精神分裂症患者颞上回的大麻素受体密度没有显著性变化

目的 近年来研究发现,在精神分裂症患者的内源性大麻素递质系统会出现异常变化,而颞上回在精神分裂症的病理生理机制中和幻听症状密切相关.因此,对照正常人群,我们研究了精神分裂症患者颞上回大麻素CB-1受体的密度变化.方法 采用定量放射自显影技术,通过[3H]SR141716A(CB-1受体选择性拮抗剂)和[3H]CP-55940(CB-1受体激动剂)检测颞上回CB-1受体密度水平.死后脑组织由澳大利亚新南威尔士州组织资源中心提供.结果 先前研究发现,精神分裂症患者与认知功能失常相关的额前叶,前、后扣带回皮质的CB-1受体密度水平有异常改变.与此相反,本研究发现在精神分裂症患者的由[3H]SR141716A和[3H]CP-55940检测的颞上回大麻素受体密度水平和对照组比较没有显著变化.结论 我们认为颞上回大麻素CB-1受体和精神分裂症患者的发病及幻听症状无关.     

精神分裂症患者颞上回的大麻素受体密度没有显著性变化

目的近年来研究发现,在精神分裂症患者的内源性大麻素递质系统会出现异常变化,而颞上回在精神分裂症的病理生理机制中和幻听症状密切相关。因此,对照正常人群,我们研究了精神分裂症患者颞上回大麻素CB-1受体的密度变化。方法采用定量放射自显影技术,通过[~3HJSR141716A(CB-1受体选择性拮抗剂)和[~3H]CP-55940(CB-1受体激动剂)检测颞上回CB-1受体密度水平。死后脑组织由澳大利亚新南威尔士州组织资源中心提供。结果先前研究发现,精神分裂症患者与认知功能失常相关的额前叶,前、后扣带回皮质的CB-1受体密度水平有异常改变.与此相反,本研究发现在精神分裂症患者的由[~3H]SR141716A和[~3H]CP-55940检测的颞上回大麻素受体密度水平和对照组比较没有显著变化。结论我们认为颞上回大麻素CB-1受体和精神分裂症患者的发病及幻听症状无关。     

Quantitative autoradiography of 5-HT1D and 5-HT1E binding sites labelled by [H]5-HT, in frontal cortex and the hippocampal region of the human brain

In human cortex and hippocampus area, [³H]5-HT (5 nM) labels 5-HT_1A, 5-HT_1D and 5-HT_1E sites. After masking 5-HT_1A receptors by 0.1 μM 8-OH-DPAT, the binding displaced by 0.1 μM 5-CT presumably represented 5-HT_1D sites and the remaining binding 5-HT_1E sites. In frontal cortex, 5-HT_1A receptors represented the main binding in layers II and VI and a lower fraction on other layers. 5-HT_1D and 5-HT_1E sites, were more homogeneously distributed in layers II to VI (21–34% of specific [³H]5-HT binding). 5-HT_1E sites were of similar affinities (K_D close to 6–8 nM) in the cortical layers II to VI. In CA1 field of hippocampus, (pyramidal layer, stratum radiatum, molecular layer), CA2 and dentate gyrus, 5-HT_1A receptors represented the major fraction, 5-HT_1D sites a significant fraction and 5-HT_1E a minor fraction of the specific [³H]5-HT binding. In CA3–CA4 fields, 5-HT_1A receptors were less densely present, 5-HT_1D sites were predominant and 5-HT_1E sites represented a significant fraction (27%). The highest densities of 5-HT_1E sites have been measured in subiculum, where 5-HT_1A, 5-HT_1D, and 5-HT_1E binding sites were equally represented and in entorhinal cortex where 5-HT_1E sites represented the major binding in layer III. They were also present in layers II and IV (29 and 24%) and, to a lesser extent, in layers V and VI. 5-HT_1A sites were predominant in layer VI, II and V and were less abundant in other layers. 5-HT_1D were homogeneously present in layers II, III, IV and were present in low amounts in other layers. No 5-HT_1E were detected in choroid plexus, where [³H]5-HT was dramatically reduced by mesulergine (5-HT_2C receptors). No significant displacement of [³H]5-HT by mesulergine was measured in other structures.     

Serotonin 2A receptor agonist binding in the human brain with [11C]Cimbi-36

[¹¹C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT_2A) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [¹¹C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT_2A receptors with [¹¹C]Cimbi-36 PET. The two-tissue compartment model using arterial input measurements provided the most optimal quantification of cerebral [¹¹C]Cimbi-36 binding. Reference tissue modeling was feasible as it induced a negative but predictable bias in [¹¹C]Cimbi-36 PET outcome measures. In five subjects, pretreatment with the 5-HT_2A receptor antagonist ketanserin before a second PET scan significantly decreased [¹¹C]Cimbi-36 binding in all cortical regions with no effects in cerebellum. These results confirm that [¹¹C]Cimbi-36 binding is selective for 5-HT_2A receptors in the cerebral cortex and that cerebellum is an appropriate reference tissue for quantification of 5-HT_2A receptors in the human brain. Thus, we here describe [¹¹C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT_2A receptors in the human brain.     

Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord

The distribution of 5-HT₁, 5-HT_1A, 5-HT_1B and 5-HT₂ receptors in the rat spinal cord was investigated with quantitative autoradiography. Receptors were labeled respectively with [³H]serotonin (5-[³H]HT],8-hydroxy-2-[N-dipropylamino-³H]tetralin (8-OH-[³H]DPAT), [¹²⁵I]iodocyanopindolol and [³H]ketanserin. It is shown that 5-HT₁, 5-HT_1A and 5-HT_1B receptors are distributed within the spinal cord according to a rostro-caudal gradient. Both 5-HT₁ and 5-HT_1A receptors are mainly present in the dorsal horn and 5-HT_1B is present throughout the spinal cord, exhibiting high densities in the caudal-most part of the dorsal in lamina X and in the sacral parasympathetic area. On the other hand, 5-HT₂ receptors are shown mostly in the thoracic sympathetic area and in the thoracic ventral horn; the dorsal horn exhibits few 5-HT₂ receptors. The differential involvement of 5-Ht through different receptors in nociception, autonomous nervous system control and motility are discussed.     

Selective loss of serotonin recognition sites in the parietal cortex in Alzheimer's disease

Numbers of serotonin (5-HT) recognition sites of total 5-HT₁, 5-HT_1A and 5-HT₂ subtypes have been measured in frontal, temporal and parietal cortex from human brain postmortem, using binding of [³H] 5-hydroxytryptamine (5-HT), [³H] 8-hydroxy-2-(di-n-propylamino) tetralin and [³H] ketanserin, respectively. A comparison has been made between 11 patients with Alzheimer's disease (AD) and 20 matched controls. Significant disease-related differences were observed only in the parietal cortex for total 5-HT₁ and 5-HT₂ recognition sites. Age-related decrease in numbers of 5-HT₂ recognition sites was seen in the controls, whereas an age-related increase in the numbers of the same site was seen in AD patients. These changes are discussed in relation to disease severity and cortical nerve cell loss in ageing and AD.     

Density and Distribution of Hippocampal Neurotransmitter Receptors in Autism: An Autoradiographic Study

Neuropathological studies in autistic brains have shown small neuronal size and increased cell packing density in a variety of limbic system structures including the hippocampus, a change consistent with curtailment of normal development. Based on these observations in the hippocampus, a series of quantitative receptor autoradiographic studies were undertaken to determine the density and distribution of eight types of neurotransmitter receptors from four neurotransmitter systems (GABAergic, serotoninergic [5-HT], cholinergic, and glutamatergic). Data from these single concentration ligand binding studies indicate that the GABAergic receptor system (³[H]-flunitrazepam labeled benzodiazepine binding sites and ³[H]-muscimol labeled GABA_A receptors) is significantly reduced in high binding regions, marking for the first time an abnormality in the GABA system in autism. In contrast, the density and distribution of the other six receptors studied (³[H]-8OH-DPAT labeled 5-HT_1A receptors, ³[H]-ketanserin labeled 5-HT₂ receptors, ³[H]-pirenzepine labled M1 receptors, ³[H]-hemicholinium labeled high affinity choline uptake sites, ³[H]-MK801 labeled NMDA receptors, and ³[H]-kainate labeled kainate receptors) in the hippocampus did not demonstrate any statistically significant differences in binding.     

Antagonism of muscarinic receptors in the rabbit iris-ciliary body by 8-OH-DPAT and other 5-HT1A receptor agonists

Summary Physiological studies have shown that serotonin and 5-HT_1A agonists can influence muscarinic function in the rabbit iris-ciliary body (ICB). The purpose of this study was to examine whether a direct interaction exists between muscarinic and 5-HT_1A receptors in the ICB. At high concentrations, the 5-HT_1A agonist 8-OH-DPAT attenuated the carbachol-induced stimulation of inositol phosphates (InsPs) production, but this was not blocked by the presence of 5-HT_1A antagonists. In contrast, serotonin failed to influence carbachol-induced InsPs formation. Moreover, 8-OH-DPAT but not serotonin displayed affinity for [³H]QNB binding sites in the ICB. The combined data suggest that activation of 5-HT_1A receptors in the ICB does not cause a modulation of muscarinic receptor-stimulated phosphoinositide turnover. The data instead suggest that, at high concentrations, 8-OH-DPAT acts as an antagonist at muscarinic receptors and in this way influences muscarinic receptor function. The mechanism of 5-HT-induced modulation of muscarinic function in the ICB therefore remains to be elucidated.     

Anti-serotonergic effects of urethane and chloral hydrate may not be mediated by a blockade of 5-HT2 receptors

Summary The general anesthetics urethane and chloral hydrate have profound anti-serotonergic effects both in the rat cortex in vivo and the rat aortic ring in vitro. The suggestion that these effects may be due to an action on 5-HT₂ receptors was tested using ex vivo and in vitro [³H]ketanserin binding assays with membrane-enriched fractions from rat brain. Urethane did not alter [³H]ketanserin binding in the ex vivo assay. In the in vitro assay, urethane, chloral hydrate, and its active metabolite 2,2,2-trichloroethanol produced slight reductions (of 16%, 9%, and 18%, respectively) of [³H]ketanserin binding. These studies suggest that anti-serotonergic effects of urethane and chloral hydrate may not be mediated by a blockade of 5-HT₂ receptors.     

Autoradiographic mapping of 5-HT1B and 5-HT1D receptors in the post mortem human brain using [H]GR 125743

The distribution of 5-HT_1B and 5-HT_1D receptors in the human post mortem brain was examined using whole hemisphere autoradiography and the radioligand [³H]GR 125743. [³H]GR 125743 binding was highest in the substantia nigra and the globus pallidus. Lower levels were detected in the striatum, with the highest densities in the ventromedial parts. In the amygdala, the hippocampus, the septal region and the hypothalamus, lower [³H]GR 125743 binding was observed, reflecting low densities of 5-HT_1B/1D receptors. In the cerebral cortex, binding was similar in most regions, although restricted parts of the medial occipital cortex were markedly more densely labeled. Binding densities were very low in the cerebellar cortex and in the thalamus. Two methods were used to distinguish between the two receptor subtypes, the first using ketanserin to block 5-HT_1D receptors and the second using SB 224289 to inhibit 5-HT_1B receptor binding. The autoradiograms indicated that in the human brain, the 5-HT_1B receptor is much more abundant than the 5-HT_1D receptor, which seemed to occur only in low amounts mainly in the ventral pallidum. Although [³H]GR 125743 is a suitable radioligand to examine the distribution of 5-HT_1B receptors in the human brain in vitro, the selectivities of ketanserin and SB 224289 are not sufficiently high to give definite evidence for the occurrence of the 5-HT_1D receptor in the human brain.     

Abnormal persistence of cerebellar serotonin-1A receptors in schizophrenia suggests failure to regress in neonates

Summary. This study investigated the neurodevelopmental basis of schizophrenia by examining an early transient population of serotonin-1A (5-HT_1A) receptors using quantitative [³H]8-OH-DPAT autoradiography on sections of frozen postmortem cerebellum. Production of an ontogenetic map showed that human neonatal cerebellum acquired dense 5-HT_1A receptors, most of which were eliminated by early childhood. Autoradiographic measurements on cerebellar vermis from 16 control adult subjects confirmed sparse 5-HT_1A receptor binding. The data show a persistence of some vermal 5-HT_1A receptors in brains from 19 adults with chronic schizophrenia in whom there may have been a slowed or arrested postnatal regression of vermal 5-HT_1A receptors. Alternatively, some 5-HT_1A receptors may have been re-expressed prior to, or subsequent to, the onset of the disease symptoms. The findings are not obviously explained by drug treatment and there are no data to explain how neuroleptics might promote expression of cerebellar 5-HT_1A receptors. We propose that the study has identified a neurotransmitter receptor population which, in schizophrenia, undergoes misdirected reshaping during brain development. The findings support neurodevelopmental hypotheses of the disease. Accepted November 3, 1997; received March 3, 1997     

Imaging cortical dopamine D1 receptors using [11C]NNC112 and ketanserin blockade of the 5-HT2A receptors

[¹¹C]NNC112 (8-chloro-7-hydroxy-3-methyl-5-(7-benzofuranyl)-2,3,4,5-tetrahydro-IH-3-benzazepine), a selective positron-emission tomography (PET) ligand for the D₁ receptor (R) over the 5-HT_2A R in vitro, has shown lower selectivity in vivo, hampering measurement of D₁ R in the cortex. [¹¹C]NNC112 PET and intravenous (i.v) ketanserin challenge were used to (1) confirm the previous findings of [¹¹C]NNC112 in vivo D₁ R selectivity, and (2) develop a feasible methodology for imaging cortical D₁ R without contamination by 5-HT_2A R. Seven healthy volunteers underwent [¹¹C]NNC112 PET scans at baseline and after a 5-HT_2A R-blocking dose of ketanserin (0.15 mg/kg, i.v.). Percent BP_ND change between the post-ketanserin and baseline scans was calculated. Irrespective of the quantification method used, ketanserin pretreatment led to significant decrease of BP_ND in the cortical (∼30%) and limbic regions (∼20%) but not in the striatum, which contains a much lower amount of 5-HT_2A R. Therefore, ketanserin allows D₁ R signal to be detected by [¹¹C]NNC112 PET without significant 5-HT_2A R contamination. These data confirm the presence of a significant 5-HT_2A R contribution to cortical [¹¹C]NNC112 signal, and call for caution in the interpretation of published [¹¹C]NNC112 PET findings on cortical D₁ R in humans. In the absence of more selective ligands, [¹¹C]NNC112 PET with ketanserin can be used for cortical D₁ R imaging in vivo.     

Autoradiographic Mapping of Brain 5-HT2A Binding Sites in P and in AA Alcohol-Preferring Rats

There is considerable evidence for an involvement of serotonergic mechanisms in the control of alcohol consumption. In the present study, an extensive 5-HT_2A receptor autoradiographic investigation was carried out in two genetically selected rat strains, P and AA alcohol-preferring rats, respectively, as well as in the corresponding NP and ANA alcohol-nonpreferring rats. The aim was to determine if there is any common pattern in 5-HT_2A binding site densities that may illuminate mechanisms of alcohol preference in these animals. For quantitating 5-HT_2A binding sites, [³H]ketanserin (2 nM) was used. Nonspecific binding was measured in the presence of methysergide 10⁻⁶ M. Results demonstrated a lower level (from 50 to 70%) of 5-HT_2A binding sites in the layer IV of prefrontal cortex, frontal cortex, parietal cortex of P rats compared to NP controls. Similarly, in the claustrum, 5-HT_2A binding density of P rats was 50% lower than that of NP rats, although this failed to achieve statistical significance. No difference was detected in the other areas investigated, including the olfactory tubercles, nucleus accumbens, caudate putamen, pyriform cortex, ventral tegmental area, temporal cortex, and entorhinal cortex. In AA rats, [³H]ketanserin binding density measured in these brain areas was very similar to that observed in ANA nonpreferring controls, and statistical analysis did not reveal any significant difference between the two rat lines. The present study confirms previous reports demonstrating lower densities of 5-HT_2A binding sites in the P rats and provides the first autoradiographic evidence showing that such an alteration does not occur in AA rats. These findings suggest that the expression of high alcohol preference in genetically selected P and AA rats is not associated with a shared neurochemical alteration of the 5-HT_2A receptor system.     

Autoradiographic evidence for the heterogeneity of 5-HT1 sites in the rat brain

The distribution of the binding sites of a new, potent agonist of serotonin (5-HT), 8-OH-N,N-dipropoyl-2-aminotetralin (PAT), was studied in the rat brain with the quantitative autoradiographic technique utilizing tritium-sensitive LKB film. The localization of [³H]PAT binding sites was very similar to that of [³H]5-HT binding sites, except in some discrete regions (choroid plexus, striatum, area preoptica lateralis, subiculum, and substantia nigra), which exhibited very low levels of labeling with [³H]PAT and high levels with [³H]5-HT. These results indicate that 5-HT₁ receptors are heterogeneous, and that [³H]PAT recognizes only a 5-HT₅ subclass (called 5-HT_1A).     

Two distinct serotonin receptors: regional variations in receptor binding in mammalian brain

Two distinct serotonin receptors in mammalian brain are labeled respectively with [³H]serotonin (5-HT₁) and [³H]spiperone (5-HT₂). In general, agonists display highest affinities for 5-HT₁ while antagonists prefer 5-HT₂ sites. To conduct regional studies of 5-HT receptors, we estimated 5-HT₂ sites with [³H]spiperone, using the 5-HT₂ specific antagonist cinanserin to displace binding to 5-HT₂ but not dopamine receptors and sulpiride to displace [³H]spiperone from dopamine but not 5-HT receptors. About 15% of cerebral cortical [³H]spiperone binding appears to involve dopamine sites and the remainder involves 5-HT₂ receptors. In the corpus striatum about 80% of [³H]spiperone binding labels dopamine receptors and the rest involves 5-HT₂ sites. [³H]mianserin binds about equally to 5-HT₂ sites are studies selectively by displacing histamine H₁-receptor binding with the H₁-antihistamine triprolidine. [³H]LSD labels both 5-HT₁ and 5-HT₂ receptors. Its binding to 5-HT₁ sites is displaced selectively with 5-HT, while its binding to 5-HT₂ receptors is displaced with cinanserin. [³H]5-HT labels only 5-HT₁ receptors. The regional distribution of the two 5-HT receptors is similar in rat, guinea pig and bovine brain. However, the regional patterns of 5-HT₁ and 5-HT₂ receptors differ considerably in all 3 species. The hippocampus is quite high in 5-HT₁ receptors but low in 5-HT₂ sites. The cerebellum contains the lowest levels of both 5-HT₁ and 5-HT₂ receptors. In bovine brain, most areas contain similar numbers of 5-HT₁ and 5-HT₂ receptors. However, the substantia nigra, the richest 5-HT₁ area inbovine brain, possesses 10 times more 5-HT₁ than 5-HT₂ sites.     

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [H]WAY-100635 and [C]WAY-100635

The distribution of 5-HT_1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT_1A receptor antagonist [³H]WAY-100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities of [³H]WAY-100635 binding, reflecting low densities of 5-HT_1A receptors. The labeling was very low in basal ganglia, such as nucleus caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT_1A receptors with [³H]WAY-100635 was antagonized by the addition of the 5-HT_1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 μM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [³H]WAY-100635 has a K_d of approximately 2.5 nM. The selective labeling of 5-HT_1A receptors with [³H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT_1A receptor subtype in vitro. [¹¹C]WAY-100635 is used for the characterization of 5-HT_1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t_1/2=20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [¹¹C]WAY-100635 gave images qualitatively similar to those of [³H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 μM), blocked all binding of [¹¹C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT_1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT_1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [¹¹C]WAY-100635. These data provide a strong basis for expecting [¹¹C]WAY-100635 to behave as a highly selective radioligand in vivo.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Atypical in vitro and in vivo binding of [3H]S-14506 to brain 5-HT1A receptors

Summary The tritiated derivative of the potent 5-HT_1A receptor agonist S-14506 {1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)piperazine} was tested for its capacity to selectively label the serotonin 5-HT_1A receptors both in vitro in the rat and the mouse brain, and in vivo in the mouse. In vitro studies showed that the pharmacological profile and the distribution of [³H]S-14506 specific binding sites (Kd=0.15 nM) in different brain regions matched perfectly those of the prototypical 5-HT_1A receptor ligand [³H]8-OH-DPAT. However, in the three regions examined (hippocampus, septum, cerebral cortex), the density of [³H]S-14506 specific binding sites was significantly higher (+ 66–90%) than that found with [³H]8-OH-DPAT. Whereas the specific binding of [³H]8-OH-DPAT was markedly reduced by GTP and Gpp(NH)p and increased by Mn²⁺, that of [³H]S-14506 was essentially unaffected by these compounds. In addition, the alkylating agent N-ethylmaleimide was much less potent to inhibit the specific binding of [³H]S-14506 than that of [³H]8-OH-DPAT. Measurement of in vivo accumulation of tritium one hour after i.v. injection of [³H]S-14506 to mice revealed marked regional differences, with about 2.5 times more radioactivity in the hippocampus than in the cerebellum. Pretreatment with 5-HT_1A receptor ligands prevented tritium accumulation in the hippocampus but not in the cerebellum. Autoradiograms from brain sections of injected mice confirmed the specific in vivo labeling of 5-HT_1A receptors by [³H]S-14506, therefore suggesting further developments with derivatives of this molecule for positron emission tomography in vivo in man.     

Identification of 5-hydroxytryptamine1 binding site subtypes in rat spinal cord

High-affinity, specific [³H]-hydroxytryptamine (5-HT) binding was analyzed in rat spinal cord homogenates. Drug competition studies were performed using 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) to complete selectively for 5-HT_1A sites. RU 24969 to complete for 5-HT_1Bsites and mesulergine to compete for 5-HT_1Csites. Competition data were analyzed by computer-assisted iterative curve-fitting analysis. The results demonstrate that 5-HT_1A, 5-HT_1Band 5-HT_1C binding sites are present in rat spinal cord. In addition, approximately 33% of total 5-HT₁ sites do not appear to represent either 5-HT_1A, 5-HT_1B or 5-HT_1C binding sites. Therefore, 5-HT_1Dand/or some other 5-HT₁ binding site subtype may also be present in rat spinal cord.     

Differential radioligand binding properties of [H]5-hydroxytryptamine and [H]mesulergine in a clonal 5-hydroxytryptamine1C cell line

[³H]5-Hydroxytryptamine ([³H]5-HT) and [³H]mesulergine were used to label 5-HT_1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [³H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P < 0.01). The K_D values of [³H]5-HT for the 2 populations are0.5±0.1nM and31±15nM, while the B_max values are400±90pmol/g protein and 3,000±600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [³H]5-HT using a centrifugation assay (K_D1 = 0.6±0.06nM, K_D2 = 60±10nM;B_max1 = 740±90pmol/g, B_max2 = 4,000±700pmol/g). By contrast, saturation analysis of [³H]mesulergine binding is monophasic (K_D = 4.7±0.7nM) with a B_max value (6,800±1,000pmol/g protein) that is significantly greater than that obtained using [³H]5-HT (P < 0.01). Drug competition studies confirm that both [³H]5-HT and [³H]mesulergine label at least 2 subpopulations of expressed 5-HT_1C receptors in NIH 3T3 cells. 10⁻⁴ M GTP eliminates the high affinity [³H]5-HT-labeled binding sites with minimal effect on the low affinity [³H]5-HT-labeled sites and no effect on [³H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT_1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.