Implication of 5-HT2A receptors in the genetic mechanisms of the brain 5-HT system autoregulation

Brain serotonin (5-HT) system has been implicated in pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT2A receptor is involved in the mechanisms of stress-induced psychopathology and impulsive behavior. Here, we investigated the role of 5-HT2A receptor in the autoregulation of the brain 5-HT system. The chronic treatment with agonist of 5-HT2A receptor DOI (1.0 mg/kg, i.p./14 days) produced considerable decrease of 5-HT2A receptor-mediated "head-twitches" in AKR/J mice indicating desensitization of 5-HT2A receptors. Chronic DOI treatment failed to alter 5-HT2A receptor gene expression in the midbrain, hippocampus and frontal cortex. At the same time, the increase in the expression of the gene encoding key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), the increase in TPH2 activity and 5-HT levels and decreased expression of serotonin transporter (5-HTT) gene was found in the midbrain of DOI-treated mice. The results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and the implication of 5-HT2A receptor in the autoregulation of the brain 5-HT system.     

Effect of p-chloroamphetamine on 5-HT1A and 5-HT7 serotonin receptor expression in rat brain

The aim of this study was to investigate if p-chloroamphetamine (PCA), which is neurotoxic to serotonin (5-HT) nerve terminals, was able to induce, like 3,4-methylenedioxymethamphetamine, a region-specific regulation of 5-HT1A receptor mRNA expression. The effect of PCA on the expression of 5-HT7 receptors, which share some pharmacological properties with 5-HT1A receptors, was comparatively studied. PCA (2 x 5 mg/kg) produced a lasting depletion of 5-HT content in the rat frontal cortex and hippocampus. In the hippocampus, the maximal 5-HT depletion was found on day 21 (-70%), whereas in the cortex, the highest 5-HT depletion was found on day 14 (-73%), with a partial but significant recovery on day 21. At the latter time point, 5-HT1A receptor mRNA expression was increased by 80% in the cortex and decreased by 50% in the hippocampus. The 5-HT1A receptor mRNA expression was also enhanced after exposure to PCA of rat cortical but not of hippocampal primary cultures. In regard to 5-HT7 receptor mRNA expression, the most remarkable change after PCA was the great increase (+200%) in the brain-stem. Binding studies to 5-HT1A receptors matched the changes in receptor mRNA expression. Gel shift assays revealed enhanced nuclear protein binding to the KB sequence with use of cortical but not hippocampal extracts of PCA-treated rats. Overall, the data show region-specific changes in 5-HT receptor-type expression that may not be entirely dependent on the neurotoxic effect of PCA on 5-HT terminals.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

The involvement of brain 5-HT(1A)-receptors in genetically determined aggressive behavior

The hypothesis was tested that one of the critical mechanisms underlying genetically determined aggressiveness involves brain serotonin 5-HT(1A)-receptors. The expression of 5-HT(1A)-receptor mRNA in brain structures and functional correlate for 5-HT(1A)-receptors identified as 8-OH-DPAT-induced hypothermia were studied in Norway rats bred over the course of 59 generations for the low and high affective (defensive) aggressiveness with respect to man and in highly aggressive (offensive) MAO A-knockout mice (Tg8 strain). Considerable differences between the aggressive and the nonaggressive animals were shown. Agonist of 5-HT(1A)-receptor 8-OH-DPAT (0.5 mg/kg for rats and 2.0 mg/kg for mice, i.p.) produced a distinct hypothermic reaction in nonaggressive rats and mice and did not affect significantly the body temperature in aggressive animals. In aggressive rats, a significant reduction of the expression of 5-HT(1A)-receptor mRNA was found in the midbrain. In Tg8 mice, 5-HT(1A)-receptor mRNA level was increased in the frontal cortex and amygdala and not changed in the hypothalamus and the midbrain. The results provide support for the idea that brain 5-HT(1A)-receptors contribute to the genetically determined individual differences in aggressiveness.     

Use of Arc expression as a molecular marker of increased postsynaptic 5-HT function after SSRI/5-HT1A receptor antagonist co-administration

An increase in central postsynaptic 5-hydroxytryptamine (5-HT) function activates expression of activity-related cytoskeletal protein (Arc). Here, Arc expression was used to test whether, in rats, co-administration of a 5-HT re-uptake inhibitor (paroxetine) and a 5-HT1A receptor antagonist (WAY 100635) increases postsynaptic 5-HT function. After pre-treatment with WAY 100635 (0.3 mg/kg s.c.), paroxetine (5 mg/kg s.c.) caused a threefold increase in 5-HT in prefrontal cortex microdialysates. In situ hybridization studies found that neither paroxetine (5 mg/kg s.c.) nor WAY 1000635 (0.3 mg/kg s.c.) altered Arc mRNA abundance in any region examined. In contrast, paroxetine (5 mg/kg s.c.) increased Arc mRNA after pre-treatment with WAY 100635 (0.3 mg/kg s.c.). This increase was apparent in cortical regions (frontal, parietal and cingulate) and caudate nucleus but was absent in hippocampus (CA1). Increases in Arc mRNA were accompanied by an increase in c-fos mRNA. The increase in Arc expression induced by paroxetine/WAY 100635 was abolished by the 5-HT synthesis inhibitor, p-chlorophenylalanine (300 mg/kg i.p., daily for two days). In conclusion, paroxetine and WAY 100635 injected in combination (but not alone) caused a region-specific, 5-HT-mediated increase in Arc expression. These data provide molecular evidence that co-administration of a 5-HT re-uptake inhibitor and 5-HT1A receptor antagonist increases 5-HT function at the postsynaptic level.     

The toadfish serotonin 2A (5-HT(2A)) receptor: molecular characterization and its potential role in urea excretion

Based on early pharmacological work, the serotonin 2A (5-HT(2A)) receptor subtype is believed to be involved in the regulation of toadfish pulsatile urea excretion. The goal of the following study was to characterize the toadfish 5-HT(2A) receptor at a molecular level, to determine the tissues in which this receptor is predominantly expressed and to further investigate the pharmacological specificity of toadfish pulsatile urea excretion by examining the effect of ketanserin, a 5-HT(2A) receptor antagonist, on resting rates of pulsatile urea excretion. The full-length toadfish 5-HT(2A) receptor encodes a 496 amino acid sequence and shares 57-80% sequence identity to 5-HT(2A) receptors of other organisms, with 100% conservation among important ligand-binding residues. Toadfish 5-HT(2A) receptor mRNA expression was highest in the swim bladder and gonad, followed by the whole brain. All other tissues tested (esophagus, stomach, anterior intestine, posterior intestine, rectum, liver, kidney, heart, muscle and gill) had mRNA expression levels that were significantly less than whole brain. Toadfish 5-HT(2A) receptor mRNA expression within the brain was highest in the hindbrain, telencephalon and midbrain/diencephalon regions. Treatment with the 5-HT(2A) receptor antagonist, ketanserin, resulted in a significant decrease in the pulsatile component of spontaneous urea excretion due to a reduction in urea pulse size with no significant change in pulse frequency. These results lend further support for the 5-HT(2A) receptor in the regulation of pulsatile urea excretion in toadfish.     

The brain 5-HT1A receptor gene expression in hibernation

Hibernation is a unique physiological state characterized by profound reversible sleep-like state, depression in body temperature and metabolism. The serotonin 5-hydroxytryptamine_1A (5-HT_1A) receptor gene sequence in typical seasonal hibernator, ground squirrel ( Spermophilus undulatus ), was specified. It was found that the fragment encoding the fifth transmembrane domain showed 93.6% of homology with the analogous fragment of the mouse and rat genes and displayed 88.5% homology with the human 5-HT_1A receptor gene. Using primers designed on the basis of obtained sequence, the expression of 5-HT_1A receptor gene in the brain regions in active, entering into hibernation, hibernating and coming out of hibernation ground squirrels was investigated. Significant structure-specific changes were revealed in the 5-HT_1A messenger RNA (mRNA) level in entry into hibernation and in arousal. An increase in the 5-HT_1A gene expression was found in the hippocampus during the prehibernation period and in ground squirrels coming out of hibernation, thus confirming the idea of the hippocampus trigger role in the hibernation. Significant decrease in 5-HT_1A receptor mRNA level in the midbrain was found in animals coming out of hibernation. There was no considerable changes in 5-HT_1A receptor mRNA level in different stages of sleep–wake cycle in the frontal cortex. Despite drastically decreased body temperature in hibernating animals (about 37°C in active and 4–5°C in hibernation), 5-HT_1A receptor mRNA level in all examined brain regions remained relatively high, suggesting the essential role of this 5-HT receptor subtype in the regulation of hibernation and associated hypothermia.     

Regulation of 5-HT1A receptor function in brain following agonist or antidepressant administration

Adaptive changes in the serotonergic system are generally believed to underlie the therapeutic effectiveness of the azapirone anxiolytics and a variety of antidepressant drugs. The serotonin-1A (5-HT(1A)) receptor has been implicated in affective disorders. Thus, studies of the regulation of 5-HT(1A) receptor function may have important implications for our understanding the role of this receptor in the mechanism of action of these therapeutic agents. This review focuses on the regulation of central 5-HT(1A) receptor function following administration of 5-HT(1A) receptor agonists or antidepressant drugs expected to increase the synaptic concentration of the neurotransmitter 5-HT. The majority of evidence supports regional differences in the regulation of central 5-HT(1A) receptor function following repeated agonist or antidepressant administration, which may be due to differences in processes involved in desensitization of the receptor at the cellular level. Region-specific differences in the regulation of 5-HT(1A) receptor function may be based on compensatory changes distal to the receptor, such as regulatory changes at the level of effector (e.g. adenylyl cyclase or ion channel), or at the level of the G protein such as changes in G protein expression, or phosphorylation of the G protein. It may be that the increase in serotonin neurotransmission, due to somatodendritic autoreceptor desensitization following agonist or antidepressant treatment, to normo-sensitive 5-HT(1A) receptors in certain brain regions (e.g. hippocampus or cortex) and to sub-sensitive 5-HT(1A) receptors in other brain regions (e.g. amygdala or hypothalamus) underlies the therapeutic efficacy of these drugs.     

Involvement of 5-HT<Subscript>2A</Subscript> receptors in genetic mechanisms of autoregulation of brain 5-HT system

The brain serotonin (5-HT) system has been implicated in the pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT_2A receptors are involved in the mechanisms of stressinduced psychopathology and impulsive behavior. In this work, we investigated the role of 5-HT_2A receptors in the autoregulation of the brain 5-HT system. Chronic treatment with DOI, a 5-HT_2A receptor agonist (1.0 mg/kg, i.p./14 days), produced a considerable decrease in the number of 5-HT_2A receptor-mediated head twitches in AKR/J mice, indicating the desensitization of 5-HT_2A receptors. Chronic DOI treatment did not affect the expression of the 5-HT_2A receptor gene in the midbrain, hippocampus and frontal cortex. At the same time, an increase in the expression of the gene encoding a key enzyme of 5-HT synthesis, tryptophan hydroxylase-2 (TPH-2), accompanied with an increase in TPH-2 activity and 5-HT levels, and decreased expression of the serotonin transporter (5-HTT) gene were observed in the midbrain of DOI-treated mice. These results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and implication 5-HT_2A receptors in the autoregulation of the brain 5-HT system.     

Plasticity of 5-HT 1A receptor-mediated signaling during early postnatal brain development

The presence of serotonin 1A receptor (5-HT(1A)-R) in the hippocampus, amygdala, and most regions of the frontal cortex is essential between postnatal day-5-21 (P5-21) for the expression of normal anxiety levels in adult mice. Thus, the 5-HT(1A)-R plays a crucial role in this time window of brain development. We show that the 5-HT(1A)-R-mediated stimulation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) in the hippocampus undergoes a transition between P6 and P15. At P6, a protein kinase C (PKC) isozyme is required for the 5-HT(1A)-R -->Erk1/2 cascade, which causes increased cell division in the dentate gyrus. By contrast, at P15, PKC alpha participates downstream of Erk1/2 to augment synaptic transmission through the Schaffer Collateral pathway but does not cause increased cell division. Our data demonstrate that the 5-HT(1A)-R -->Erk1/2 cascade uses PKC isozymes differentially, first boosting the cell division to form new hippocampal neurons at P6 and then undergoing a plastic change in mechanism to strengthen synaptic connections in the hippocampus at P15.     

Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice

The aim of the present studies was to determine the effects of reduced or absent serotonin (5-HT) transporters (5-HTTs) on 5-HT2A and 5-HT2C receptors. The density of 5-HT2C receptors was significantly increased in the amygdala and choroid plexus of 5-HTT knockout mice. On the other hand, the density of 5-HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5-HTT knockout mice. However, 5-HT2A mRNA was not changed in any brain region measured. 5-HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5-HT2A receptors was evaluated by hormonal responses to (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5-HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5-HTTs changes the density of 5-HT2A and 5-HT2C receptors in a brain region-specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5-HTT knockout mice.     

Differential effect of lithium on 5-HT1 receptor-linked system in regions of rat brain.

The effect of chronic administration (0.4% for 30 days) of lithium carbonate (Li2CO3) on 5-HT1 receptor-linked second messenger system was studied in regions of rat brain. We observed that chronic treatment of Li2CO3, significantly decreased the density of [3H]5-HT binding sites in cortex (62%), hippocampus (64%) and striatum (65%), compared to the control levels. The affinity of [3H]5-HT to 5-HT1 binding sites was significantly decreased in all the regions. A significant decrease in the density of high affinity 5-HT1A receptor sites was observed in cortex (81%) and hippocampus (42%), without change in the affinity of [3H]8-OH-DPAT for 5-HT1A sites in these regions. 5-HT-stimulated, but not basal, adenylyl cyclase activity was significantly increased in all the regions after Li treatment. The present study concludes that the increase in the 5-HT-stimulated adenylyl cyclase activity might be attributed to the functional downregulation of 5-HT1 receptors, as these are negatively coupled to adenylyl cyclase, suggesting the involvement of 5-HT1 receptor mediated response in the therapeutic efficacy of lithium.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.     

大鼠中央杏仁核5-HT3受体参与胸腺功能调制

本研究旨在探讨大鼠中央杏仁核(central amygdala,CeA)内5-HT3受体激动之后,对丝裂原刀豆球蛋白A(concanavalin A,ConA)刺激的胸腺细胞增殖反应的影响,及其潜在的神经内分泌调节环路.分别经大鼠腹腔(intraperitoneal,i.p.)、双侧侧脑室(intracerebroventricle,i.c.v.)和双侧CeA(intracentral amygdala,i.c.a.)注射选择性5-HT3受体激动剂1-phenylbiguanide(PBG),同时制备正常大鼠胸腺细胞悬液与不同浓度PBG(1×10-8～1×10-5 mol/L)体外共同孵育.经MTT法测定显示,无论有无ConA刺激,正常大鼠离体胸腺细胞在与PBG(1×10-8～1×10-5 mol/L)体外共同孵育时其增殖反应均不受后者影响;PBG i.p.(每天0.5 mg/kg,连续5 d)对ConA刺激的胸腺细胞的增殖反应亦无影响,而PBG i.c.v.(每天10 μg/侧,连续5 d)则显著增强之;当PBG i.c.a.(每天1.0 μg/侧,1 d或连续3、5、7 d)时,ConA刺激的胸腺细胞的增殖反应于给药后第1天即开始增强且日益显著,第5天达到高峰,第7天则趋于减弱.在给予PBG 5 min前相同给药部位先给予5-HT3受体拮抗剂tropisetvon(TRP)预处理可逆转PBG的促胸腺细胞增殖效应.免疫组织化学SABC法检测显示,PBG(1.0 μg/侧,i.c.a.)单次给药后各脑区可相继出现大量c-Fos阳性细胞(CeA1 h;海马及皮层1～2 h;下丘脑4 h;中脑导水管周围灰质8 h),并迅速达到各自高峰(CeA1 h;海马及皮层2 h;下丘脑4 h),与相应的生理盐水对照组及TRP预处理组相比均有显著性差异.随后,这一表达在各脑区中逐步减弱并消失(CeA4 h;海马、皮层及下丘脑8 h).由此推论,大鼠CeA内5-HT3受体至少可部分通过边缘系统-皮层-下丘脑-中脑导水管周围灰质这一神经内分泌环路调制胸腺细胞功能.     

Dose-dependent dual effect of corticosterone on cerebral 5-HT metabolism

The action of 1.0 and 10.0 mg/kg (i.p.) of corticosterone on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents and on serotonin turnover, measured by an MAO-inhibitor method, was studied at 30 and 120 min after administration. A 1.0 mg/kg dose of corticosterone increased the serotonin content and turnover in the hypothalamus and mesencephalon 30 min after administration; however, it was ineffective on dorsal hippocampus and frontal and parietal cortex. 5-HIAA content did not change significantly in any of the brain areas studied. A 10.0 mg/kg dose of corticosterone decreased the serotonin content and turnover in the hypothalamus and mesencephalon; it was ineffective in other brain areas investigated. 5-HIAA content significantly decreased in the hypothalamus while it increased in the mesencephalon and dorsal hippocampus. In the parietal and frontal cortex, 5-HIAA content did not change following administration of 10.0 mg/kg of corticosterone. At 120 min after corticosterone administration, neither 5-HT content and turnover nor 5-HIAA content showed any change in the brain areas investigated. The results suggest that corticosteroids might change the activity of the brain serotoninergic system in a dose- and time-dependent manner, and in this way the serotoninergic system might play an important role in mediation of the corticosteroid effect exerted on brain function.     

Circadian and seasonal rhythms of 5-HT receptor subtypes, membrane anisotropy and 5-HT release in hippocampus and cortex of the rat.

Specific serotonin binding (5-HT1, 5-HT1A, and 5-HT2 subtypes) and membrane anisotropy were measured at 2 h intervals over a 24 h period in the hippocampus and cortex of Wistar WU rats, housed under a 12 h light-dark cycle, with lights on at 07.00. All experiments were performed both in March and December. In the hippocampus significant circadian rhythms could be ascertained for 5-HT1 binding sites in March and December while for 5-HT1A (subtype of 5-HT1) binding sites the circadian rhythm was only significant in March. The membrane anisotropy also showed significant variations only in March. Circadian rhythms were also found in the cortex for 5-HT1 (December) and 5-HT2 (March and December) binding sites as well as for the membrane anisotropy (December). A correlation was found between membrane anisotropy and 5-HT1 and 5-HT2 binding sites in hippocampus and cortex, respectively. A circadian rhythmicity was also observed for serotonin release as measured by in vivo voltammetry in both brain areas. The results obtained on the diurnal variations of serotonin receptor subtypes and serotonin release and the probable inverse relationship of these two parameters may be relevant in understanding the coupling of pre- and postsynaptic activity.     

Inactivation of 5-HT1A and [3H]5-HT Binding Sites by N-Ethoxycarbonyl-2-Ethoxy-1, 2-Dihydroquinoline (EEDQ) in Rat Brain

Inactivation of 5-HT_1A and [³H]5-HT binding sites by N-Ethoxycarbonyl-2-ethoxy-1, 2-dihydro-quinoline (EEDQ) was studied in regions of rat brain. After exposure to EEDQ (4 mg/kg body wt.) for 7 days, it is observed that the density of 5-HT₁ receptor sites was decreased by nearly 20% in both cortex and hippocampus. The decrease, however, in 5-HT_1A sites was more significant (70%) in both the regions. The affinity of [³H]5-HT to 5-HT₁ sites was decreased significantly in both cortex and hippocampus after exposure to EEDQ, without affecting the Kd of 5-HT_1A sites. Displacement studies suggested that EEDQ has high affinity to 5-HT₁ sites with a Ki of 42.9 ± 2.4 nM. After exposure neither basal nor 5-HT stimulated adenylyl cyclase activity was changed in cortex. The results of this study suggest that EEDQ decreases the density of 5-HT₁ and 5-HT_1A receptor sites but does not cause functional downregulation of these sites in rat brain.