Chronic administration of a glycine partial agonist alters the expression of N-methyl-D-aspartate receptor subunit mRNAs.

Both acute and chronic treatments with the glycine partial agonist 1-aminocyclopropanecarboxylic acid (ACPC) are neuroprotective in animal models of focal, global and spinal ischemia. After a chronic regimen of ACPC, brain and plasma levels were undetectable at the time of ischemic insult, which suggests that the neuroprotective effects of acute and chronic ACPC are mediated by different mechanisms. To investigate the possibility that chronic administration of ACPC alters N-methyl-D-aspartate (NMDA) receptor composition, the levels of mRNAs encoding zeta and epsilon subunits were quantified by in situ hybridization histochemistry with 35S-labeled riboprobes. Chronic ACPC administered to mice (200 mg/kg for 14 days) increased the level of epsilon-1 mRNA in the hippocampus (particularly CA1 and CA2 regions) and cerebral cortex (frontal, parietal and occipital regions), without altering levels in cerebellum. In contrast, this regimen decreased epsilon-3 subunit mRNA levels in the hippocampus (especially CA1 and dentate gyrus) and frontal and occipital cortices. Decreases in epsilon-2 subunit mRNA levels in cerebral cortex (especially frontal and parietal cortices) were also observed without accompanying alterations in the cerebellum, hippocampus or dentate gyrus. The levels of zeta subunit mRNA (determined with a probe that detects all splice variants) were not altered in any brain areas examined. Based on studies in recombinant receptors, these region-specific changes in mRNAs produced by a chronic regimen of ACPC could result in NMDA receptors with reduced affinities for glycine and glutamate. It is hypothesized that such alterations in NMDA receptor subunit composition may explain the neuroprotective effects produced by chronic ACPC.

Cloning of the cDNA for the human NMDA receptor NR2C subunit and its expression in the central nervous system and periphery.

Several overlapping cDNA clones containing 3995 nucleotides of the human 2C NMDA receptor subunit (NR2C) were isolated from human hippocampal and cerebellar cDNA libraries. The nucleic acid sequence of the overlapping cDNA clones displays 85% identity to that of rat NR2C. The predicted protein sequence is 1233 amino acids long and has 88% identity to the amino acid sequence of the rat NR2C, Northern blot analysis has demonstrated a wide distribution pattern of the NR2C transcript in the brain. While the predominant expression is in the cerebellum, as observed in the rat, readily detectable levels are present in the hippocampus, amygdala, caudate nucleus, corpus callosum, subthalamic nuclei and thalamus. NR2C was also detected in the heart, skeletal muscle and pancreas. Distribution of the mouse NR2C NMDA receptor subunit homologue was investigated in mouse brain by in situ hybridization histochemistry using exonic genomic probes. Expression of the transcript was principally in the cerebellum, but is also detected in the hippocampus, dentate gyrus, thalamic and subthalamic nuclei, vestibular nuclei and olfactory bulb. These results demonstrate a widespread expression pattern of the NR2C gene, both in the CNS and in the periphery.

Role of CRH in the effects of 5-HT-receptor agonists on food intake and metabolic rate.

Two series of experiments were conducted to investigate the role of corticotropin-releasing hormone (CRH) in the effects of 5-hydroxytryptamine (5-HT) on energy intake and energy expenditure. The first set of experiments was carried out to confirm the influence of 5-HT1A-, 5-HT1B-, 5-HT2A/2C-receptor agonists on the activation of the hypothalamic-pituitary-adrenal axis. Plasma corticosterone levels were measured, and a double-immunolabeling procedure was used to determine whether the neuronal activity marker, c-Fos protein (Fos), could be found within brain neurons containing CRH after treatments with 5-HT1A-, 5-HT1B-, 5-HT2A/2C-receptor agonists. The second series of experiments was conducted to assess the involvement of CRH in the effects of 5-HT on food intake and metabolic rate (VO2). The effects of the 5-HT1A-, 5-HT1B-, 5-HT2A/2C-receptor agonists on food intake and VO2 were measured in rats treated with the CRH antagonist, alpha-helical CRH-(9-41). In both experiments rats were intraperitoneally injected with either a vehicle (NaCl 0.9%), the 5-HT1A-receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), the 5-HT1B-receptor agonist 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU-24969), or the 5-HT2A/2C-receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI). Fos immunoreactivity was detectable within the CRH-containing neurons of the paraventricular nucleus of the hypothalamus (PVH) after injection of each of the 5-HT-receptor agonists used. The CRH antagonist alpha-helical CRH-(9-41) attenuated the increases in metabolic rate induced by DOI and 8-OH-DPAT. alpha-Helical CRH did not, however, prevent the effects of RU-24969 and DOI on either nocturnal metabolic rate or food intake. The present results provide further evidence for a role of CRH in 5-HT-mediated thermogenic effect, which likely involves the 5-HT2A/2C receptor during the day and the 5-HT1A receptor during the night. Moreover, these results do not support a role for CRH in 5-HT anorectic effects, which likely involves 5-HT1B and 5-HT2A/2C receptors. Finally, the results of this study indicate that the stimulation of CRH-containing neurons located in the PVH does not necessarily predict changes in food intake and energy expenditure.

Fenfluramine-induced activation of the immediate-early gene c-fos in the striatum: possible interaction between serotonin and dopamine.

DL-Fenfluramine, a serotonin (5-HT) releasing agent, induces rapid expression of Fos-like immunoreactivity (Fos-LI) in the striatum as well as in other brain structures receiving a dense 5-HT innervation. Fenfluramine-induced Fos-LI expression in the striatum may result directly from the activation of 5-HT receptors or may be the result of interactions between dopamine (DA) and 5-HT neurotransmitter systems. To discriminate between these two possibilities, various groups of rats were pretreated with different 5-HT antagonists or a DA D1 antagonist, 20 min before fenfluramine administration. Animals were killed 60 min later. In the striatum, fenfluramine-induced expression of Fos-LI was almost completely blocked by SCH 23390, methysergide and S(-)-propranolol. The immediate-early gene response to fenfluramine was only slightly affected by pretreatment with the 5-HT2A/2C antagonist ritanserin. Fenfluramine was also administered to sham-operated and to unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. In the 6-OHDA-lesioned rats, fen-fluramine-induced Fos-LI was decreased by 60% on the DA denervated side compared to the intact side and to sham-operated rats. To further probe the possibility of a direct activation of Fos-LI by 5-HT receptor subtypes, we evaluated the expression of Fos-LI after the administration of different 5-HT agonists. Our results demonstrate that neither 8-OH-DPAT, CGS-12066B, RU 24969 nor phenyl-biguanide was able to reproduce the effects of fenfluramine. Only a high dose of DOI (8.5 mg/kg) produced a moderate expression of Fos-LI in the dorsomedial part of the striatum. This contrasted with the Fos-LI expression in other brain areas where 8-OH-DPAT and DOI (2.5 and 8.5 mg/kg) reproduced the effects of the 5-HT releasing agent. Our results suggest that the release of 5-HT by fenfluramine induced Fos-LI expression predominantly in a striatal region related to associative functions and, that this c-fos response may be under the control of both 5-HT and DA. Moreover, the mechanism by which fenfluramine induces c-fos expression in the striatum differs from other brain regions.

Effect of dexfenfluramine on the transcriptional activation of CRF and its type 1 receptor within the paraventricular nucleus of the rat hypothalamus.

1. The present study investigated the effect of intraperitoneal (i.p.) administration of the indirect 5-hydroxytryptamine (5-HT) receptor agonist, dexfenfluramine, on the transcriptional activity of corticotropin-releasing factor (CRF) and its type 1 receptor in the brains of conscious male Sprague-Dawley rats via in situ hybridization histochemistry (ISHH) using both intronic and exonic probe technology. 2. The immediate early gene (IEG) c-fos mRNA was also used as index of cellular activity, whereas localization between CRF-immunoreactive (ir) perikarya and the IEG was accomplished to determine the site of CRF neuronal activation in the brain of dexfenfluramine-treated rats. 3. Thirty minutes, 1, 3, and 6 h after a single injection of either dexfenfluramine (10 mg kg-1) or the vehicle solution, adult male rats (230-260 g) were deeply anaesthetized and rapidly perfused with a 4% paraformaldehyde-borax solution (PF). The brains were removed from the skull, postfixed, and placed in a solution of 4% PF-10% sucrose overnight at 4 degrees C. Frozen brains were mounted on a microtome and cut from the olfactory bulb to the medulla in 30-microns coronal sections. 4. Dexfenfluramine induced a general neuronal activation as indicated by the strong signal of c-fos mRNA in several structures of the brain, including the parietal cortex, caudate putamen, circumventricular organs, medial preoptic area, bed nucleus of the stria terminalis, choroid plexus, choroidal fissure, supraoptic nucleus, paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus, central nucleus of the amygdala, dorsomedial nucleus of the hypothalamus, laterodorsal tegmental nucleus, locus coeruleus, and several subdivisions of the dorsal vagal complex. In most of these structures, the signal was maximal at 30 min, still strong and positive at 60 min, largely decreased at 3 h, and had completely disappeared 6 h after injection. 5. In the parvocellular division of the PVN, the large majority of CRF-ir perikarya displayed a positive signal for the mRNA encoding c-fos, indicating a profound CRFergic activation within this neuroendocrine nucleus after dexfenfluramine administration. 6. Colocalization between CRF-ir neurones and c-fos positive cells was not detected in any other regions. This selective activation of PVN CRF neurones was also confirmed by the presence of CRF primary transcript; 30 min after i.p. injection of the indirect 5-HT agonist, a positive signal for CRF hnRNA was observed, specifically in the parvocellular PVN. 7. Transcription of the gene encoding the type 1 receptor for CRF was highly stimulated in the PVN following 5-HT activation. Although this hypothalamic nucleus exhibited a barely detectable signal under basal conditions, dexfenfluramine induced a strong signal of CRF1 receptor mRNA in the parvocellular PVN. Interestingly, CRF-ir neurones displayed a positive signal for the mRNA encoding the CRF1 receptor, 3 and 6 h after systemic treatment with dexfenfluramine. 8. These results indicate that although dexfenfluramine can generate a wide neuronal activation throughout the brain, this 5-HT agonist triggers the activity of CRF neurones selectively in the parvocellular division of the PVN, a mechanism possibly related to the activity of hypothalamic-pituitary-adrenal axis. Induction of CRF1 receptor mRNA in CRF cells of the PVN indicates that neuroendocrine CRF neurones can be targeted by CNS CRF under 5-HT stimulation.

Lesion of central nucleus of amygdala promotes fat gain without preventing effect of exercise on energy balance.

Male Wistar rats with intact or lesioned central nucleus of amygdala (CeA) were kept at rest or subjected to a treadmill running program for 21 consecutive days. Food intake and body weight were monitored throughout the exercise training program. At the end of the program, rats were killed and their carcasses processed for analysis of the contents in energy, fat, and protein. Exercise and CeA lesions induced opposite effects on energy balance; exercise delayed gains in body energy and fat, whereas CeA lesions promoted them. Total energy intake was lower in exercised rats than in sedentary ones over the 12 and 24 h that followed exercise. Food intake was higher in lesioned rats than in intact animals over the second half of the 12-h period that followed exercise. There was no interaction effect of exercise and CeA lesions on energy balance and intake and on body composition. Plasma levels of adrenocorticotropin hormone and corticosterone were higher in exercised rats than in sedentary ones, but there was no difference between lesioned and intact rats. This study, as well as confirming the effect of exercise on energy balance, indicates that CeA lesions may promote energy deposition in rats. Above all the present results provide evidence that CeA does not represent a necessary neuroanatomic structure in the effect of exercise on energy balance.

Functional assessment of the 5-HT 1A-, 1B-, 2A/2C-, and 3-receptor subtypes on food intake and metabolic rate in rats.

The 5-hydroxytryptamine (5-HT) agonists (+/-)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), RU-24969, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), and 1-phenylbiguanide were administered to male Wistar rats to assess the respective involvement of the 5-HT 1A-, 1B-, 2A/2C-, and 3-receptor subtypes in the control of food intake and metabolic rate (VO2). Four series of experiments were carried out, each series addressing the effects of four doses (including saline or dose 0) of each of the agonists selected. The drugs were intraperitoneally injected in spontaneously fed animals. Injections were performed during the first 15 min of either the diurnal or the nocturnal phases of the light-dark daily cycle. Food intake and VO2 measurements were carried out over the 12-h periods ensuing after the agonist injections. The two highest doses of the 5-HT1A-receptor agonist 8-OH-DPAT led to a quickly appearing but transient elevation of diurnal VO2. During the night, VO2 was higher when the rats were treated with 8-OH-DPAT than when they were treated with saline. There was no significant effect of 8-OH-DPAT on either diurnal or noctural food intake. The highest dose of RU-24969 induced a significant increase in diurnal VO2, whereas all doses of RU-24969 blunted the nocturnal rise in metabolic rate characteristically observed in rats kept under a daily light-dark cycle. Importantly, RU-24969 induced marked diurnal and nocturnal hypophagia.(ABSTRACT TRUNCATED AT 250 WORDS)