Treatment of Pregnant Alcohol-Consuming Rats with Buspirone: Effects on Serotonin and 5-Hydroxyindoleacetic Acid Content in Offspring

This laboratory previously demonstrated that in utero ethanol exposure markedly impairs the development of the serotonergic system in rat brain. Developmental abnormalities could be detected as early as G15 in the brainstem and G19 in the cortex. Because of the importance of fetal serotonin (5-HT) and 5-HT_1A receptors for the normal development of 5-HT containing neurons, we initiated studies to determine whether administration of a 5-HT_1A agonist, buspirone, to pregnant rats could overcome the adverse effects of in utero ethanol exposure on the developing serotonergic system in offspring. Female, Sprague-Dawley rats were given daily subcutaneous injections of buspirone (4.5 mg/kg) from gestational day 13 (G13) to G20. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cortex and cortical regions. These experiments demonstrated that the ethanol-associated abnormalities in the development of the serotonergic system can be partially overcome by in utero exposure to buspirone. Specifically, whereas untreated ethanol rats had a deficiency of 5-HT and/or 5-HIAA in whole cortex on PN5, and in the motor cortex on PN19 and 35, no significant differences were detected in these regions of the age-matched offspring of buspirone-treated, ethanol-fed rats. In contrast, the 5-HT and 5-HIAA deficiency in the somatosensory cortex of 19-dayold offspring of ethanol-fed rats was not corrected by in utero buspirone treatment. These results suggest that the abnormal development of cortical projections of serotonergic neurons may be due in part to the low fetal 5-HT content in ethanol-exposed rats and may potentially be overcome by in utero treatment with a 5-HT_1A agonist. However, additional studies are needed to elucidate the importance of dose and timing of buspirone administration, and the effects of in utero buspirone exposure on other components of serotonergic and nonserotonergic neurons.     

Effects of Chronic Ethanol Consumption and Aging on 5-HT2A Receptors and 5-HT Reuptake Sites

The serotonergic system in brain is adversely affected by both aging and chronic ethanol consumption. The present study examined the combined effects of aging and chronic ethanol consumption on two components of the serotonergic system. Serotonin (5-HT) reuptake sites and 5-HT_2A receptors were quantitated in brain areas of 5-, 14-, and 24-month-old male Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet on a chronic basis. The regions examined include those containing the cell bodies and projections of serotonergic neurons. These experiments demonstrated the sensitivity of the serotonergic system of male Fischer 344 rats to both aging and chronic ethanol consumption. In control rats, aging was associated with a decline in the concentration of 5-HT_2A receptors in the nucleus accumbens and four cortical regions: frontal, parietal, piriform, and cingulate cortex. 5-HT_2A receptors were also reduced in the frontal, parietal, and cingulate cortex of aged ethanol-fed rats. In contrast, 5-HT reuptake sites were increased in older rats in the frontal cortex, nucleus accumbens, amygdala, and CA3 region of the hippocampus. If comparable changes in 5-HT_2A receptors and 5-HT reuptake sites occur in elderly humans, they may contribute to ethanol consumption, and lead to cognitive and other age-related problems. These changes may also alter the effectiveness of serotonergic drugs used in the treatment of alcoholism and mental disorders. The effects of chronic ethanol consumption were more limited. The only significant ethanol effect was an increase of 5-HT_2A receptors in the nucleus accumbens of 5-month-old ethanol-fed rats.     

Effects of Maternal Ethanol Consumption and Buspirone Treatment on Dopamine and Norepinephrine Reuptake Sites and D1 Receptors in Offspring

Previously, it was shown that in utero ethanol exposure results in decreased serotonin (5-HT) and altered concentrations of 5-HT reuptake sites and 5-HT_1A receptors in fetal and/or postnatal rats. Because fetal 5-HT is an essential trophic factor, this laboratory previously investigated the hypotheses that the early ethanol-associated 5-HT deficit contributed to subsequent development abnormalities in the serotonergic system and that the effects of the fetal 5-HT deficit could be prevented by maternal treatment with buspirone, a 5-HT_1A receptor agonist. The present report determined the effects of maternal treatment with buspirone on two other neurotransmitter systems in the developing offspring of ethanol-fed dams: dopamine (DA) and norepinephrine reuptake sites and D1 receptors in postnatal day 19 offspring of control and ethanol-fed dams, that received daily injections of saline or 4.5 mg/kg buspirone. These investigations found that in utero ethanol exposure significantly decreased norepinephrine reuptake sites in the dorsomedial hypothalamic nucleus and anteroventral thalamic nucleus. There was also an ethanol effect in the dorsal raphe. D1 receptors were moderately increased (5–10% increase) in the striaturn, and DA reuptake sites were unchanged in PN19 ethanol-exposed offspring. No other significant ethanol-related effects were noted. Maternal buspirone treatment did not adversely affect the concentration of DA reuptake sites or D1 receptors in control rats. Thus, whereas buspirone exerts protective effects on the developing 5-HT system of ethanol-exposed rats, it does not appear to damage the development of the DA system. Maternal buspirone produced only one significant abnormality in control offspring; it resulted in a significant reduction of norepinephrine reuptake sites in the DR.     

Ethanol Fails to Modify [3H]GR65630 Binding to 5-HT3 Receptors in NCB-20 Cells and in Rat Cerebral Membranes

Low concentrations of ethanol have been found to enhance the electrophysiologic effect of serotonin (5-HT) acting at 5-HT3 receptors on NCB-20 cells. To determine whether this action of ethanol reflects a change in the agonist-receptor interaction, the effect of ethanol (100 mM) on agonist and antagonist binding to 5-HT3 receptor was studied in vitro in membrane from NCB-20 cells and from cortex plus hippocampus of rat. The antagonist [3H]GR65630 was used to label 5-HT3 recognition sites. Ethanol did not change the characteristics of saturable [3H]GR65630 binding in either membrane preparation. In competition studies, the agonists 5-HT and 2-methyl-5-HT completely inhibited the binding of [3H]GR65630 to NCB-20 cell membranes, while in brain membranes the maximum displacement of specific [3H]GR65630 binding by 5-HT was approximately 30%. Ethanol decreased the affinity of the receptor for 2-methyl-5-HT, but not to 5-HT in NCB-20 cells, and had no effect on agonist binding in brain membranes. The results indicate that enhancement of 5-HT responses at 5-HT3 receptors by ethanol is not a result of changes in the equilibrium binding characteristics of the agonist.     

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Contraction, relaxation, inositol 1,4,5-triphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. 5-HT binding was completely inhibited by 5-HT and partially by 5-HT_2A (ketanserin), 5-HT₄ (SDZ-205,557), and 5-HT_1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP₃, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT_2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT_2A receptors mediating contraction and 5-HT₄ receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT₄ receptors are closely similar or identical to 5-HT_1p receptors.     

5-HT4 and 5-HT2 receptors antagonistically influence gap junctional coupling between rat auricular myocytes

5-hydroxytryptamine-4 (5-HT₄) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT₄ receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT₄ receptors were present and real-time RT-PCR analysis revealed that 5-HT_4b was the predominant isoform. Serotonin (1 μM) significantly reduced cAMP concentration unless a selective 5-HT₄ inhibitor (GR113808 or ML10375, both 1 μM) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT₄ inhibitor but strongly reduced when 5-HT_2A and 5-HT_2B receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT₄, 5-HT_2A and 5-HT_2B were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT₄ (mainly 5-HT_4b), 5-HT_2A and 5-HT_2B receptors coexisted in auricular myocytes of newborn rat, that 5-HT₄ activation reduced cAMP concentration, I_CaL and intercellular coupling whereas 5-HT_2A or 5-HT_2B activation conversely enhanced GJIC.     

Role of the 5-HT2AReceptor and α1-adrenoceptor in the Contractile Response of Rat Pulmonary Artery to 5-HT in the Presence and Absence of Nitric Oxide

This study investigated the role of 5-HT_2Areceptors andα₁ -adrenoceptors in the contractile response to 5-HT in the first branch pulmonary artery of the rat and their interaction with endogenous nitric oxide. 5-HT and phenylephrine induced concentration-dependent contractions. Theα₁ -adrenoceptor antagonists prazosin, HV723 and phentolamine produced concentration-dependent rightward shifts of the 5-HT concentration-response curves (CRC) consistent with an action at α₁-adrenoceptors. The 5-HT₂receptor antagonists ritanserin, ketanserin and methysergide produced rightward shifts that were less than would have been predicted for an action solely at 5-HT_2Areceptors. 5-HT and phenylephrine CRCs were shifted to the left by -NAME. Endothelium denudation also increased the tissue sensitivity to 5-HT. In the presence of -NAME, ketanserin produced greater antagonism of the 5-HT CRC but not the phenylephrine CRC. Ketanserin also produced greater antagonism of the 5-HT CRC in endothelium denuded rings compared with endothelium intact rings. These findings indicate (a) that both theα₁ -adrenoceptor class and the 5-HT_2Areceptor is involved in the contractile response to 5-HT; (b) in the presence of endogenous nitric oxide the contractile response to 5-HT is mediated predominently byα₁ -adrenoceptors; (c) inhibition of endogenous nitric oxide potentiates the 5-HT_2Areceptor-mediated component of the contraction.     

Genetic deletion of MAO-A promotes serotonin-dependent ventricular hypertrophy by pressure overload

The potential role of serotonin (5-HT) in cardiac function has generated much interest in recent years. In particular, the need for a tight regulation of 5-HT to maintain normal cardiovascular activity has been demonstrated in different experimental models. However, it remains unclear how increased levels of 5-HT could contribute to the development of cardiac hypertrophy. Availability of 5-HT depends on the mitochondrial enzyme monoamine oxidase A (MAO-A). Therefore, we investigated the consequences of MAO-A deletion on ventricular remodeling in the model of aortic banding in mice. At baseline, MAO-A deletion was associated with an increase in whole blood 5-HT (39.4 ± 1.9 μM vs. 24.0 ± 0.9 μM in KO and WT mice, respectively). Cardiac 5-HT_2A, but not 5-HT_2B receptors were overexpressed in MAO-A KO mice, as demonstrated by real-time PCR and Western-blot experiments. After aortic banding, MAO-A KO mice demonstrated greater increase in heart wall thickness, heart to body weight ratios, cardiomyocyte cross-section areas, and myocardial fibrosis compared to WT. Exacerbation of hypertrophy in KO mice was associated with increased amounts of 5-HT in the heart. In order to determine the role of 5-HT and 5-HT_2A receptors in ventricular remodeling in MAO-A KO mice, we administered the 5-HT_2A receptor antagonists ketanserin (1 mg/kg/day) or M100907 (0.1 mg/kg/day) during 4 weeks of aortic banding. Chronic administration of these antagonists strongly prevented exacerbation of ventricular hypertrophy in MAO-A KO mice. These results show for the first time that regulation of peripheral 5-HT by MAO-A plays a role in ventricular remodeling via activation of 5-HT_2A receptors.     

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo

Objective

Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT₇ receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation.

Methods

Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2–4 rats/sample) to evaluate 5-HT₇ receptor expression.

Results

Topical 5-HT (1–10 nmols) or the 5-HT_1/7 receptor agonist, 5-carboxamidotryptamine (10–30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT₇ receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT₇ receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT₇ receptors.

Conclusions

5-HT₇ receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.     

Functional 5-HT receptor subtypes in the isolated canine common carotid artery

Summary The vascular responses to 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT, a selective 5-HT₁-like receptor agonist), alphamethyl-5-HT (-M-5-HT, a relatively selective 5-HT₂ receptor agonist), noradrenaline (NA), and KCl were examined in isolated, cannulated, and perfused canine common carotid arterial preparations. They caused strong vasoconstrictions. The rank order of vasoconstrictive potency was 5-HT > -M-5-HT NA > 5-CT >> KCl. The 5-HT-induced vasoconstriction was significantly depressed by methysergide (a 5-HT₁ and 5-HT₂ receptor antagonist), ketanserin (a selective 5-HT₂ receptor antagonist), and spiperone (a selective 5-HT₂ receptor antagonist). The 5-CT- and -M-5-HT-induced vasoconstrictions were also significantly inhibited by methysergide, spiperone, and ketanserin. The NA-induced vasoconstriction was readily inhibited by bunazosin (an -adrenoceptor antagonist) and ketanserin but not significantly inhibited by spiperone and methysergide. KCl has a weak potency for producing a vasoconstriction of the canine common carotid artery. A relatively large dose of diltiazem (a calcium channel blocker) did not modify 5-HT-induced vasoconstrictions. From these results, we conclude that (a) the canine common carotid artery contains abundant 5-HT receptors; (b) there are no functional 5-HT₁ receptors, but 5-HT₂ receptors are prominent; (c) 5-CT-induced vasoconstrictions might be due to activation of 5-HT₂ but not to 5-HT₁ receptors; (d) 5-HT-induced vasoconstriction might not involve -adrenoceptors; and (e) the vasoconstriction related to 5-HT in the common carotid artery is not significantly mediated via activation of calcium ion channels of smooth muscle cells, but may be induced by calcium ions from intracellular stores.     

Serotonin receptor subtypes influence prolactin secretion in the turkey

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D₁ dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(−)-DOI hydrochloride (DOI), a selective 5-HT_2A eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT_2A receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D₁ DA receptors in the INF were blocked by the D₁ DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT_2A receptor subtype. The DOI-induced PRL increase did not occur when (±)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T_1A receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT_2A receptors are activated, they influence the release of DA to the INF. When 5-HT_1A receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT_2A receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D₂ DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT_1A receptors, could effectively inhibit PRL secretion.     

Effects of Chronic Alcohol Consumption and Aging on Dopamine D1 Receptors in Fischer 344 Rats

The present study examined the hypothesis that chronic alcoholism augments the age-related loss of dopamine D₁ receptors. This hypothesis was investigated because previous studies reported that both aging and chronic alcoholism produce significant changes in dopaminergic systems, and because chronic alcoholism potentiates some age-related CNS losses. In addition, this study investigated the effects of aging on D₁ receptors in animals 1 and 7 days after withdrawal from chronic ethanol. Quantitative autoradiography was used to measure [³H]SCH 23390 binding to D₁ receptors in brain areas associated with both the nigrostriatal and mesocorticolimbic dopamine systems. Receptors were assessed in 5-, 14-, and 24-month-old male Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet for 6 weeks. The results of these studies demonstrated that aging is associated with a significant decline in D₁ receptors in the rostral and caudal striatum, and substantia nigra of both control and ethanol-fed rats. These receptor changes in the nigrostriatal system may be associated with motor abnormalities. In addition, there was an age-related decline in D₁ receptors in two brain areas of the mesocorticolimbic system: the nucleus accumbens and frontal cortex. The latter findings may be important because of the involvement of this system with the rewarding properties of ethanol and other drugs of abuse. There were no age-related differences in the response of D₁ receptors to ethanol withdrawal in the caudal and rostral striatum, substantia nigra, and nucleus accumbens. Consumption of the 6.6% (v/v) ethanol-containing liquid diet for 6 weeks did not augment any of the age-related losses of D, receptors. Nonetheless, we cannot eliminate the possibility that either a more prolonged ethanol exposure or consumption of a higher dose of ethanol would have had more pronounced effects on the young to aged rats.     

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HT_apAC1, stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HT_apAC1 expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HT_apAC1 as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.     

5-HT 2A Receptor and 5-HT Degradation Play a Crucial Role in Atherosclerosis by Modulating Macrophage Foam Cell Formation,Vascular Endothelial Cell Inflammation,and Hepatic Steatosis

Aim: Previously, we found that diabetes-related liver dysfunction is due to activation of the 5-HT _2A receptor (5-HT _2A R) and increased synthesis and degradation of 5-HT. Here, we investigated the role of 5-HT in the development of atherosclerosis. Methods: The study was conducted using high-fat diet-fed male ApoE ^−/− mice, THP-1 cell-derived macrophages, and HUVECs. Protein expression and biochemical indexes were determined by Western blotting and quantitative analysis kit, respectively. The following staining methods were used: oil red O staining (showing atherosclerotic plaques and intracellular lipid droplets), immunohistochemistry (showing the expression of 5-HT _2A R, 5-HT synthase, and CD68 in the aortic wall), and fluorescent probe staining (showing intracellular ROS). Results: In addition to improving hepatic steatosis, insulin resistance, and dyslipidemia, co-treatment with a 5-HT synthesis inhibitor and a 5-HT _2A R antagonist significantly suppressed the formation of atherosclerotic plaques and macrophage infiltration in the aorta of ApoE ^−/− mice in a synergistic manner. Macrophages and HUVECs exposed to oxLDL or palmitic acid in vitro showed that activated 5-HT _2A R regulated TG synthesis and oxLDL uptake by activating PKCε, resulting in formation of lipid droplets and even foam cells; ROS production was due to the increase of both intracellular 5-HT synthesis and mitochondrial MAO-A-catalyzed 5-HT degradation, which leads to the activation of NF-κB and the release of the inflammatory cytokines TNF-α and IL-1β from macrophages and HUVECs as well as MCP-1 release from HUVECs. Conclusion: Similar to hepatic steatosis, the pathogenesis of lipid-induced atherosclerosis is associated with activation of intracellular 5-HT _2A R, 5-HT synthesis, and 5-HT degradation.     

Decrease in Gastric Sensitivity to Distension by 5-HT1A Receptor Agonists in Rats

Using an in vivo model for evaluation of gastricsensitivity in awake rats, we aimed to determine whether5-hydroxytryptamine 1A (5-HT_1A) agonistsmodify pain threshold and gastric compliancespecifically through 5-HT_1A receptors. Isobaricgastric distensions were performed with a barostat usingsteps of 5 mm Hg in male rats equipped with a gastricballoon and electrodes implanted in the neck muscles.Gastric distension at 15 or 20 mm Hg induced a typicalposture associated with contractions of the neckmuscles. Rats received drugs 30 min before gastricdistension. The 5-HT receptor agonist8-hydroxy-2-(di-n-propylamino)tetra1A lin (8-OH-DPAT),administered intraperitoneally (0.5 mg/kg) increasedgastric pain threshold and gastric tone. These effectswere reproduced when administered centrally (0.05 mg/kg) and blocked by intracerebroventricularadministration of the 5-HT_1A antagonist WAY100635. Flesinoxan (4 mg/kg, intraperitoneally), another5-HT_1A agonist reproduced the effects of8-OH-DPAT on pain threshold and gastric tone and the alpha-receptorantagonist yohimbine did not modify the action of8-OH-DPAT. Our results indicate that activation of5-HT_1A receptors at the level of the centralnervous system increases gastric tone and decreases gastric sensitivityto distension.     

5-HT transporter sites and 5-HT1A and 5-HT3 receptors in Fawn-Hooded rats: a quantitative autoradiography study

BACKGROUND: The neuroanatomical profiles of 5-HT1A receptors, 5-HT3 receptors, and 5-HT transporters (5-HTT) in the brain of the Fawn-Hooded (FH) rat, particularly mesocorticolimbic regions, are not fully elucidated. METHODS: By means of in vitro quantitative autoradiography, we used [3H]citalopram, [3H]8-OH-DPAT, and [3H]GR65630 to label 5-HTT, 5-HT1A receptors, and 5-HT3 receptors in the brain of alcohol-na?ve FH rats, Wistar-Kyoto (WKY) rats, and FH rats given free access to 5% ethanol and/or after 24 to 48 hr withdrawal. RESULTS AND CONCLUSIONS: In alcohol-na?ve rats, FH rats displayed significantly higher (p < 0.05) densities of [3H]citalopram binding in the nucleus accumbens (+30%), lateral septum (+37%), ventral pallidum (+21%), and ventral tegmental area (+24%), as well as an increased binding of [3H]8-OH-DPAT to 5-HT1A receptors in the frontal and parietal cortex (+33%), occipital and temporal cortex (+25%), and hippocampal CA3 region (+31%), compared with WKY rats, whereas both strains exhibited comparable [3H]GR65630 binding to 5-HT3 receptors. Compared with control FH (naive) rats, chronic ethanol consumption significantly decreased (p < 0.(15)[3H]8-OH-DPAT binding in the frontal and parietal cortex (-15%) but significantly increased binding (p < 0.05) in the entorhinal cortex (+25%), retrosplenial granular cortex (+20%), and hippocampal CA1 (+14%) and CA3 regions (+18%). Moreover, ethanol withdrawal induced the same extent of increased [3H]8-OH-DPAT binding in the entorhinal and retrosplenial cortex as seen in FH (chronic) rats. In contrast, [3H]8-OH-DPAT binding in the hippocampal CA1 and CA3 regions was decreased by -9% and -20% from the level of chronic ethanol-treated FH rat (p < 0.05) and returned to the control level seen in FH (na?ve) rats. SIGNIFICANCE: The elevated 5-HT transporters and 5-HT1A receptors in the mesocorticolimbic areas in FH rats may reflect a potential innate altered transmission at serotonergic synapses, which possibly may affect the high intake of alcohol in FH rats. The region-specific alterations of 5-HT1A receptors in FH rat brain after ethanol challenges suggest that 5-HT1A receptors are sensitive to ethanol challenges, whereas 5-HTT are apparently insensitive.     

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [³H]-5-HT-moduline bound specifically to rat brain sections with high affinity (K_d = 0.77 nM and B_max = 0.26 dpm/mm²). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT_1B receptor knock-out mice, the specific binding of [³H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT_1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT_1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT_1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT_1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT_1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT_1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.     

Effects of Prenatal Ethanol Exposure on Phospholipase C-β1 and Phospholipase A2 in Hippocampus and Medial Frontal Cortex of Adult Rat Offspring

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A₂ (PLA₂) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-β1 and PLA₂ activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-β1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca²⁺-dependent PLA₂ activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA₂ form. Cytosolic PLA₂ activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca²⁺-dependent PLA₂ and PLC-β1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.     

The role in spawning of a putative serotonin receptor isolated from the germ and ciliary cells of the gonoduct in the gonad of the Japanese scallop, Patinopecten yessoensis

Serotonin (5-hydroxytriptamin, 5-HT) triggers germinal vesicle breakdown (GVBD) of oocytes and the transporting of the mature oocyte through the gonoduct via cilia motility in bivalves. The 5-HT receptor in the oocyte membrane of the Japanese scallop, Patinopecten yessoensis, has been pharmacologically characterized as a mixed profile of 5-HT₁/5-HT₂ and is induced by estradiol-17β (E₂). Here we report the isolation, cloning, and tissue expression of the 5-HT receptor from the gonad of the Japanese scallop. A full-length cDNA (1818 bp) encoding a putative 5-HT receptor (5-HT_py) of 454 amino acid residues was isolated from the ovary and shared 53.3% and 40.2% homology with the Aplysia 5-HT_1ap and mouse 5-HT_1A, respectively. The 5-HT_py sequence possessed typical characteristics of 5-HT₁, including seven transmembrane domains, a long third inner loop, and a short fourth inner terminal. Phylogenetic analysis suggested that 5-HT_py was classified into the 5-HT₁ subtype as well as other invertebrate 5-HT₁ receptors. Semi-quantitative RT-PCR showed the expression of the 5-HT_py gene in both the nervous system and peripheral tissues and the induction of expression by E₂ in the ovarian tissue. In situ hybridization revealed a strong 5-HT_py signal in the oocytes, spermatids, and ciliary epithelium of the gonoducts in the ovary and testis. These results suggest that the effects of 5-HT on the induction of oocyte maturation, sperm motility, and transport of mature oocytes and sperm through the ciliated epithelium of the gonoducts are mediated by 5-HT_py.