GABA and serotonin (5-HT) pattern in the supraependymal fibers of the rat epithalamus: Combined radioautographic and immunocytochemical studies. Effect of 5-HT content on [H]GABA accumulation

Numerous studies have suggested the serotoninergic nature of the supraependymal plexuses; moreover, several supraependymal fibers are also able to take up [³H]GABA and could be GABA-containing fibers. In this approach, by combined immunocytochemistry and radioautography, we analyzed and compared the distribution of endogenous and exogenous GABA and 5-HT in the supraependymal layer, after inhibition of their respective catabolisms. The majority of the supraependymal fibers are reactive to GABA and 5-HT antisera which indicates that they could contain both GABA and 5-HT. Furthermore it is possible to show that endogenous 5-HT containing fibers are able to accumulate [³H]GABA and conversely. These data point to a functional role for both neurotransmitters in these nerve elements. On the other hand, GABA and 5-HT contents may be connected sincep-chlorophenylalaninetreatment which inhibits 5-HT synthesis increased [³H]GABA labelling of these plexuses. Finally, several supraependymal fibers are also able to take up [³H]glutamate (but not [³H]glutamine); this compound might be accumulated as GABA precursor and/or as neurotransmitter.     

Oestradiol Microimplants in the Ventromedial Preoptic Area Inhibit Secretion of Luteinizing Hormone via Dopamine Neurones in Anoestrous Ewes

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.     

大鼠脊髓内5-HT2C,5-HT3,5-HT6和5-HT7受体亚型mRNAs的表达

目的:观察5-HT2C,5-HT3,5-HT6和5-HT7受体亚型mRNAs在大鼠脊髓不同节段的表达.方法:反转录PCR方法.结果:5-HT2C受体亚型mRNA在颈、胸、腰、骶段脊髓的背角(DH)和前角(VH)均有较强的表达;5-HT3受体mRNA在各节段脊髓DH的表达水平较高,而在VH则较低;与5-HT3受体亚型相反,5-HT6受体亚型mR-NA在脊髓VH的表达水平高于DH;5-HT7受体mRNA在脊髓的表达则与5-HT3受体相似,在各节段的DH均有较高水平的表达.不同的受体亚型在脊髓同一节段以及同一受体亚型在不同脊髓节段的表达水平存在差异.结论:本研究结果表明,上述四种5-HT受体亚型在脊髓具有不同的表达特点,提示它们在脊髓水平可能发挥着不同的生理作用,并为深入探讨5-HT受体参与伤害性感受和运动的调节机制提供了依据.     

Involvement of Cholecystokinin in Food Intake: III. Oestradiol Potentiates the Inhibitory Effect of Cholecystokinin Octapeptide on Food Intake in Ovariectomized Rats

The role of Cholecystokinin in a model of hypophagia, oestradiol-treated Ovariectomized rats, was investigated. Implantation of oestradiol-filled constant-release implants in rats made obese by ovariectomy potentiated the inhibitory effect of intraperitoneal injection of Cholecystokinin octapeptide on food intake after 24 h of food deprivation. The alterations in the concentration of Cholecystokinin in pjasma and of cholecystokinin-like immunoreactivity in cerebrospinal fluid produced by deprivation of food for 24 h and subsequent food intake for 1 h were unaffected by the oestradiol treatment as was the amount of food consumed during 1 h. Oestradiol-treated rats deprived of food for 6 h, however, consumed less food during a 15-min test than controls. Treatment with oestradiol blunted the decrease in the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid in response to 6 h of food deprivation. No alterations in the concentration of Cholecystokinin in plasma occurred after this period of food deprivation and subsequent feeding during 15 min in either oestradiol-treated or control rats. Thus, treatment with oestradiol enhances responsivity to exogenous Cholecystokinin octapeptide and changes the response of endogenous levels of cholecystokinin-like immunoreactivity in the cerebrospinal fluid to a short period of food deprivation. It is suggested that these effects are caused by an action of oestradiol on Cholecystokinin pathways in the brain. The results support the suggestion that hunger in the rat is inversely related to the decrease in the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid.     

5-HT2A/C receptors mediate the antipsychotic-like effects of alstonine

The purpose of this study was to determine the effects of alstonine, an indole alkaloid with putative antipsychotic effects, on working memory by using the step-down inhibitory avoidance paradigm and MK801-induced working memory deficits in mice. Additionally, the role of serotonin 5-HT_2A/C receptors in the effects of alstonine on mouse models associated with positive (MK801-induced hyperlocomotion), negative (MK801-induced social interaction deficit), and cognitive (MK801-induced working memory deficit) schizophrenia symptoms was examined. Treatment with alstonine was able to prevent MK801-induced working memory deficit, indicating its potential benefit for cognitive deficits now seen as a core symptom in the disease. Corroborating previously reported data, alstonine was also effective in counteracting MK801-induced hyperlocomotion and social interaction deficit. Ritanserin, a 5-HT_2A/C receptor antagonist, prevented alstonine's effects on these three behavioral parameters. This study presents additional evidence that 5-HT_2A/C receptors are central to the antipsychotic-like effects of alstonine, consistently seen in mouse models relevant to the three dimensions of schizophrenia symptoms.     

The role of the hippocampus and 5-HT/GABA interaction in the central effects of benzodiazepine receptor ligands

Summary. The effects of an intrahippocampal administering of a non-selective full (midazolam), a partial benzodiazepine (BDZ) receptor agonist (bretazenil), and a BDZ₁ selective (zolpidem) receptor ligand were examined in the open field test (OFT) of neophobia and Vogel's test (VT) of conflict behavior in rats. Moreover, the influence of local injections of a non-competitive GABA_A receptor antagonist, picrotoxin, on the anxiolytic-like effect of serotonin (5-HT) depletion (p-chlorophenylalanine, p-CPA) in the Vogel test was studied. It was found that in the OFT only midazolam (0.1 μg/site) given to the hippocampus (HP) disinhibited rat exploratory behavior, whereas all the examined compounds inhibited animal motor activity when injected locally at 10.0 μg/site, the highest dose used in the tests. In the VT, again, only midazolam disinhibited rat conflict behavior on a dose-dependent basis. Picrotoxin administered to the HP produced a tendency to increase locomotor activity in rats, and significantly attenuated the anti-conflict action of serotonin depletion without changing the pain threshold and spontaneous drinking of the animals. p-CPA induced potent, dose-dependent and selective 5-HT and 5-hydroxyindoleacetic acid decrease in the HP after administering the dose used in the behavioral experiment. Thus, the present data provide evidence for the lack of selective anxiolytic activity of a partial non-selective agonist and a full selective agonist at the BDZ₁ receptor after their administration to the HP. The model of intra-HP drug injections appeared effective in discriminating the anxiolytic spectrum of activity of new psychotropic compounds. Moreover, the obtained results indicate that the dorsal HP is one of the central sites important for GABA /5-HT interaction that modulates rat emotional behavior. Received January 12, 1998; accepted September 2, 1998     

Synaptically released 5-HT modulates the activity of tonically discharging neuronal populations in the rostral ventral medulla (RVM)

There is substantial evidence for an important modulating role of monoamines (catecholamines and serotonin, 5-HT) in the rostral ventral medulla (RVM), a region which plays an important role in cardiovascular and nociceptive functions. We investigated in slices the role of endogenous monoamines in the synaptic control of the activity of rat RVM neuronal populations using intracellular recordings in the lateral RVM plus lateral aspect of nucleus paragigantocellularis lateralis. A triple-labelling protocol allowed us to identify the location of impaled neurons and their eventual monoaminergic phenotype within the serotonergic and catecholaminergic populations of the RVM. Focal electrical stimulation revealed the existence of a functional monoaminergic input onto RVM neurons which was mediated by endogenous 5-HT acting at inhibitory 5-HT1A receptors but did not involve noradrenergic neurotransmission. The slow 5-HT-mediated inhibitory postsynaptic potential (IPSP) was only observed in the regularly discharging neurons, which were found to be neither catecholaminergic nor serotonergic. The synaptic release of 5-HT was, itself, under an inhibitory control involving GABAA (gamma-aminobutyric acid) receptors. Moreover, we characterized the effect of the 5-HT-releasing agent fenfluramine on this functional 5-HT-mediated synaptic transmission. Our results show that the effect of fenfluramine is biphasic consisting of an initial prolongation of the serotonergic IPSP followed by a decrease in amplitude. Our data provide a basis for the previously reported inhibitory effects of exogenously applied serotonin agonists/antagonists on the autonomic functions controlled by the RVM. This 5-HT pathway, which functionally links the serotonergic and catecholaminergic regions, might play an important role in cardiovascular and nociceptive functions.     

Effect of Naloxone on the Pulsatile Secretion of Luteinizing Hormone in Gonadectomized Male and Female Ferrets Before and After Oestradiol Replacement

Intravenous infusions of the opioid receptor antagonist, naloxone, caused a significant rise in luteinizing hormone (LH) pulse frequency in male and female ferrets which had been gonadectomized 10 days earlier while in breeding condition; mean LH concentrations and LH pulse amplitudes were not affected. By contrast, naloxone failed to stimulate LH pulse frequency, or other LH parameters in gonadectomized ferrets of either sex which received daily injections of a low dose of oestradiol. Our results for the ferret, in which the female is an induced ovulator, resemble those previously obtained in another induced ovulator, the rabbit. They contrast, however, with the results of numerous studies using spontaneously ovulating species in which sex steroids, if anything, facilitate the ability of naloxone to stimulate LH secretion.     

5-HT(1B) receptors in nucleus accumbens efferents enhance both rewarding and aversive effects of cocaine

Whether serotonin-1B (5-HT(1B)) receptor activation enhances or diminishes the reinforcing properties of psychostimulants remains unclear. We have previously shown that increased expression of 5-HT(1B) receptors in nucleus accumbens (NAcc) shell neurons sensitized rats to the locomotor-stimulating and rewarding properties of cocaine. In this study we further examined the contribution of 5-HT(1B) receptors on the effect of cocaine under conditions intended to selectively influence either conditioned place preference or avoidance (CPP or CPA, respectively). Viral-mediated gene transfer techniques were used to overexpress 5-HT(1B) receptors in medial NAcc shell medium spiny neurons projecting to the ventral tegmental area. Animals were then conditioned to associate place cues with the effects of either a low (5 mg/kg) or moderately high (20 mg/kg) dosage of cocaine immediately or 45 min after intraperitoneal cocaine administration. Animals with increased 5-HT(1B) expression showed cocaine-induced CPP immediately after administration of the low 5 mg/kg dose of cocaine, but a CPA 45 min after administration of the high 20 mg/kg dose. Control animals showed no preference at the 5 mg/kg dose and a significant preference at 20 mg/kg. Given this, we believe that increased 5-HT(1B) receptor activation in NAcc shell projection neurons intensifies both the rewarding and negative properties of cocaine use.     

Nicotine regulates 5-HT(1A) receptor gene expression in the cerebral cortex and dorsal hippocampus

The 5-HT(1A) receptor has previously been shown to be important in mediating the behavioural effects of nicotine. It is possible that nicotine administration might regulate the levels of 5-HT receptors in limbic and cortical regions, and such regulations may underlie adaptive responses to nicotine in the central nervous system. The effects of acute and chronic systemic (--)-nicotine administration on 5-HT(1A) receptor gene expression were measured by in situ hybridization, in the rat cerebral cortex, dorsal hippocampus and lateral septum. In the cortex, acute nicotine (0.5 mg/kg i.p.) significantly increased the expression of 5-HT(1A) receptor mRNA 2 h and 24 h after injection. Similarly, acute nicotine significantly increased 5-HT(1A) receptor mRNA in the dentate gyrus (DG), CA3 and CA1 regions of the dorsal hippocampus 2 h and 24 h after injection. Acute nicotine was without effect in the lateral septum. Chronic nicotine (0.5 mg/kg i.p; twice daily for 7 days) significantly decreased 5-HT(1A) receptor mRNA in the cortex 2 h after the final injection, but was without effect at 24 h or 72 h. Chronic nicotine caused no changes in 5-HT(1A) mRNA in the lateral septum or dorsal hippocampus. These data demonstrate that nicotine regulates 5-HT(1A) receptor gene expression in the cortex and hippocampus. The rapid regulation of expression of 5-HT(1A) receptor mRNA leads to the hypothesis that nicotine-induced 5-HT release may alter the postsynaptic sensitivity to 5-HT.     

Morphometry of Cytoarchitecture of the Lateral Hypothalamic Area in the Rat

Abstract: The cytoarchitecture of the lateral hypothalamic area (LHA) in rats was studied with cresyl violet-stained coronal celloidin sections and with sections of the brain impregnated by a Golgi method. Unimodality was established in the frequency distribution histogram of both the somatic cross-sectional area of the LHA neurons and somatic shape (elongation and circularity). The predominant somatic orientation was in the dorsomedial-ventrolateral direction:bimodality of the frequency distribution of somatic orientation was denied. These findings suggest that the LHA neurons examined in the present study are not subdivisible on the basis of the somatic area, shape or orientation. Although the neurons were classified into eight types based upon the dendritic pattern, those in the LHA largely consisted of only three of them; Type III (dendrites extending in two directions along the long axis of soma), Type IV (three directions) and Type VIII (four directions) jointly accounted for 97.1 percent of the total neurons examined. This finding suggests that the parameter of dendritic pattern serves an important purpose in the typing of the rat LHA neurons. The orientation of intrinsic dendrites and intrinsic axons in the LHA has also been described.     

Differential pulse voltammetry in the dorsal horn of the spinal cord of the anesthetized rat: Are the voltammograms related to 5-HT and/or to 5-HIAA?

Treated carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vitro and in vivo determination of indoleamines. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Treated carbon fiber microelectrodes respond in vitro linearly over a large range of concentrations of 5-hydroxytryptamine (5-HT) and of 5-hydroxyindoleacetic acid (5-HIAA), but are 5-8 times more sensitive to 5-HT than to 5-HIAA. In vivo, the question remains as to the exact nature of the peak because the oxidation potentials of 5-HT and 5-HIAA are close together and cannot be monitored separately. Pharmacological investigations were hence carried out in order to characterize the electrochemical signal detected at 300 mV in the dorsal horn of the lumbar spinal cord of chloral hydrate-anesthetized rats. Using 250 micron long carbon fiber microelectrodes, the electrochemical signal stabilizes at 30-90 min and the peak remains constant for up to 210 min. Administration of the monoamine oxidase inhibitor (MAOI) clorgyline produced a progressive decrease of the signal which reached a decrease of 33% of control at 180 min after injection. At this time biochemical measures demonstrated a 117% increase in 5-HT and a 32% decrease in 5-HIAA in the dorsal half of the spinal cord. Reserpine provoked an increase of 20% in the electrochemical peak and the 5-HIAA outflow blocker probenecid gave rise to a sustained plateau of about 60% above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist

A complete transection of the spinal cord at a low thoracic level induces a paraplegic syndrome that is accompanied by a loss of spinal cord serotonin content. Former experimental data suggest that the central pattern generator for locomotion, located in the lumbar segments of the spinal cord, might be able to generate rhythmic motor outputs (similar to automatic walking under certain circumstances) involving exteroceptive stimulations and activation of serotonergic receptors. In the present study, we investigated the effects of a chronic treatment using a serotonergic agonist, delivered continuously to the sublesionned spinal cord, and its effect on motor function recovery. The data obtained from behavioural, kinematic and electromyographic measurements suggest that the chronic stimulation of 5-HT2 type receptors allows motor function recovery. Behavioural measurements show a clear improvement in motor performances when compared to spinal animals (confirmed by kinematic observations): alternating steps and foot placement is recovered in these animals. However, electromyographic data demonstrate that the pattern of activation of the muscles is only restored partially.     

Neurotensin-lmmunoreactive Neurons in the Rat Medial Preoptic Area are Oestrogen-Receptive

The identity of neurons influenced by oestrogen is critical for the understanding of ovarian steroid actions in the brain. The medial preoptic area (MPOA) contains one of the largest oestrogen-receptive cell populations in the rat brain and participates in several oestrogen-dependent functions, including the regulation of luteinizing hormone (LH) secretion and sexual behaviour. Using double immunostaining procedures with antibodies specific for the oestrogen receptor and neurotensin, a neuropeptide implicated in the regulation of LH secretion within this area, we found that approximately half of the neurotensin-immunoreactive neurons in the MPOA also displayed immunoreactivity for the oestrogen receptor. We estimate that oestrogen-receptive neurotensin neurons represent 5% to 12% of all oestrogen receptor-positive cells in the MPOA. Our results provide morphological evidence that neurotensin mediates oestrogen-dependent mechanisms within the brain and suggest that oestrogen may act through preoptic neurotensin neurons to aid in the generation of the LH surge.     

Progesterone stimulates sexual behaviour in female rats by increasing 5-HT activity on 5-HT2 receptors

We have investigated the possibility that there is a correlation between female sexual receptivity and CNS serotonergic activity. In ovariectomized rats primed with a submaximal steroid regime of 2 micrograms oestradiol benzoate (OB) plus 0.2 mg progesterone (P), so that only a proportion were receptive, the 5-hydroxyindoleacetic acid:5-hydroxytryptamine (5-HT) ratio in the hypothalamus was greater in the receptive compared to the non-receptive rats. When rats were primed with 10 micrograms OB alone, there was no difference in 5-HT activity in the receptive and non-receptive animals. Depletion of hypothalamic 5-HT levels by p-chlorophenylalanine methyl ester (PCPA) inhibited behaviour normally induced by OB plus P and this could be reversed by 5-hydroxytryptophan. PCPA had no effect in animals made receptive by OB alone. This indicates that P, but not OB, exerts its stimulatory effect on sexual behaviour via increasing 5-HT activity. Administration of 5-HT into the lateral, but not 3rd ventricle stimulated sexual behaviour and systemic injection of the selective 5-HT1 and 5-HT2 agonists, Ru 24969 and MK 212, inhibited and stimulated behaviour, respectively. It is suggested that 5-HT has a dual effect on female sexual receptivity acting via different systems, the inhibitory tract acting on 5-HT1 and the stimulatory tract on 5-HT2 receptors.     

5HT1A-receptor binding in the brain of cyclic and ovariectomized female rats

Although it has been reported that hypothalamic 5HT1A-receptor functioning is modulated by oestrogen and that this modulation contributes to the regulation of female sexual behaviour, there have been no reports up to now showing changes in numbers of these receptors during the oestrus cycle and after oestrogen treatment. We therefore analysed 5HT1A-receptors in eight brain areas of female rats at different stages of the oestrus cycle, and in ovariectomized (OVX) females without and with oestrogen replacement. In-vitro receptor autoradiography with the agonist 3H-8-OH-DPAT(3H-8-hydroxy-2-[di-n-propylamino]tetralin) was used to determine numbers and affinities of 5HTA1A-receptors. To evaluate the hormonal state of the animals, serum concentrations of oestradiol, progesterone, luteinizing hormone (LH), and prolactin were also measured. Hormone determinations confirmed the expected endocrine states of the animals. In the ventromedial hypothalamic nucleus, the number of 3H-8-OH-DPAT binding sites (Bmax-value) during oestrus was increased compared to dioestrus yielding significant differences when using ANOVA statistics. In OVX females, the number of binding sites was decreased compared to pro-oestrus and oestrus, and after oestrogen replacement, it was as high as during oestrus. All other brain areas analysed (medial preoptic area, bed nucleus of the stria terminalis, lateral septum, cingulate cortex, amygdala, hippocampal region CA1, and layers V and VI of the occipital cortex) showed no significant changes in 3H-8-OH-DPAT binding site numbers. Also the affinity of 3H-8-OH-DPAT binding sites did not change during the oestrus cycle, but in the medial preoptic area, oestradiol-treated OVX animals showed a tendency for increased affinity compared to untreated OVX females. This was indicated by a change in Kd which appeared to be significant when groups were compared with the t-test. We conclude from our data, that in the ventromedial hypothalamic nucleus, which is involved in the regulation of sexual function, 5HT1A-receptors are up-regulated during oestrus, that ovariectomy reduces the receptor numbers, and that oestradiol replacement counteracts the effect of ovariectomy. Since the ventromedial hypothalamic nucleus contains a high number of oestrogen receptive cells, our data indicate that oestrogen up-regulates 5HT1A-receptor expression in this nucleus.     

Altered regulation of the 5-HT system in the brain of MAO-A knock-out mice

Genetic deficiency of monoamine oxidase-A (MAO-A) induces major alterations of mood and behaviour in human. Because serotonin (5-HT) is involved in mood regulation, and MAO-A is responsible for the catabolism of 5-HT, we investigated 5-HT mechanisms in knock-out mice (2-month-old) lacking MAO-A, using microdialysis, electrophysiological, autoradiographic and molecular biology approaches. Compared to paired wild-type mice, basal extracellular 5-HT levels were increased in ventral hippocampus (+202%), frontal cortex (+96%) and dorsal raphe nucleus (DRN, +147%) of MAO-A mutant mice. Conversely, spontaneous firing rate of 5-HT neurons in the DRN (recorded under chloral hydrate anaesthesia) was approximately 40% lower in mutants. Acute 5-HT reuptake blockade by citalopram (0.2 and 0.8 mg/kg i.v.) produced a much larger increase in extracellular 5-HT levels (by approximately 4 fold) and decrease in DRN neuronal firing (with a approximately 4.5 fold decrease in the drug's ED50) in MAO-A knock-out mice, which expressed lower levels of the 5-HT transporter throughout the brain (-13 to -34% compared to wild-type levels). The potency of the 5-HT1A agonist 8-OH-DPAT to produce hypothermia and to reduce the firing of DRN serotoninergic neurons was significantly less in the mutants, indicating a desensitization of 5-HT1A autoreceptors. This was associated with a decreased autoradiographic labelling of these receptors (-27%) in the DRN. Altogether, these data indicate that, in MAO-A knock-out mice, the enhancement of extracellular 5-HT levels induces a down-regulation of the 5-HT transporter, and a desensitization of 5-HT1A autoreceptors which allows the maintenance of tonic activity of 5-HT neurons in the DRN.