Negative versus positive allosteric modulation of metabotropic glutamate receptors (mGluR5): indices for potential pro-cognitive drug properties based on EEG network oscillations and sleep-wake organization in rats.

RATIONALE: Evidence is emerging that positive and negative modulation of the metabotropic glutamate (mGluR5) receptors has the potential for treating cognitive deficits and neuroprotection associated with psychiatric and neurodegenerative diseases, respectively. Sleep and synchronisation of disparate neuronal networks are critically involved in neuronal plasticity, and disturbance in vigilance states and cortical network connectivity contribute significantly to cognitive deficits described in schizophrenia and Alzheimer's disease. Here, we examined the circadian changes of mGluR5 density and the functional response to modulation of mGluR5 signaling. METHODS: The current study carried out in Sprague-Dawley rats quantified the density of mGluR5 across the light-dark cycle with autoradiography. The central activity of mGluR5 negative allosteric modulators (2-methyl-6-(phenylethynyl)pyridine (MPEP) and [(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and positive allosteric modulators (S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone (ADX47273) and (7S)-3-tert-butyl-7-[3-(4-fluoro-phenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine (LSN2814617) was examined on sleep-wake architecture. The functional effect of mGluR5 modulation on cortical networks communication was described in freely moving animals. RESULTS: The density of mGluR5 in the striatal, cortical, hippocampal and thalamic structures was unchanged across the light-dark cycle. Allosteric blockade of mGluR5 consistently consolidated deep sleep, enhanced sleep efficiency and elicited prominent functional coherent network activity in slow theta and gamma oscillations. However, allosteric activation of mGluR5 increased waking, decreased deep sleep and reduced functional network connectivity following the activation of slow alpha oscillatory activity. CONCLUSION: This functional study differentiates the pharmacology of allosteric blockade of mGluR5 from that of allosteric activation and suggests that mGluR5 blockade enhances sleep and facilitates oscillatory network connectivity, both processes being known to have relevance in cognition processes.

Preclinical evaluation of [(18)F]JNJ42259152 as a PET tracer for PDE10A.

Phosphodiesterase-10A (PDE10A) is implicated in several neuropsychiatric disorders involving basal ganglia neurotransmission, such as schizophrenia, obsessive-compulsive disorder and Huntington's disease. To confirm target engagement and exposure-occupancy relationships of clinical candidates for treatment, and to further explore the in vivo biology of PDE10A, non-invasive imaging using a specific PET ligand is warranted. Recently we have reported the in vivo evaluation of [(18)F]JNJ41510417 which showed specific binding to PDE10A in rat striatum, but with relatively slow kinetics. A chemically related derivative JNJ42259152 was found to have a similar in vivo occupancy, but lower lipophilicity and lower PDE10A in vitro inhibitory activity compared to JNJ41510417. (18)F-labeled JNJ42259152 was therefore evaluated as a potential PDE10A PET radiotracer. Baseline PET in rats and monkey showed specific retention in the PDE10A-rich striatum, and fast wash-out, with a good contrast to non-specific binding, in other brain regions. Pretreatment and chase experiments in rats with the selective PDE10A inhibitor MP-10 showed that tracer binding was specific and reversible. Absence of specific binding in PDE10A knock-out (KO) mice further confirmed PDE10A specificity. In vivo radiometabolite analysis using high performance liquid chromatography (HPLC) showed presence of polar radiometabolites in rat plasma and brain. In vivo imaging in rat and monkey further showed faster brain kinetics, and higher striatum-to-cerebellum ratios for [(18)F]JNJ42259152 compared to [(18)F]JNJ41510417. The arterial input function corrected for radiometabolites was determined in rats and basic kinetic modeling was established. For a 60-min acquisition time interval, striatal binding potential of the intact tracer referenced to the cerebellum showed good correlation with corresponding binding potential values of a Simplified Reference Tissue Model and referenced Logan Plot, the latter using a population averaged reference tissue-to-plasma clearance rate and offering the possibility to generate representative parametric binding potential images. In conclusion we can state that in vivo imaging in PDE10A KO mice, rats and monkey demonstrates that [(18)F]JNJ42259152 provides a PDE10A-specific signal in the striatum with good pharmacokinetic properties. Although presence of a polar radiometabolite in rat brain yielded a systematic but reproducible underestimation of the striatal BPND, a Logan reference tissue model approach using 60 min acquisition data is appropriate for quantification.

Pharmacokinetic-pharmacodynamic modelling of fluvoxamine 5-HT transporter occupancy in rat frontal cortex.

BACKGROUND AND PURPOSE: The pharmacokinetic-pharmacodynamic (PK-PD) correlation of fluvoxamine 5-HT transporter (SERT) occupancy was determined in rat frontal cortex ex vivo. EXPERIMENTAL APPROACH: Rats (n=47) with permanent arterial and venous cannulas received a 30 min intravenous infusion of fluvoxamine (1 or 7.3 mg kg(-1)). At various time points after dosing, brains were collected for determination of fluvoxamine concentration and SERT occupancy. In addition, the time course of fluvoxamine concentration in plasma was determined up to the time of brain collection. In a separate study (n=26), the time course of fluvoxamine concentration in brain extracellular fluid (ECF) and plasma was determined. The results of the investigations were interpreted by nonlinear mixed effects modeling. KEY RESULTS: Highest SERT occupancy was reached at the first time point (10 or 15 min) and maintained for 1.5 and 7 h after 1 and 7.3 mg kg(-1), respectively. Thereafter, SERT occupancy decreased linearly at a rate of 8% h(-1). SERT occupancy could be directly related to plasma, brain ECF and brain tissue concentrations by a hyperbolic function (Bmax model). Maximal SERT occupancy (Bmax) was 95%. Estimated concentrations at half-maximal SERT occupancy (EC50) in plasma, ECF and brain tissue were 0.48, 0.22 and 14.8 ng mL(-1) respectively. The minimum value of the objective function decreased 12 points for ECF and brain tissue concentrations relative to plasma (P<0.01), presumably as a result of nonlinear brain distribution. CONCLUSIONS AND IMPLICATIONS: The proposed PK-PD model constitutes a useful basis for prediction of the time course of ex vivo SERT occupancy in behavioural studies with selective serotonin reuptake inhibitors.

Effects of chronic infusion of neurotensin and a neurotensin NT1 selective analogue PD149163 on amphetamine-induced hyperlocomotion.

Neurotensin (NT) has been proposed as an endogenous antipsychotic based in part on the similarity in behavioural effects to antipsychotic drugs, for example, attenuation of both amphetamine-induced hyperlocomotion (AH) and amphetamine disrupted pre-pulse inhibition in the rat. However, there is some evidence that repeated administration of NT or an analogue produces behavioural tolerance to such effects. The present experiments sought to confirm and extend these findings by testing the effects on AH of 7 days central administration of NT and the NT1 selective analogue PD 149163 and the effects of 21 days central administration of NT. NT and PD149163 continuously administered for 7 days produced no effect on AH (in contrast to attenuation with a single injection here and previously reported), whereas 21 days of NT administration potentiated AH. Together, these studies report that the effects of NT or a NT analogue on AH depends on the duration of administration of peptide. The results are discussed in comparison with the reported antipsychotic properties of acute administration of NT and possible mechanisms involving NT1 receptors.

Synthesis and biodistribution of [11C]R107474, a new radiolabeled alpha2-adrenoceptor antagonist.

R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective alpha(2)-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for halpha(2A)- and halpha(2C)-adrenoceptors (K(i) = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the halpha(2B)-adrenoceptors and 5-hydroxytryptamine(7) (h5-HT(7)) receptors (K(i) = 1 and 5 nM, respectively). R107474 interacted weakly (K(i) values ranging between 81 and 920 nM) with dopamine-hD(2L), -hD(3) and -hD(4), h5-HT(1D)-, h5-HT(1F)-, h5-HT(2A)-, h5-HT(2C)-, and h5-HT(5A) receptors. The compound, tested up to 10 microM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo alpha(2A)- and alpha(2C)-adrenoceptor occupancy was measured by ex vivo autoradiography 1h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the alpha(2A)- and alpha(2C)-adrenoceptors with an ED(50) (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-(11)C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([(11)C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central alpha(2)-adrenoceptors. [(11)C]R107474 was obtained via a Pictet-Spengler reaction with [(11)C]formaldehyde in 33 +/- 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/micromol. The biodistribution of [(11)C]R107474 in rats revealed that the uptake of [(11)C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of alpha(2)-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 +/- 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 +/- 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 +/- 0.45, 30 min pi) and entorhinal cortex (3.43+/-0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective alpha(2)-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [(11)C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [(11)C]R107474 deserves further investigation as a potential radioligand for studying alpha(2)-adrenoceptors using PET.

Bilateral control of brain activity by dopamine D1 receptors: evidence from induction patterns of regulator of G protein signaling 2 and c-fos mRNA in D1-challenged hemiparkinsonian rats.

Recent reports show that striatal dopamine D1-type receptors from one side of the normal rat brain can control brain activity (as measured by c-fos induction) on both sides of the brain. However, this phenomenon has not yet been studied in the presence of sensitized dopamine D1-type receptors. Here we address this issue by investigating the extent to which dopamine D1-type receptors control brain activation in rats with unilaterally sensitized dopamine D1-type receptors. Gene induction assays were used to identify activated regions from midbrain to forebrain in unilaterally 6-hydroxydopamine lesioned (hemiparkinsonian) rats challenged with the full dopamine D1-type agonist SKF82958 (3 mg/kg, 0.5 and 2 h). The genes used are c-fos, the proven neuronal activity marker, and Regulator of G protein Signaling 2, a gene we propose as a marker of signaling homeostasis. SKF82958-mediated induction of both genes is greatly enhanced in hemiparkinsonian rats compared with shams, in both the lesioned and the intact hemisphere. For example, in the denervated caudate-putamen at 2 h postinjection, this enhancement is more than 80-fold for c-fos and up to 20-fold for Regulator of G protein Signaling 2; for the intact side this is 35-fold for c-fos and 27-fold for Regulator of G protein Signaling 2. Cortical induction of c-fos and Regulator of G protein Signaling 2 was generalized to most neocortical regions and was essentially equivalent in both the denervated and intact hemispheres. Interestingly, hippocampal structures also showed strong bilateral induction of both genes. This overall pattern of brain activation can be accounted for by the basal-ganglia thalamocortical and hippocampal circuits which both contain hemisphere-crossing connections and which can be initially activated in the lesioned hemisphere. Some regions, such as the intact striatum or the CA1 region, showed relatively low c-fos induction and relatively high Regulator of G protein Signaling 2 induction, possibly indicating that these regions are engaged in unusually strong signaling regulation activities. Our results show that, besides basal ganglia-thalamocortical circuits, dopamine D1-type-mediated brain activation in hemiparkinsonian rats also involves hippocampal circuits.

Synthesis and biodistribution of [(11)C]R116301, a promising PET ligand for central NK(1) receptors.

N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(11)C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) ligand for investigation of central neurokinin(1) (NK(1)) receptors. 1-Bromo-3,5-di(trifluoromethyl)benzene was converted in three steps into 3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl chloride, which was reacted with N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzylhexahydro-4-pyridinyl]piperazino}acetamide providing [(11)C]R116301 in 45-57% decay-corrected radiochemical yield. The total synthesis time, from end of bombardment (EOB) to the formulated product, was 35 min. Specific activity (SA) was 82-172 GBq/micromol (n=10) at the end of synthesis. N1-([4-(3)H]-2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(3)H]R116301) was also synthesized (SA: 467 GBq/mmol). The B(max) for [(3)H]R116301 measured in vitro on Chinese hamster ovary cell membranes stably transfected with the human NK(1) receptor was 19.10+/-1.02 pmol/mg protein with an apparent dissociation constant of 0.08+/-0.01 nM. Ex vivo, in vivo and in vitro autoradiography studies with [(3)H]R116301 in gerbils demonstrated a preferential accumulation of the radioactivity in the striatum, olfactory tubercule, olfactory bulb and locus coeruleus. In vivo, the biodistribution of [(11)C]R116301 in gerbils revealed that the highest initial uptake is in the lung, followed by the liver and kidney. In the brain, maximum accumulation was found in the olfactory tubercules (1.10+/-0.08 injected dose (ID)/g 20 min post injection (p.i.)) and the nucleus accumbens (1.00+/-0.12ID/g 10 min p.i.). Tissue/cerebellum concentration ratios for striatum and nucleus accumbens increased with time due to rapid uptake followed by a slow wash out (1.29 and 1.64, respectively, 30 min p.i.). A tissue to cerebellum ratio of 1.33 and 1.62 was also observed for olfactory bulb and olfactory tubercules, respectively (20 min p.i.). In summary, [(11)C]R116301 appears to be a promising radioligand suitable for the visualization of NK(1) receptors in vivo using PET.

Acute wake-promoting actions of JNJ-5207852, a novel, diamine-based H3 antagonist.

1 1-[4-(3-piperidin-1-yl-propoxy)-benzyl]-piperidine (JNJ-5207852) is a novel, non-imidazole histamine H3 receptor antagonist, with high affinity at the rat (pKi=8.9) and human (pKi=9.24) H3 receptor. JNJ-5207852 is selective for the H3 receptor, with negligible binding to other receptors, transporters and ion channels at 1 microm. 2 JNJ-5207852 readily penetrates the brain tissue after subcutaneous (s.c.) administration, as determined by ex vivo autoradiography (ED50 of 0.13 mg kg(-1) in mice). In vitro autoradiography with 3H-JNJ-5207852 in mouse brain slices shows a binding pattern identical to that of 3H-R-alpha-methylhistamine, with high specific binding in the cortex, striatum and hypothalamus. No specific binding of 3H-JNJ-5207852 was observed in brains of H3 receptor knockout mice. 3 In mice and rats, JNJ-5207852 (1-10 mg kg(-1) s.c.) increases time spent awake and decreases REM sleep and slow-wave sleep, but fails to have an effect on wakefulness or sleep in H3 receptor knockout mice. No rebound hypersomnolence, as measured by slow-wave delta power, is observed. The wake-promoting effects of this H3 receptor antagonist are not associated with hypermotility. 4 A 4-week daily treatment of mice with JNJ-5207852 (10 mg kg(-1) i.p.) did not lead to a change in body weight, possibly due to the compound being a neutral antagonist at the H3 receptor. 5 JNJ-5207852 is extensively absorbed after oral administration and reaches high brain levels. 6 The data indicate that JNJ-5207852 is a novel, potent and selective H3 antagonist with good in vitro and in vivo efficacy, and confirm the wake-promoting effects of H3 receptor antagonists.

Detailed localization of regulator of G protein signaling 2 messenger ribonucleic acid and protein in the rat brain.

Regulator of G protein signaling (RGS) proteins are a recently identified family of proteins which dampen G protein-coupled receptor-mediated signaling by accelerating the intrinsic GTPase activity of Galpha subunits of heterotrimeric G proteins. More than 20 different RGSs have been identified and at least 10 are expressed in the CNS. The present study describes in detail the localization in the rat brain of one member of this family, RGS2. The distribution of RGS2 mRNA and protein has been studied in parallel by performing in situ hybridization and immunoautoradiography on adjacent rat brain sections. Our localization study reveals that RGS2 mRNA and protein are widely expressed in the brain. Protein and mRNA are mostly colocalized such as in neocortex, piriform cortex, caudate-putamen, septum, hippocampus, locus coeruleus. Some mismatches were also observed such as presence of mRNA but not protein in the paraventricular nucleus, the substantia nigra pars compacta and the red nucleus, suggesting that RGS2 protein is present in neuronal projections. Previous reports describing an induction of RGS2 mRNA in the rat striatum after psychostimulants (amphetamine, cocaine) led us to focus on the distribution of RGS2 in the basal ganglia circuitry. The absence of RGS2 mRNA and protein in the globus pallidus suggests that RGS2 would play its regulatory role more in the direct (striatonigral) than in the indirect (striatopallidal) striatal output pathway. In addition, to delineate the implication of RGS2 in pre- and/or postsynaptic functions in the basal ganglia, we performed lesions of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) and striatal quinolinic acid lesions. The 6-OHDA lesion did not modify RGS2 mRNA or protein levels in the caudate-putamen whereas the intrastriatal quinolinic acid infusion caused a marked reduction of RGS2 mRNA and protein in the lesioned zone. These data indicate that RGS2 is predominantly expressed in intrinsic striatal neurons. Moreover, the absence of detectable change in RGS2 expression after injections of 6-OHDA suggests also that RGS2 is not primarily involved in the hypersensitization of postsynaptic dopamine receptors observed after lesion of the nigrostriatal pathway.

The differential effects of atypical antipsychotics on prolactin elevation are explained by their differential blood-brain disposition: a pharmacological analysis in rats.

All atypical antipsychotics avoid extrapyramidal side-effects yet differ in their propensity to cause other side-effects, like prolactin elevation. We proposed that the atypical antipsychotics with a propensity for prolactin elevation would show a higher pituitary versus striatal D2 receptor occupancy. To investigate this hypothesis, we tested four atypical antipsychotics, two that are commonly associated with prolactin elevation (amisulpride and risperidone) and two that are less frequently associated (quetiapine and olanzapine). In particular, we calculated their ED(50) values to increase plasma prolactin and block peripheral pituitary D2 receptors to their ED(50) values to antagonize apomorphine-induced stereotypy and occupy central striatal D2 receptors. All antipsychotics dose dependently increased prolactin levels and antagonized apomorphine-induced stereotypy. However, the central to peripheral potency (ED(50) for apomorphine antagonism to ED(50) for prolactin elevation) differed remarkably across these drugs: amisulpride (21764), risperidone (14), quetiapine (10), and olanzapine (1.7). Compounds displaying a higher peripheral potency brought about higher prolactin levels for a given level of functional central antagonism. This dissociation between central and peripheral effects was explained by the differential occupancy of D2 receptors in the striatum versus in the pituitary [ratio of striatal/pituitary ED(50) values (milligram per kilogram) for D2 occupancy): amisulpride (17/0.026 = 654), risperidone (0.89/0.081 = 14), quetiapine (24/4.1 = 6), olanzapine (0.30/0.43 = 0.7). These results indicate that dissociation between central and peripheral D2 receptor occupancy is a major determinant of the degree of prolactin elevation observed at therapeutic doses.

Use of the beta-imager for rapid ex vivo autoradiography exemplified with central nervous system penetrating neurokinin 3 antagonists.

The neurokinin 3 (NK3) receptor antagonists represent a novel class of pharmacological agents, which are currently under evaluation for the treatment of psychiatric disorders. An efficient brain penetration is one of the main prerequisites to further evaluate compounds displaying high potency to bind the NK3 receptor. The present report describes a method for determining the in vivo occupancy of central NK3 receptors after peripheral administration of drugs. An ex vivo measurement of NK3 receptor occupancy by quantitative autoradiography employing [3H]senktide as the radioligand has been developed. The speed of the method, which is usually considered low due to the time dedicated to film exposure (from weeks to months), has been considerably increased by the use of the beta-imager. The high sensitivity of this new radioimager was used to visualize and quantitatively analyze the [3H]senktide binding sites in brain sections within hours. Using this method, we have demonstrated that the reference NK3 antagonist SR142801 dose dependently occupied the NK3 receptors in the gerbil brain after subcutaneous administration with an ED50 of 0.85 mg/kg. The less active enantiomer SR142806 occupied the NK3 receptors only by 25% at the highest used dose of 10 mg/kg. These values are in accordance with the reported behavioral effects of the compounds. Our results indicate that ex vivo receptor occupancy measurements can be dependently used to predict the central activity of NK3 antagonists. More generally, the combination of ex vivo receptor autoradiography with the beta-imager detection constitutes a new and fast method to evaluate the brain penetration of drug candidates.

Detailed distribution of Neurokinin 3 receptors in the rat, guinea pig and gerbil brain: a comparative autoradiographic study.

The neurokinin 3 (NK3) receptor is predominantly expressed in the central nervous system (CNS). Species differences in neurokinin 3 (NK3) receptor pharmacology have led to the preferential use of guinea pigs and gerbils in the characterization of non-peptide NK3 antagonists. Little is known about the central localization of NK3 receptors in the CNS of these species. To study this, [(3)H]senktide and [(3)H]SR 142801 were used in autoradiography experiments to visualize the NK3 receptors in the guinea pig and gerbil brain and compared to with the distribution of [(3)H]senktide binding sites in the rat brain. In the three species, the NK3 receptor was similarly distributed within the cerebral cortex, the zona incerta, the medial habenula, the amygdaloid complex, the superior colliculus and the interpeduncular nucleus. Outside of these structures, our study has revealed that each species displayed a specific distribution pattern of central NK3 receptors. The rat was the only species where NK3 receptors could be visualized in the striatum, the supraoptic nucleus and the paraventricular nucleus of the hypothalamus. The guinea pig differed mainly from the two other species by the absence of detectable binding sites in the substantia nigra pars compacta and the ventral tegmental area. A specific localization of NK3 receptors in the anterodorsal and anteroventral thalamic nuclei characterized the gerbil. This last species is also unique by in the higher level of NK3 receptors in the dorsal and median raphe nuclei. All these differences suggest that the NK3 receptor mediates different functions in different species.

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain.

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.

Mapping of serotonin 5-HT(4) receptor mRNA and ligand binding sites in the post-mortem human brain.

The anatomical localization of 5-HT(4) receptor mRNA and 5-HT(4) receptor protein was examined in sections of post-mortem human brain by in situ hybridization histochemistry and radioligand receptor autoradiography. In the in situ hybridization study, the highest levels of 5-HT(4) receptor mRNA were found in caudate nucleus, putamen, nucleus accumbens, and in the hippocampal formation. No 5-HT(4) receptor mRNA was detected in globus pallidus and substantia nigra. For receptor autoradiography, two new and highly selective radioligands were compared: [(3)H]prucalopride, which preferentially labels the G-protein coupled fraction of receptors, and [(3)H]R116712, which labels the entire receptor population at subnanomolar concentrations. [(3)H]Prucalopride and [(3)H]R116712 binding was performed on human brain hemisphere sections. The highest densities for both radioligands were found in the basal ganglia (caudate nucleus, putamen, nucleus accumbens, globus pallidus, substantia nigra). Moderate to low densities were detected in the hippocampal formation and in the cortical mantle. Mismatches between 5-HT(4) receptor mRNA and binding sites in the globus pallidus and the substantia nigra suggested that the binding sites may be localized on axonal projections originating from the striatum. To compare densities of binding sites, concentration binding curves with [(3)H]prucalopride, [(3)H]R116712 and [(3)H]GR113808 were performed on membranes from homogenates of several human brain regions. Comparison of B(max)-values obtained with [(3)H]prucalopride and [(3)H]R116712 indicated that the G-protein coupled fraction of 5-HT(4) receptors in the substantia nigra was exceptionally high (54%) in comparison with percentages (16-27%) found in the frontal cortex, the striatum and the hippocampus. Such a high percentage (40%) of [(3)H]prucalopride vs. [(3)H]R116712 binding was also observed in the substantia nigra in the receptor autoradiography experiments. The [(3)H]prucalopride binding was GppNHp-sensitive, whereas [(3)H]R116712 and [(3)H]GR113808 was not. These data indicate that in the substantia nigra 5-HT(4) receptors are more strongly coupled to their signal transduction pathway than in other brain regions.

Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain.

The 5-HT1A and 5-HT1B receptors of serotonin play important roles as auto- and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5-HT1A) and in the globus pallidus and substantia nigra (5-HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5-HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane-associated 5-HT1A receptors could be estimated to be at least 30-40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5-HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5-HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5-HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto- and as heteroreceptors. The localization of 5-HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS.

Dominant role of the cytosolic C-terminal domain of the rat 5-HT1B receptor in axonal-apical targeting.

The 5-HT(1A) and 5-HT(1B) receptors for serotonin exhibit a different membrane localization to either soma and dendrites (5-HT(1A)R) or axons and terminals (5-HT(1B)R) of neurons in the CNS. The mechanisms responsible for their differential targeting were investigated previously by transfecting various 5-HT(1A)R/5-HT(1B)R chimeras in the epithelial Lilly pork kidney (LLC-PK1) cell line. This first study suggested that a specific targeting signal is located in the C-terminal portion (comprising the last two transmembrane and the cytoplasmic C-terminal domains) of the 5-HT(1A)and/or 5-HT(1B) receptors. In the present study, the role of the cytosolic C-terminal tail of the receptors was further investigated by transfecting truncated receptors and 5-HT(1A)R/5-HT(1B)R chimeras in both the epithelial LLC-PK1 cells and rat hippocampal neurons in primary culture. Confocal microscopic analysis of immunofluorescence with specific anti-5-HTR antibodies and anti-microtubule-associated protein 2 or anti-neurofilament 200k antibodies showed that substitution of the cytosolic C-terminal tail of the 5-HT(1B)R in the 5-HT(1A)R addressed the resulting chimera to the axon of neurons and to the apical domain of LLC-PK1 cells. Therefore, the short tail of the 5-HT(1B)R presents an apical targeting signal that can also act as an axonal targeting signal. In addition, a domain within the third intracytoplasmic loop of the 5-HT(1B)R, responsible for its Golgi sequestration in LLC-PK1 cells, appeared to act as another axonal targeting signal in hippocampal neurons.

Humanization of mouse 5-hydroxytryptamine1B receptor gene by homologous recombination: in vitro and in vivo characterization.

We replaced the coding region of the murine 5-hydroxytryptamine (5-HT)1B receptor by the human 5-HT1B receptor using homologous recombination in embryonic stem cells and generated and characterized homozygous transgenic mice that express only the human (h) 5-HT1B receptor. The distribution patterns of h5-HT1B and murine (m) 5-HT1B receptor mRNA and binding sites in brain sections of transgenic and wild-type mice were identical as measured by in situ hybridization histochemistry and radioligand receptor autoradiography. When measured in parallel under identical conditions, the h5-HT1B receptor expressed in mouse brain had the same pharmacological characteristics as that in human brain. Stimulation by 5-HT1B agonists of [35S]guanosine-5'-O-(3-thio)triphosphate binding in brain sections demonstrated the functional coupling of the h5-HT1B receptor to G proteins in mouse brain. In tissue slices from various brain regions, electrically stimulated [3H]5-HT release was not modified by 5-HT1B agonists in tissue from either transgenic and wild-type mice; a 5-HT1B antagonist enhanced electrically stimulated [3H]5-HT release in wild-type mouse brain, but was ineffective in the transgenics. The centrally active 5-HT1A/5-HT1B agonist RU24969 induced hypothermia but did not increase locomotor activity in the transgenic mice. The ineffectiveness of RU24969 in the transgenic mice could be due to the lower affinity of the compound for the h5-HT1B receptor compared with the m5-HT1B receptor. The present study demonstrates a complete replacement of the mouse receptor by its human receptor homolog and a functional coupling to G proteins. However, modulation of [3H]5-HT release could not be shown. Furthermore, behavioral effects were not clearly observed, which may be due to a lack of appropriate tools.

Differential membrane targeting and pharmacological characterization of chimeras of rat serotonin 5-HT1A and 5-HT1B receptors expressed in epithelial LLC-PK1 cells.

The serotonin 5-HT1A and 5-HT1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT1A and 5-HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).

Co-localization of 5-HT1B- and 5-HT1D receptor mRNA in serotonergic cell bodies in guinea pig dorsal raphé nucleus: a double labeling in situ hybridization histochemistry study.

In order to provide further details on the cellular localization of 5-HT1B- and 5-HT1D receptor mRNA in the dorsal raphé nucleus, we performed, in the same sections of guinea-pig dorsal raphé nucleus, double labeling in situ hybridization histochemistry for: (1) 5-HT1B receptor mRNA and 5-HT1D receptor mRNA, (2) 5-HT1B receptor mRNA and 5-HT transporter (5-HTT) mRNA as marker for serotonergic neurons and (3) 5-HT1D receptor mRNA and 5-HTT mRNA. The 5-HT1B receptor mRNA was present in all cells containing 5-HT1D receptor mRNA. Similarly, both 5-HT1B- and 5-HT1D receptor mRNA was present in all 5-HTT mRNA positive cells. The present study demonstrates that 5-HT1B- and 5-HT1D receptor mRNA is co-localized in serotonergic cell bodies of the guinea pig dorsal raphé nucleus.

Light and electron microscopic immunocytochemical visualization of 5-HT1B receptors in the rat brain.

Specific antipeptide antibodies were used for the immunohistochemical visualization of 5-HT1B receptors in the rat brain. A dense, specific 5-HT1B receptor-like immunoreactivity was found in the globus pallidus, the dorsal subiculum and the substantia nigra. At the light microscope level, immunostaining was diffuse within the neuropil but absent from cell bodies. Observations at the electron microscope level in the substantia nigra showed immunoperoxidase staining in fine unmyelinated axons and nerve terminals.