[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Binding of the Novel Serotonin Agonist 8-Hydroxy-2-(Di-n-Propylamino) Tetralin in Normal and Alzheimer Brain

Binding of [3H]8-hydroxy-2-(di-n-propylamino) tetralin, a putative ligand for the 5-hydroxytryptamine (5-HT, serotonin) 1A recognition site, was measured in neocortex from postmortem human brain. The substance was found to bind to a saturable site with a KD value and pharmacological profile similar to that of rat. Binding to membranes from normal human temporal cortex was found to significantly correlate (inversely) with age. A significant reduction in binding, reflecting decreased density of recognition sites, was observed in the frontal cortex of patients with Alzheimer's disease (48% loss). This region in the dement brains showed unaltered presynaptic 5-HT function (5-HT and 5-hydroxyindoleacetic acid content) whereas 5-HT concentration was reduced in the temporal cortex.     

Brain Serotonin2 and Serotonin1A Receptors Are Altered in the Congenitally Hyperammonemic Sparse Fur Mouse

In previous studies we documented an increase in the levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label serotonin2 sites and 8-[3H]hydroxy(di-n-propylamino)tetralin to label serotonin1A sites in cortical membrane homogenates. The capacity (Bmax) for [3H]ketanserin binding was significantly lower (-21%; p less than 0.05) in sparse fur animals than in control animals; there was no change in affinity (KD). In contrast, the capacity for 8-[3H]hydroxy(di-n-propylamino)tetralin binding was significantly greater (26%; p less than 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin2 receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p less than 0.005). Hypothermia elicited by administration of 8-hydroxy(di-n-propylamino)tetralin was studied as a physiologic response mediated by serotonin1A receptors. Although there were not overall group differences in the dose-response data, there was a significant increase in the hypothermia induced by 8-hydroxy(di-n-propylamino)tetralin in sparse fur compared with control mice (p less than 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.     

Anatomical and functional evidence for a stress-responsive,monoamine-accumulating area in the dorsomedial hypothalamus of adult rat brain

The dorsomedial hypothalamus (DMH) plays an important role in relaying information to neural pathways mediating neuroendocrine, autonomic, and behavioral responses to stress. Evidence suggests that the DMH is a structurally and functionally diverse integrative structure that contributes to both facilitation and inhibition of the hypothalamo-pituitary-adrenal axis, depending on the nature of the stimulus and the specific neural circuits involved. Previous studies have determined that stress or stress-related stimuli elevate tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine, and noradrenaline selectively within the DMH. In order to determine the specific region of the rat DMH involved, we used high-performance liquid chromatography with electrochemical detection to measure tissue concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline within five different subregions of the DMH in adult female Lewis and Fischer rats immediately or 4 h following a 30-min period of restraint stress. Compared to unrestrained control rats, restrained rats had elevated concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline immediately after a 30-min period of restraint and had elevated concentrations of 5-HT 4 h following the onset of a 30-min period of restraint stress. These effects were confined to a specific region that included medial portions of the dorsal hypothalamic area and dorsal ependymal, subependymal, and neuronal components of the periventricular nucleus. Furthermore, these effects were observed in Lewis rats, but not Fischer rats, two closely related rat strains with well-documented differences in neurochemical, neuroendocrine, autonomic, and behavioral responses to stress. These data provide support for the existence of a stress-responsive, amine-accumulating area in the DMH that may play an important role in the differential stress responsiveness of Lewis and Fischer rats.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.     

Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5-HT1A Receptors and Carbachol-Stimulated Phosphoinositide Turnover in the Rat Hippocampus

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Administration of 8-Hydroxy-2-(Di-n-Propylamino)tetralin in Raphe Nuclei Dorsalis and Medianus Reduces Serotonin Synthesis in the Rat Brain: Differences in Potency and Regional Sensitivity

Following administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.04-5.0 micrograms/0.5 microliter) in the raphe nucleus dorsalis (DR) or medianus (MR), the synthesis of serotonin (5-HT), as assessed by the accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition, was measured in various regions of the rat CNS. At all doses, 8-OH-DPAT in the DR significantly reduced 5-HTP accumulation in the striatum, nucleus accumbens, cortex, and prefrontal cortex, whereas even the highest dose had no effect in the hippocampus, hypothalamus, and spinal cord. One microgram of 8-OH-DPAT in the MR significantly reduced 5-HTP accumulation in the nucleus accumbens and prefrontal cortex, and 5 micrograms had an effect in all the areas except the striatum and spinal cord. One and 5 micrograms of 8-OH-DPAT, administered in either the DR or MR, did not significantly modify the accumulation of dihydroxyphenylalanine in the striatum and nucleus accumbens. The results confirm that DR and MR have different sensitivities to 5-HT1A receptor agonists, and that activation of 5-HT1A receptors in these nuclei produces different effects on 5-HT synthesis in different brain regions.     

Pertussis Toxin Attenuates 5-Hydroxytryptamine1A Receptor-Mediated Inhibition of Forskolin-Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes

The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet-activating protein (IAP; pertussis toxin). In vehicle-treated animals, 5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30-55% the 5-HT1A agonist inhibition of adenylate cyclase activity via 5-HT1A receptors. The data indicate that the inhibitory guanine nucleotide-binding protein or Go (a similar GTP-binding protein of unknown function purified from brain) mediates the 5-HT1A agonist inhibition of hippocampal adenylate cyclase.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Differential Regulation of Somatodendritic Serotonin 5-HT1A Receptors by 2-Week Treatments with the Selective Agonists Alnespirone (S-20499) and 8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract : Single treatment with the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) and alnespirone (S-20499) reduces the extracellular 5-HT concentration (5-HT_ext) in the rat midbrain and forebrain. Given the therapeutic potential of selective 5-HT_1A agonists in the treatment of affective disorders, we have examined the changes in 5-HT_1A receptors induced by 2-week minipump administration of alnespirone (0.3 and 3 mg/kg/day) and 8-OH-DPAT (0.1 and 0.3 mg/kg/day). The treatment with alnespirone did not modify baseline 5-HT_ext but significantly attenuated the ability of 0.3 mg/kg s.c. alnespirone to reduce 5-HT_ext in the dorsal raphe nucleus (DRN) and frontal cortex. In contrast, the ability of 8-OH-DPAT (0.025 and 0.1 mg/kg s.c.) to reduce 5-HT_ext in both areas was unchanged by 8-OH-DPAT pretreatment. Autoradiographic analysis revealed a significant reduction of [³H]8-OH-DPAT and [³H]WAY-100635 {³H-labeled N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexanecarboxamide · 3HCl} binding to somatodendritic 5-HT_1A receptors (but not to postsynaptic 5-HT_1A receptors) of rats pretreated with alnespirone but not with 8-OH-DPAT. In situ hybridization analysis revealed no change of the density of the mRNA encoding the 5-HT_1A receptors in the DRN after either treatment. These data indicate that continuous treatment for 2 weeks with alnespirone, but not with 8-OH-DPAT, causes a functional desensitization of somatodendritic 5-HT_1A receptors controlling 5-HT release in the DRN and frontal cortex.     

Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5-, 14-, and 24-month-old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5-HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5-HIAA/5-HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5-month-old ethanol-fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5-HT and 5-HIAA contents in the VTA and reduced DOPAC and 5-HIAA levels in the VP. In addition, ethanol blunted the normal age-related increase in 5-HIAA/5-HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age-related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol-associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5-HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.     

Differential effects of (R)-, (R, S)- and (S)-8-hydroxy-2-(di-n-propylamino)tetralin on hippocampal serotonin release and induction of hypothermia in awake rats

The effects of (R)- and (S)-optical isomers of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and of the racemate (R,S)-8-OH-DPAT on serotonin (5-HT) release in the ventral hippocampus of awake rats and on induction of the whole-body hypothermia were studied. Extracellular 5-HT levels were determined by a newly developed high-sensitive HPLC method based on derivatization with benzylamine and fluorescence detection. The basal levels of 5-HT in 20 min microdialysates from rats perfused with Ringer solution or with Ringer solution containing 1 microM citalopram were 6.3 +/- 1.3 fmol/20 microl and 36.1 +/- 4.2 fmol/20 microl (n=20), respectively. The reduction of hippocampal 5-HT levels induced by subcutaneous (s.c.) administration of (R,S)-8-OH-DPAT (0.3 mg/kg) was significantly attenuated by the presence of 5-HT reuptake inhibitor citalopram in Ringer solution only at its peak value at 40 min (maximal reduction to 60% compared to 46% of control values in Ringer-perfused rats), whereas the overall effects were comparable at both experimental conditions. Injection of (R)-8-OH-DPAT (0.3 mg/kg s.c.) caused further reduction of 5-HT levels, to 49% and 41%, respectively, whereas (S)-8-OH-DPAT (0.3 mg/kg s.c.) caused maximal reduction of 5-HT levels only to 74% of controls in both perfusion groups. Similar pattern and time-courses were observed in rats with hypothermia induced by injection of 8-OH-DPAT enantiomers, where (R,S), (R)-forms were about two-times more potent than the (S)-isomer. It is concluded that the acute systemic dose of (R)-, (S)- and (R,S)-8-OH-DPAT enantiomers exerted enantiomer-specific effects on 5-HT(1A) receptor-mediated function both at the presynaptic and postsynaptic sites as revealed by monitoring hippocampal 5-HT levels and body temperature.     

The effects of serotonin on glucocorticoid receptor binding in rat raphe nuclei and hippocampal cells in culture

The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamus-pituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (+/-)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropiona mide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.     

Evidence for the Presence of Serotonin Receptors Negatively Coupled to Adenylate Cyclase in the Rabbit Iris-Ciliary Body

Serotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin-, isoproterenol-, and vasoactive intestinal peptide-induced stimulation of cyclic AMP levels in a dose-dependent manner. Serotonergic, cholinergic, muscarinic, alpha-adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical application of a serotonin/pargyline solution to the living eye causes desensitisation of the serotonin response in the iris-ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5-HT1A [5-hydroxytryptamine (serotonin) type 1A] receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin and buspirone mimic the serotonin response in reducing the forskolin-stimulated cyclic AMP levels, as do the indole derivatives 5-methoxytryptamine, 5-hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5-HT1A agonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5-HT1A receptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin receptor to the adenylate cyclase catalytic unit.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity

Abstract: To clarify the effects of adenosine receptor subtypes (A₁, A₂, and A₃) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5-HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyladenosine, decreased extracellular 5-HT levels, whereas an adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and caffeine increased these levels. A selective A_2A receptor agonist (CGS-21680), an adenosine A₂ receptor agonist (PD-125944), an adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and an adenosine A₃ receptor agonist, N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5-HT levels. When the adenosine A₁ receptor was blocked by CPT, the hippocampal extracellular 5-HT level was increased by adenosine, CGS-21680, and PD-125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A₁ and A₂ receptors were blocked by CPT and DMPX, the extracellular 5-HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5-HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A₁ receptor was blocked, irrespective of whether the adenosine A₂ receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5-HT levels, during both adenosine A₁ and A₂ receptor blockade, were inhibited by selective 5-HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A₂ receptor and the inhibitory effects of the A₃ receptor on hippocampal extracellular 5-HT levels are masked by the inhibitory effects of the adenosine A₁ receptor.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Effects of the Serotonin1A Agonist, 8-Hydroxy-2-(Di-n-Propylamino)tetralin on Neurochemical Responses to Stress

Abstract: The effects of intracerebroventricular administration of the 5-hydroxytryptamine (5-HT)_1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.1 pmol) on adrenocortical and neurochemical responses to stress were examined in conscious male rats. The following stress paradigms were used: acoustic stimulation (105 dB for 2 min); footshock (0.2 mA, five shocks over 5 min); conditioned fear (animals placed in a footshock chamber for 5 min, 24 h after footshock); restraint (5 min); intraperitoneal (i.p.) injection of recombinant human interleukin-1α (rHu-IL-1α, 20 µg/kg); and injection of cocaine hydrochloride (20 mg/kg, i.p.). As previously shown, 8-OH-DPAT was able to attenuate the adrenocortical response to acoustic stress, conditioned fear, rHu-IL-1α, and cocaine administration. Cocaine decreased 5-hydroxyindoleacetic acid (5-HIAA)/5-HT and dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratios and norepinephrine (NE) concentration in the prefrontal cortex, hypothalamus, and brainstem in all experiments, and 8-OH-DPAT reversed the changes in DOPAC/DA ratio without affecting 5-HIAA/5-HT ratios or NE content. 8-OH-DPAT alone had no effect on these parameters, although it decreased NE content in the prefrontal cortex in several experiments, and in the brainstem in one experiment. Significant decreases in NE content were observed in some brain regions following some of the stressors, but these changes were not generally affected by 8-OH-DPAT. Increases in the 5-HIAA/5-HT and DOPAC/DA ratios were also observed in some brain sites following some stressors, but these changes were not affected by 8-OH-DPAT except in the case of the increased 5-HIAA/5-HT ratio in the prefrontal cortex following the conditioned fear response. These results indicate that although 8-OH-DPAT is able to decrease plasma corticosterone responses following acoustic stress, conditioned fear, rHu-IL-1α, and cocaine administration, these effects do not appear to be related to an action of the 5-HT_1A agonist on biogenic amine metabolism. This observation indicates that the predominant effect of 8-OH-DPAT on adrenocortical responses is mediated at postsynaptic sites not involved in the regulation of cerebral biogenic amine metabolism.