Inhibition of neuronal 5-HT uptake by ketamine, but not halothane, involves disruption of substrate recognition by the transporter

The effects of halothane and ketamine on (1) serotonin (5-hydroxytryptamine; 5-HT) uptake and (2) paroxetine binding to the 5-HT transporter in neuronal membranes were determined in rat brain. Both anesthetics inhibited the uptake of [3H]5-HT by synaptosomes, but only ketamine affected binding of [3H]paroxetine to the 5-HT transporter. Saturation analysis indicated that ketamine inhibition of [3H]paroxetine binding was competitive (Ki = 18.8 microM). These results indicate that halothane and ketamine inhibit 5-HT transport by different mechanisms.     

5-HT2A and muscarinic receptors in schizophrenia: a postmortem study

Although evidence suggests that 5-HT(2A) and muscarinic M1/M4 receptors are implicated in the pathology of schizophrenia, the results are not conclusive. In the present study we tested the hypothesis that binding of 5-HT(2A) and M1/M4 receptors is altered in the postmortem brain of schizophrenia subjects. Quantitative autoradiography was employed to measure [(3)H]ketanserin binding to 5-HT(2A) receptors and [(3)H]pirenzepine binding to both M1 and M4 receptors in Brodmann's area 9 (BA9), caudate/putamen, and the hippocampal formation from six schizophrenic and six control subjects. A significant reduction in the density of 5HT(2A) receptors in BA 9 of schizophrenic subjects was observed (p=0.036). No significant difference was observed in the density of 5HT(2A) receptors in the hippocampus or caudate/putamen between the two groups. No significant changes in the density of M1/M4 receptors was observed in these three regions between the two groups. These findings support a possible involvement of the serotonergic system in the pathology of schizophrenia.     

Aging but not estrogen alters regional [3H]5-HT binding in the CNS of female Fischer 344 rats

Age-related changes in serotonergic regulation of neuroendocrine function were investigated in female Fischer 344 rats; serotonin ([3H]5-HT) binding sites were characterized in several brain regions. Neither the number (Bmax) nor the affinity (Kd) of [3H]5-HT sites were altered in the frontal cortex of reproductively young and senescent groups. However, a significant decline in receptor affinity was observed in the hypothalamus and midbrain dorsal raphe nucleus. An increase in the density of binding sites was also observed in the hypothalamus with advancing age. Acute 48 h exposure to estrogen failed to influence [3H]5-HT binding site characteristics in these brain regions. In summary, these results suggest that age-related changes in [3H]5-HT binding are regionally specific. Moreover, the observed changes in hypothalamic 5-HT function may underlie neuroendocrine aging events.     

Visualization of a novel serotonin recognition site (5-HT1D) in the human brain by autoradiography

The localization of a novel serotonin (5-hydroxytryptamine, 5-HT) recognition site named 5-HT1D was studied by autoradiography in human postmortem brain material. Serotonin-1 sites were labeled with [3H]5-HT. The different subpopulations of 5-HT1 sites were investigated by the use of unlabeled selective compounds. 8-OH-DPAT (8-hydroxy-2-[N,N-di-n-propyl-amino]tetralin) was used to block [3H]5-HT binding to 5-HT1A, the beta-blocker (-)-21 009 (4-[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester) to 5-HT1B and the ergoline mesulergine to 5-HT1C recognition sites. 5-HT1D sites were defined as the binding sites remaining when both 8-OH-DPAT and mesulergine were added to the incubation medium. Under these conditions, 5-HT1D sites represented a high proportion of total [3H]5-HT binding sites in the basal ganglia and substantia nigra. Lower proportions were observed in other brain areas such as the hippocampal formation and raphé nuclei. 5-HT1D sites thus represent the majority of 5-HT1 binding sites in the striatonigral pathway in man. This localization suggests an involvement of these sites in the mediation of serotoninergic mechanisms in basal ganglia functions and a possible role in brain diseases where these areas are known to be involved.     

Quantitative autoradiography of [3H]norharman [( 3H]beta-carboline) binding sites in the rat brain

The anatomical distribution of [3H]norharman binding sites was determined by quantitative autoradiography in rat brain slices. They are enriched in hypothalamic, thalamic, accumbens and amygdaloid nuclei as well as in hippocampal, neocortical and olfactory-related structures. The distribution pattern differs from that of other previously described receptors or binding sites (e.g. monoamine oxidase, benzodiazepine, tryptamine, 5-hydroxytryptamine receptors (5-HT1A, 5-HT1B, 5-HT1C, 5HT2], which suggests that a unique class of [3H]norharman binding sites exists in the rat brain. The findings are consistent with previous experiments which showed high affinity binding sites for [3H]norharman in rat brain membranes (KD 1.552 nM; autoradiography KD 5.5 nM). A correspondence in the displacing activity of drugs was found for both methods (crude membrane fraction: harman much greater than tryptamine much greater than 5-hydroxytryptamine greater than N-methyl-beta-carboline-3-carboxamide (FG 7142) = diazepam; autoradiography: harman much greater than tryptamine much greater than FG 7142 greater than 5-hydroxytryptamine greater than diazepam). Provided that the binding sites represent functional receptors, the present anatomical findings may explain the biological effects of norharman, e. g. pro-conflict behaviour (limbic-hypothalamic structures), tonic-clonic convulsions (limbic-cortical structures) and alterations of locomotor activity (accumbens nucleus).     

In vivo autoradiography of [3H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists

The purpose of this study was to examine the receptor occupancy of D1/D5 antagonists for D1-like dopamine receptors in rat brain using [3H]SCH 39166, a highly selective D1/D5 antagonist with low affinity for 5HT2 receptors. A single concentration of triated SCH 39166 was administered to rats, with or without competing doses of the Dl/D5 antagonist SCH 23390 and unlabeled SCH 39166. the D2-like antagonists haloperidol or the 5-HT, antagonist ketanserin. The bound radioactivity in the cortex, striatum, nucleus accumbens and olfactory tubercle was then quantified using an in vivo autoradiographic procedure. The results indicated that [3H]SCH 39166 was dose dependently displaced by the Dl/D5 antagonists in regions associated with both the nigro-striatal pathway and the mesolimbic dopamine pathway, particularly the nucleus accumbens. Neither haloperidol nor ketanserin displaced [3H]SCH 39166 in any of the regions examined. The data were compared with previously published data examining the in vivo binding of [3H]SCH 39166 in rat brain homogenates. The relative values obtained were comparable to values detected in rat brain homogenates after in vivo binding of [3H]SCH 39166.     

Differential interactions of "prosexual" drugs with 5-hydroxytryptamine1A and alpha 2-adrenergic receptors

Radioligand binding studies were used to analyze the interactions of six "prosexual" drugs with 5-hydroxytryptamine1A (5-HT1A) and alpha 2-adrenergic receptors in rat brain membranes. Three drugs that facilitate seminal emissions and/or ejaculations [8-hydroxy-2-n-propylaminotetralin (8-OH-DPAT), 5-methoxy-dimethyltryptamine, and RDS-127] are potent and selective inhibitors of [3H]8-OH-DPAT binding to 5-HT1A receptors. By contrast, three drugs with primary sexual arousal effects (yohimbine, imiloxan, and idazoxan), are potent agents at alpha 2-adrenergic receptors labeled by [3H]yohimbine. These data suggest that radioligand binding analysis of prosexual agents may elucidate the pathophysiology of sexual behavior.     

Differential response of subtypes of 5-HT1 recognition sites in the rat hippocampus to partial denervation

[3H]5-HT uptake and [3H]5-HT binding to 5-HT1 receptor subtypes (5-HT1A and non-5-HT1A sites, having high and low affinity to spiperone, respectively) were studied in the rat hippocampus ten days after two types of electrocoagulative lesions. The lesion of supracallosal area and subtotal lesion of the septum destroy supra- and subcallosal hippocampal afferents, respectively. Both types of afferents carry serotonergic fibers to the hippocampus. A significant decrease of [3H]5-HT uptake (by about one half of the control) was observed after both lesions. [3H]5-HT binding to 5-HT1A sites, comprising ca 60% of the total number of 5-HT1 sites, remained unchanged after both lesions, while the binding to non-5-HT1A sites decreased significantly (by ca 20%) but only after the lesion of the septum. The results point to a postsynaptic localization of 5-HT1A and of the bulk of non-5-HT1A sites; the decrease of the proportion of non-5-HT1A sites after septal lesion may be due to their presynaptic localization on the subcallosal pathway and/or may reflect receptor alterations in consequence of transsynaptic events in the hippocampus caused by septal lesion. Differential response of serotonin receptor subtypes to lesions of supracallosal and septal areas may underlie the differential functional responses to those lesions.     

Inactivation of presynaptic 5-HT autoreceptors by lithium in rat hippocampus

The effect of lithium ion on the electrically stimulated 5-[3H]hydroxytryptamine (5-HT) release from the rat hippocampal slices preloaded with [3H]5-HT was studied. Electrically stimulated [3H]5-HT release decreased when the slices were exposed to 5-HT in a concentration-dependent manner. Lithium (2.5 mM) did not affect [3H]5-HT release when added alone to the superfusion medium. However, the inhibitory effect of 5-HT (1 microM) on [3H]5-HT release was abolished by lithium. The results suggest that lithium may inhibit the regulation of 5-HT release via presynaptic 5-HT autoreceptors in rat hippocampus.     

Long-term treatment with zimelidine leads to a reduction in 5-hydroxytryptamine neurotransmission within the central nervous system of the mouse and rat

Male rats and mice received a 14 day peroral treatment with zimelidine, a novel antidepressant drug. Zimelidine produced a 70 and 60% reduction in the number of high affinity [3H]5-hydroxytryptamine (5-HT) and [3H]D-lysergic acid diethylamide (LSD) binding sites, respectively, and induced low affinity binding sites for [3H]5-HT and for [3H]D-LSD. The head twitch behaviour induced by 5-hydroxytryptophan and 5-methoxydimethyltryptamine was attenuated and reductions of prolactin and growth hormone were observed. These findings give further evidence that long-term treatment with zimelidine can produce a reduction of 5-HT neurotransmission in several brain regions.     

Preservation of 5-hydroxytryptamine1A receptor-G protein interactions in the cerebral cortex of patients with Alzheimer's disease.

The coupling of 5-hydroxytryptamine1A (5-HT1A) receptors to guanine nucleotide binding (G) proteins was investigated in membranes prepared from frontal and parietal cortices of control and Alzheimer's disease brains by characterising the effect of guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) on [3H]8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]8-OH-DPAT) binding parameters. In the absence of guanine nucleotides, [3H]8-OH-DPAT bound to a single high affinity binding site in all membrane types. The number of [3H]8-OH-DPAT binding sites was significantly decreased in the parietal cortex of Alzheimer's disease samples compared with controls, whereas in the frontal cortex the number of binding sites remained unchanged. Gpp[NH]p reduced the [3H]8-OH-DPAT binding affinity and the number of binding sites to the same degree in both regions in control and Alzheimer's disease cases. [3H]8-OH-DPAT binding was inhibited in a concentration dependent manner with an IC50 value of approximately 1 microM in all cases. These results suggest that the 5-HT1A receptor-G protein complex is functionally intact in these regions in Alzheimer's disease brain.     

Radioligand binding analysis of knockout mice reveals 5-hydroxytryptamine(7) receptor distribution and uncovers 8-hydroxy-2-(di-n-propylamino)tetralin interaction with alpha(2) adrenergic receptors

In the present autoradiographic study, we took advantage of 5-hydroxytryptamine(7) (5-HT(7)) receptor knockout mice to analyze the brain distribution of 5-HT(7) receptor binding sites using [(3)H]5-carboxamidotryptamine (5-CT; a 5-HT(1A/1B/1D/5/7) receptor ligand) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT(1A/7) receptor ligand). Low to moderate densities of [(3)H]5-CT (2 nM) binding sites insensitive to pindolol (10 microM, for 5-HT(1A/1B) receptor blockade) and GR-127935 (1 microM; for 5-HT(1D) receptor blockade) were observed in wild-type mice (mainly in thalamus and hypothalamus) but not in 5-HT(7) receptor knockout mice. Surprisingly, moderate to high densities of [(3)H]8-OH-DPAT (10 nM) binding sites insensitive to pindolol (10 microM) remained in 5-HT(7) receptor knockout mouse brain. These non-5-HT(1A), non-5-HT(7) binding sites were found to be adrenergic alpha(2A) receptor binding sites. In alpha(2A) receptor knockout mice low to moderate densities of [(3)H]8-OH-DPAT binding sites insensitive to pindolol but sensitive to the selective 5-HT(7) receptor antagonist SB-269970 (300 nM) were observed mainly in thalamus and hypothalamus. Therefore, in addition to 5-HT(1A) and 5-HT(7) binding sites, [(3)H]8-OH-DPAT also binds to alpha(2A) receptor binding sites in wild-type mouse brain. [(3)H]8-OH-DPAT (in the presence of pindolol and 1 microM RX-821002 for alpha(2) receptor blockade) and [(3)H]5-CT (in the presence of pindolol and GR-127935) bind to a similar receptor binding population corresponding to 5-HT(7) binding sites. Detailed anatomical mapping of 5-HT(7) receptor binding sites in wild-type mouse brain was then performed using both radioligands in the presence of suitable pharmacological agents for non-5-HT(7) receptor binding sites blockade. The mapping revealed binding sites consistent with the mRNA distribution with the highest densities found in anterior thalamic nuclei.     

Identification of a 5-HT1 recognition site in human brain membranes different from 5-HT1A, 5-HT1B and 5-HT1C sites

In human caudate and cortex membranes, [3H]serotonin ([3H]5-HT) labels 5-HT1A and 5-HT1C recognition sites which show nanomolar affinity for 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) and mesulergine respectively, whereas no 5-HT1B binding could be identified. However, the majority of the sites labelled by [3H]5-HT (greater than or equal to 60% in cortex, 90% in caudate) are different from 5-HT1A, 5-HT1B and 5-HT1C sites. Competition experiments were performed in human caudate membranes incubated with [3H]5-HT in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine. Under those conditions, [3H]5-HT labelled an apparently homogeneous population of 5-HT1-like sites which display nanomolar affinity for tryptamines (5-carboxamido-tryptamine, (5-CT) greater than 5-HT greater than or equal to 5-methoxytryptamine (5-MeOT) greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin). The pharmacological profile of these sites is different from that of 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2 and 5-HT3 sites but is consistent with the pharmacology of a 5-HT1-like receptor. It is very similar to that of the 5-HT1D site recently described in bovine brain by Heuring and Peroutka.     

On the mechanism of action of the antidepressant drugs amitriptyline and nortriptyline. Evidence for 5-hydroxytryptamine receptor blocking activity

Effects of amitryptyline (AMI) and nortriptyline (NOR) were studied on the [5-³H] hydroxytryptamine ([5-³H]HT) and D-[³H]lysergic acid diethylamide-(D-[³H] LSD) binding in cerebral cortex of rats and on behaviours depedent upon 5-hydroxtrryptamine (5-HT) receptor activity. AMI and NOR were found to have affinity for some of the D-LSD binding sites in the cerebral cortex but no or very weak affinity for the 5-HT binding site. AMI and NOR blocked the 5-hydroxytryptophan (5-HTP) and D-LSD induced head twitches in mice and the D-LSD induced extensor reflex activity. This 5-HT receptor blocking activity of AMI and NOR may in part mediate their antidepressant actions.     

Changes in serotonin receptors in different brain regions after light exposure of dark-reared rats

Male rats dark-reared from birth until 50 days of age and then exposed to light for 3 h show significant increases in specific [³H]serotonin (5-[³H]HT) binding to P₂ membranes from visual and motor cortex and superior colliculus (25, 65 and 23% respectively) as compared with normal and dark-reared littermates. These increases are transient and return to normal levels after 7 days. The role of 5-HT as a transmitter in the visual system is discussed.     

Region specific changes in forebrain 5-hydroxytryptamine1A and 5-hydroxytryptamine2A receptors in isolation-reared rats: an in vitro autoradiography study

The neurochemical correlates of the behavioural consequences of isolation rearing of rats are complex and involve many neurotransmitters, including the serotonergic system. Impaired functioning of the ascending serotonergic system has been implicated in many neuropsychiatric syndromes, including attention deficit hyperactivity disorder and schizophrenia. In the present investigation serotonergic function was assessed using in vitro receptor autoradiography. The 5-hydroxytryptamine(2A) (5-HT(2A)) receptor antagonist [(3)H]ketanserin and the 5-HT(1A) receptor antagonist, [(3)H]WAY100, 635 were used to compare 5-HT receptor subtype densities in the forebrains of socially and isolation-reared rats. Regions of highest receptor density were observed in the frontal cortex for 5-HT(2A) receptors and in the frontal cortex, dorsal hippocampus and lateral septum for 5-HT(1A) receptors. In isolation-reared rats, 5-HT(2A) receptor binding site densities were significantly increased by between 36 and 67% in the prelimbic, motor and cingulate cortices compared with socially reared controls. By contrast, 5-HT(1A) receptor binding site densities were significantly reduced by 22% in the prelimbic cortex, and significantly increased by between 10 and 50% in the motor cortex, somatosensory cortex, dentate gyrus and CA fields of the hippocampus.These data demonstrate that isolation-rearing produces significant effects on forebrain 5-HT(1A) and 5-HT(2A) receptor densities in the adult rat. It is hypothesised that altered serotonergic function, particularly in the hippocampus and prefrontal cortex, may underlie some of the behavioural abnormalities associated with isolation-rearing.     

Effect of hydroxycitric acid on serotonin release from isolated rat brain cortex.

There is evidence that hydroxycitric acid (HCA), an extract of dried fruit rind of South Asian trees of the genus Garcinia cambogia, can reduce food intake in experimental animals. In the present study, we investigated the effect of HCA on basal and potassium-depolarization evoked increase in radiolabeled serotonin ([3H]-5-HT) release from rat brain cortex slices in vitro. HCA (10 microM-1 mM) altered the baseline of spontaneous tritium efflux but had no significant effect on potassium-evoked release of [3H]-5-HT. When applied on its own, HCA (10 microM-1 mM) elicited a concentration-dependent increase in efflux of [3H]-5-HT reaching a maximum at 300 microM. We conclude that HCA can increase the release of radiolabeled 5-HT from the isolated rat brain cortex.     

The distribution of serotonin binding sites in the hippocampal region of the rat brain. An autoradiographic study

The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autoradiographic evidence of serotonin1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord

Spinal serotonin1 (5-HT1)(labelled by [3H]5-HT), 5-HT1A (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT)), mu- (labelled by [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]naloxone) and delta-opiate (labelled by [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr [( 3H]DSTLE] receptor binding sites were studied in adult rats using quantitative autoradiography after either neonatal treatment with capsaicin or unilateral cervical dorsal rhizotomy. Both treatments produced a significant loss of 5-HT (-20 to -30%) and opiate (-30 to -45%) binding sites within the superficial layers of the dorsal horn, suggesting they are partly located presynaptically on primary afferent fibres. Thus, 5-HT, as well as opiates, might generate analgesia by acting--at least partly--on primary afferent nociceptive fibres at the spinal level.     

Limited inhibition of 5-HT1A receptor expression in the rat brain by antisense RNA and oligodesoxynucleotides

The efficiency of antisense approaches to produce a selective regional inhibition of the expression of brain 5-HT1A receptors was tested in the rat. In vivo ICV injections of modified antisense oligodesoxynucleotides yielded at most an 18% specific decrease in 5-HT1A receptor expression in the hippocampus only, as measured by [3H]8-OH-DPAT autoradiographic labeling. In vitro, when 5-HT1A receptors were transiently expressed in LLC-PK1 cells, co-transfection with antisense RNA encoding plasmids resulted in a marked reduction (50-70%) in the density of 5-HT1A binding sites. In vivo stereotaxic injections of the same constructs into the hippocampus, but not in the raphe, which contains 5-HT1A autoreceptors, were shown to produce a approximately 20% reduction in local 5-HT1A receptor density. These data show that antisense strategies could be used to inhibit 5-HT1A receptors expression in the rat hippocampus, but with a limited efficacy.