining the proportion and distribution of cholinergic septal cells which project to the rat hippocampal formation. Injections of WGA-HRP were placed into different regions of the hippocampal formation and sections through the septal complex were processed for the simultaneous demonstration of the retrogradely transported marker and for choline acetyltransferase (ChAT) immunoreactivity. Preliminary analysis of adjacent normal series prepared either for the demonstration of ChAT or stained by the Nissl method demonstrated several distinct cell groups in the classically defined medial septal nucleus and vertical limb of the nucleus of the diagonal band. The groups of cells ranged from almost entirely ChAT-positive to entirely noncholinergic. On the basis of shape and size of the constituent cells, the ChAT-positive cells of the septal complex were divided into dorsal, intermediate, and ventral subdivisions. The proportion of retrogradely labeled cells that were also ChAT positive ranged from 22.8% to 77.4% in different experiments. When only the hippocampus and dentate gyrus are considered, this variation can largely be accounted for by the topographic organization of the septohippocampal projection. The medial, noncholinergic half of the medial septal nucleus projects primarily to the rostral or septal portions of the dentate gyrus and hippocampus, whereas the lateral half, in which the dorsal ChAT group is located, projects heavily to more temporal levels. Rostral portions of the hippocampus and dentate gyrus receive most of their cholinergic input from the ventral ChAT cell group which forms a major component of the vertical limb of the nucleus of the diagnoal band. While some ChAT-positive cells in the intermediate group project to the hippocampal formation, they are generally less numerous than those from the dorsal and ventral groups. However, in a control experiment in which the WGA-HRP injection was placed into the cingulate cortex overlying the rostral hippocampal formation, the intermediate ChAT group accounted for 71.2% of the double-labeled cells.     

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity choline transport and acetylCoA production in brain and their roles in the regulation of acetylcholine synthesis

This review describes recent advances made in the understanding of the regulation of acetylcholine synthesis in brain with regard to the availability of its two precursors, choline and acetylCoA. Choline availability appears to be regulated by the high affinity choline transport system. Investigations of the localization and inhibition of this system are reviewed. Procedures for measuring high affinity choline transport and their shortcomings are described. The kinetics and effects of previous in vivo and in vitro treatments on high affinity choline transport are reviewed. Kinetic and direct coupling of the transport and acetylation of choline are discussed.Recent investigations of the source of acetylCoA used for the synthesis of acetylcholine are reviewed. Three sources of acetylCoA have recently received support: citrate conversion catalyzed by citrate lyase, direct release of acetylCoA from mitochondria following its synthesis from pyruvate catalyzed by pyruvate dehydrogenase, and production of acetylCoA by cytoplasmic pyruvate dehydrogenase. Investigations indicating that acetylCoA availability may limit acetylcholine synthesis are reviewed. A model for the regulation of acetylcholine synthesis which incorporates most of the reviewed material is presented.     

The hypothalamohypophyseal system in vitro: Electrophysiology of the pars intermedia and evidence for both excitatory and inhibitory inputs

The purpose of this study was to better assess the function of catecholamine-containing nerve terminals in the pituitary pars intermedia lobe. Hypothalamohypophyseal explants, which included the intact mediobasal hypothalamus (MBH), median eminence, infundibular stalk and the neurointermediate lobe, were obtained from 2-3-week-old male and female albino rats. The tissue was placed in a perfusion chamber and maintained under physiological conditions for up to 12 h. A set of bipolar stimulating electrodes was positioned on the surface of the median eminence, infundibular stalk or the rostroventral arcuate nucleus of the MBH. A microelectrode recorded electrical activity in the pars intermedia gland. Two types of spontaneous action potentials were found; fast 2-4 ms duration neural fiber type spikes and slower 7-10 ms duration spikes probably derived from non-neural endocrine cells. Single-pulse electrical stimulation at all 3 sites evoked both kinds of potentials, while trains of stimuli (0.1-20 Hz) decreased or completely inhibited the basal firing rate of the slower ones. Application of the neuroleptic. L-sulpiride (0.01, 0.1 or 1.0 mumol), to the perfusion medium increased the spontaneous endocrine cell activity and blocked the stimulus-induced inhibition in the explants but had no effect on the activity in isolated pituitaries. Dopamine (0.1 mumol), which is known to inhibit the secretion of pro-opiomelanocortin peptides, reversibly suppressed the spontaneous endocrine cell potentials. These observations support a hypothesis for the presence of a functional tuberohypophyseal dopamine inhibitory system and a possible, but as yet unidentifiable, excitatory system in the pars intermedia. Thus, hypothalamohypophyseal explants can be used to elucidate specific information on this type of neuroendocrine axis.     

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.     

Effects in vitro of New Growth Hormone Releasing Peptide (GHRP-1) on Growth Hormone Secretion from Ovine Pituitary Cells in Primary Culture

Continuous perifusion of pituitary cells was used to study the effects of a newly synthesized GHRP (GHRP-1 or KP 101) on growth hormone (GH) secretion from ovine pituitary cells and these have been compared to effects of growth hormone-releasing factor (GRF) and the original growth hormone-releasing peptide (GHRP-6). GH was continuously released at a constant rate during perifusion and secretion was increased by KP 101, GHRP-6 and GRF in a dose-dependent manner. The half-maximal effective dose of KP 101 and GHRP-6 was 10^?7 M, an order of magnitude higher than that for GRF. The maximal effects of KP 101 and GHRP-6 were similar but significantly less than the maximal effect of GRF. Blockade of calcium channels with Cd²⁺ (2 mM) totally and reversibly abolished the releasing effects of all three peptides. Like GHRP-6, the GH release induced by KP 101 was not affected by a GRF antagonist ([Ac-Tyr¹, D-Arg²]-GRF 1–29, 1 μM) which significantly reduced the effect of GRF on GH release. For each peptide, the response to a second application (1 h after the first application) was lower than the first response. When GRF (or KP 101, GHRP-6) was applied first and then KP 101 or GHRP-6 (or GRF) given 1 h later, the second response was not attenuated. Only a small additive effect on the release of GH by GRF was obtained by the co-administration of either KP 101 or GHRP-6. This result was achieved with maximal doses of the peptides, but not with half-maximal doses. These data indicate that KP 101 has similar potency and GH releasing properties to GHRP-6 but both are less potent than GRF. There is no synergistic effect of the peptides with GRF and only a small additive effect of KP 101 or GHRP-6 on GRF-stimulated GH release from ovine pituitary cells in vitro. KP 101 stimulates GH release by a mechanism that involves a common step employed also by GRF and GHRP-6, an increase in calcium influx. In addition, our data strongly suggest that KP 101 like GHRP-6 do not act through the GRF receptor and that there is no cross-desensitization the GRF elicited response.