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Notes: Abstract: The expression and distribution of mRNA encoding pre-proatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA Oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus (“anteroventral/third ventricle region”) and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CAl pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta. and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CAl pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.

Notes: Abstract: The binding of [3H]spiperone, a neuroleptic/dopamine receptor ligand, to membranes of the ventral tegmental area of the rat was studied in vitro and found to be rapid, saturable, reversible, and of high affinity. Specific binding was displaced by the dopaminergic agonists dopamine, apomorphine, and 2-amino-6,7-dihydroxytetralin, and stereospecifically by the neuroleptic drugs butaclamol and flupenthixol. Bromocryptine and other ergots displaced the binding, as did the D-2 antagonists domperidone, molindone, metoclopramide, and sulpiride. Noradrenergic, histaminergic, and serotonergic components of the binding were not detected in displacement studies with various agonists and antagonists. These data are consistent with the hypothesis that [3H]spiperone labels dopamine receptors in the ventral tegmental area that are not linked to adenylate cyclase and are therefore likely to be of the D-2 type.

Notes: Abstract: The rat ventral tegmentum (containing somata and dendrites of mesolimbic dopaminergic neurones) contained 1.3 μmnol/g wet weight of glycine. Slices of ventral tegmentum accumulated exogenous [3H]glycine by an energy-, temperature- and sodium-dependent mechanism. The uptake was mediated by two different transport systems; one system with relatively low affinity for glycine (Km∼400 μm) and the other a higher affinity for glycine (Km∼ 10 μm). Small amino acid analogues of glycine inhibited the uptake process, the most potent being taurine and β-alanine (47% and 44% inhibition, respectively, at 1 mm). Release of exogenous [3H]glycine by elevated potassium and by protoveratrine A was calcium-dependent and tetrodotoxin-sensitive. Glycine (500 μm-2 mm) potentiated the protoveratrine A-induced release of exogenous [3H]dopamine from slices of ventral tegmentum; this potentiation was blocked by strychnine (10 μm). A convulsant dose of strychnine elevated the concentration of 3,4-dihydroxyphenylacetic acid in the ventral tegmentum. Glycine is likely to be a transmitter in the ventral tegmentum and to have a role regulating the activity of somatodendritic regions of mesolimbic dopaminergic neurones.

Notes: Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (– LI), [125I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [125I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80–170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p 〈 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3–5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10–12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.

Notes: The distribution of receptors for the sulphated octapeptide cholecystokinin 26–33 (CCK-8S) in rat brain was investigated by radioligand binding in conjunction with autoradiography using the novel iodinable, non-oxidizable, amino- and thiolendopeptidase-resistant CCK analogue, d-Tyr25(Nle28,31)-CCK 25–33S. Labelling of the peptide was achieved by synthesis utilizing Na125l and Chloramine-T. [125l]D-Tyr25(Nle26,31)-CCK 25–33S (100 pM) bound rapidly and reversibly to a single population of sites on slide-mounted coronal sections of rat forebrain with a dissociation constant of 34 pM. Specific binding was fully inhibited by CCK-8S, CCK-8, CCK-4, L-365,260 and L-364,718, with inhibition constants 2.7, 9.8, 35, 7.0 and 130 nM, respectively. These inhibition data may indicate that the [125l] ligand binds preferentially to a CCKB subtype of receptor, but may also reflect the relative paucity of CCKA receptors in the rat forebrain. Optimum conditions for autoradiography combined the preincubation of brain sections in unlabelled 10 pM d-Tyr25(Nle28,31)-CCK 25–33S with a 60-min wash after incubation with the [125l] ligand. Analyses of the autoradiograms obtained from the use of coronal and horizontal brain sections were aided by the high levels of specific binding (80–90%), and revealed that CCK receptors were topographically distributed through the neuroaxis. High densities of receptor-associated silver grains were found in the olfactory bulb (internal plexiform layer), neocortex (layer III), nucleus accumbens, parasubiculum, subbrachial nucleus, parabigeminal nucleus, dorsal vagal complex, area postrema and the A2 region. Moderate labelling was observed in many telencephalic and diencephalic nuclei. The majority of these receptors were of the CCKB subtype, as shown by the use of subtype-selective antagonists, although CCKA receptors were present in moderate to high densities in the A2 area, area postrema and nucleus tractus solitarii, and at low density in the interpeduncular nucleus and central amygdala. These findings provide further evidence for the widespread, topographic distribution of CCK receptors and indicate that [125l]d-Tyr25(Nle28,31)-CCK 25–33S is very suitable for autoradiographic investigations because of its low non-specific binding.

Notes: Chromogranin/secretogranins are a family of acidic, soluble proteins with a widespread distribution in secretory vesicles of endocrine and nervous tissues. The effects of experimental stimuli of differing duration and intensity on chromogranin B and secretogranin II mRNA levels in relevant areas of the rat brain were examined by in situ hybridization histochemistry using 35S-labelled oligonucleotides. Effects of two ‘chronic stimulation’ paradigms were studied—the effect of 4 days of water or food deprivation on mRNA levels in the hypothalamus and the effect of unilateral cervical vagotomy on transcript levels in the dorsal vagal complex 1, 2 and 7 days after surgery. After 4 days of water deprivation secretogranin II mRNA was significantly increased in the supraoptic nucleus (366 ± 21% of control, P 〈 0.01), the magnocellular paraventricular nucleus (209 ± 20% of control, P 〈 0.01) and the parvocellular paraventricular nucleus (147 ± 6% of control, P 〈 0.01). Conversely, the level of secretogranin II mRNA in the supraoptic nucleus was decreased (61 ± 13% of control, P 〈 0.05) after 4 days of food deprivation. Seven days after unilateral cervical vagotomy, secretogranin II and chromogranin B mRNA levels were markedly decreased in the ipsilateral dorsal motor nucleus of the vagus (25 ± 4 and 47 ± 8% of contralateral values respectively, P 〈 0.01). Rapid changes in chromogranin mRNA were also detected following shorter duration ‘acute stimulation’—in the hypothalamus after hypertonic saline injection, in the hippocampus after electrical stimulation-induced kindled seizures, and in the cerebral cortex after unilateral craniotomy. A large increase in secretogranin II mRNA was detected in the supraoptic nucleus (202 ± 25% of control, P 〈 0.01) and the magnocellular paraventricular nucleus (168 ± 29% of control, P 〈 0.05) 3 h after a single intraperitoneal injection of hypertonic (1.8 M) saline. Markedly increased levels of secretogranin II (125–160% of control) and chromogranin B (140–230% of control) mRNA were observed in granule cells of the dentate gyrus 0.5–2 h after amygdaloid stimulation-induced seizures. A moderate increase in secretogranin II mRNA (144 ± 11% of contralateral side, P 〈 0.01) was found in the underlying cerebral cortex 2.5 h after unilateral craniotomy. These results indicate that measurement of changes in chromogranin mRNA, particularly secretogranin II, is a useful means of assessing both rapid and long-lasting increases and decreases in neuronal activity and, in contrast to immediate early gene mRNA levels, may better reflect specific changes in neuronal secretory activity associated with transmitter/peptide release.

Notes: Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P〈0.05 and 261±34% of control, P〈0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P〈0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment-induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH-diaphorase histochemistry, and NADPH-diaphorase staining was observed in neurones both positive and negative for oxytocin-like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity-dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.

Notes: Neurones of the supraoptic nucleus (SON) and the magnocellular and parvocellular divisions ofthe paraventricular nucleus (PVN) express galanin and [125I]galanin binding sites. Although the precise role(s) of galanin in these different cell populations is still unknown, it has been shown to regulate the electrophysiological, neurochemical and secretory activity of magnocellular neurones.In light of the well-described effects of hyperosmotic stimuli, such as salt-loading on magnocellular neurone activity and galanin synthesis and release, and the recent identification of multiple galanin receptors in brain, this study assessed the possible regulation of galanin receptor subtype expression in the PVN/SON of salt-loaded, dehydrated and food-deprived rats. Gal-R1 mRNA was abundant in the SON (and magnocellular PVN) of control rats and levels were increased in these same cells after 4 days of salt-loading (2% NaCl solution as drinking water) or water deprivation. The density of specific [125I]galanin(1–29) binding and the intensity of Gal-R1-like immunostaining were also increased in the characteristically enlarged, magnocellular neurones of the PVN and SON after these treatments. Gal-R2 mRNA was detected in the parvocellular PVN, but levels were not altered by the hyperosmotic stimuli. In contrast, food deprivation (4 days), which has been shown to reduce levels of several neurochemical markers in magnocellular neurones, produced a significant reduction in Gal-R1 (and galanin) mRNA levels in the SON, but no consistent change in neurone size, [125I]galanin binding levels, or Gal-R1 immunostaining. Along with previous findings from this and other laboratories, these data suggest that the expression of galanin and Gal-R1 receptors is regulated in parallel with functional and morphological changes in hypothalamic magnocellular neurones. Furthermore, Gal-R1 immunoreactivity was primarily detected in somatodendritic areas and thus galanin may influence the activity of these cells, particularly vasopressin synthesis/release, via autocrine or paracrine activation of Gal-R1 receptors, especially during long-lasting stimulation.

Notes: Summary Muscimol, a potent GABA receptor agonist, produced increases in DOPAC and dopamine concentrations in the rat hypothalamus. GABAergic receptors, therefore, modulate hypothalamic dopaminergic neurones.