Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Visualization of 5-HT Receptors Using Radioligand-Binding Autoradiography.

Described in this unit are techniques to visualize the majority of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes in sections of frozen brain tissue using receptor autoradiography. Protocols for brain extraction and sectioning, radioligand exposure, autoradiogram generation, and data quantification are provided, as are the optimal incubation conditions for the autoradiographic visualization of receptors using agonist and antagonist radioligands. © 2016 by John Wiley & Sons, Inc.

Cartography of 5-HT1A and 5-HT2A Receptor Subtypes in Prefrontal Cortex and Its Projections.

Since the development of chemical neuroanatomical tools in the 1960s, a tremendous wealth of information has been generated on the anatomical components of the serotonergic system, at the microscopic level in the brain including the prefrontal cortex (PFC). The PFC receives a widespread distribution of serotonin (5-hydroxytryptamine, 5-HT) terminals from the median and dorsal raphe nuclei. 5-HT receptors were first visualized using radioligand autoradiography in the late 1980s and early 1990s and showed, in contrast to 5-HT innervation, a differential distribution of binding sites associated with different 5-HT receptor subtypes. Due to the cloning of the different 5-HT receptor subtype genes in the late 1980s and early 1990s, it was possible, using in situ hybridization histochemistry, to localize cells expressing mRNA for these receptors. Double in situ hybridization histochemistry and immunohistochemistry allowed for the chemical characterization of the phenotype of cells expressing 5-HT receptors. Tract tracing technology allowed a detailed cartography of the neuronal connections of PFC and other brain areas. Based on these data, maps have been constructed that reflect our current understanding of the different circuits where 5-HT receptors can modulate the electrophysiological, pharmacological, and behavioral functions of the PFC. We will review current knowledge regarding the cellular localization of 5-HT1A and 5-HT2A receptors in mammalian PFC and their possible functions in the neuronal circuits of the PFC. We will discuss data generated in our laboratory as well as in others, focusing on localization in the pyramidal and GABAergic neuronal cell populations in different mammalian species using molecular neuroanatomy and on the connections with other brain regions.

Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action.

The prefrontal cortex (PFC) is involved in higher brain functions altered in schizophrenia. Classical antipsychotic drugs modulate information processing in cortico-limbic circuits via dopamine D2 receptor blockade in nucleus accumbens (NAc) whereas atypical antipsychotic drugs preferentially target cortical serotonin (5-HT) receptors. The brain networks involved in the therapeutic action of atypical drugs are not fully understood. Previous work indicated that medial PFC (mPFC) pyramidal neurons projecting to ventral tegmental area express 5-HT2A receptors suggesting that atypical antipsychotic drugs modulate dopaminergic activity distally, via 5-HT2A receptor (5-HT2A-R) blockade in PFC. Since the mPFC also projects heavily to NAc, we examined whether NAc-projecting pyramidal neurons also express 5-HT2A-R. Using a combination of retrograde tracing experiments and in situ hybridization we report that a substantial proportion of mPFC-NAc pyramidal neurons in rat brain express 5-HT2A-R mRNA in a layer- and area-specific manner (up to 68% in layer V of contralateral cingulate). The functional relevance of 5-HT2A-R to modulate mPFC-NAc projections was examined in dual-probe microdialysis experiments. The application of the preferential 5-HT2A-R agonist DOI into mPFC enhanced glutamate release locally (+66 ± 18%) and in NAc (+74 ± 12%) indicating that cortical 5-HT2A-R activation augments glutamatergic transmission in NAc. Since NAc integrates glutamatergic and dopaminergic inputs, blockade of 5-HT2A-R by atypical drugs may reduce cortical excitatory inputs onto GABAergic neurons of NAc, adding to dopamine D2 receptor blockade. Together with previous observations, the present results suggest that atypical antipsychotic drugs may control the activity of the mesolimbic pathway at cell body and terminal level.

Chronic effects of corticosterone on GIRK1-3 subunits and 5-HT1A receptor expression in rat brain and their reversal by concurrent fluoxetine treatment.

Dysregulation of the serotonergic system and abnormalities of the hypothalamic-pituitary-adrenal axis have been demonstrated in major depression. Animal studies indicate that 5-HT1A receptor expression may be reduced by long-term administration of corticosterone. However, similar studies on the regulation of GIRK channels, one of the most important effectors of the neuronal 5-HT1A receptor, are limited. In order to address these issues, slow-release corticosterone pellets were implanted subcutaneously to adrenal intact male rats (200mg pellets, 35 days release). Starting on day 15, animals were treated for 21 days with fluoxetine (5mg/kg/day, i.p.), or vehicle. Using in situ hybridization histochemistry and receptor autoradiography, we found that chronic corticosterone treatment was accompanied by a significant decrease on the mRNAs coding for mineralocorticoid receptors in hippocampal areas. Under these conditions, 5-HT1A receptor mRNA expression decreased in dorsal raphe nucleus and dentate gyrus. However, 5-HT1A receptor levels, as measured by [(3)H]-8-OH-DPAT binding, diminished significantly only in dentate gyrus. It is noteworthy that chronic treatment with fluoxetine reversed the alterations on 5-HT1A receptor mRNA levels only in dorsal raphe. Finally, chronic corticosterone treatment produced an increase on the mRNA coding for the GIRK2 subunit in several hypothalamic and thalamic areas, which was reversed by fluoxetine. Measurements of cell density and volume of the granular layer of the dentate gyrus did not reveal significant changes after corticosterone or corticosterone plus fluoxetine treatments. These data are relevant for a better understanding of the differential regulation of pre- and postsynaptic 5-HT1A receptors by corticosterone flattened rhythm.

Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions.

RATIONALE: Identifying the etiological factors in anxiety and depression is critical to develop more efficacious therapies. The inhibitory serotonin(1A) receptors (5-HT(1A)R) located on 5-HT neurons (autoreceptors) limit antidepressant responses and their expression may be increased in treatment-resistant depressed patients. OBJECTIVES: Recently, we reported that intranasal administration of modified small interference RNA (siRNA) molecules targeting 5-HT(1A)R in serotonergic neurons evoked antidepressant-like effects. Here we extended this finding using marketed siRNAs against 5-HT(1A)R (1A-siRNA) to reduce directly the 5-HT(1A) autoreceptor expression and evaluate its biological consequences under basal conditions and in response to stressful situations. METHODS: Adult mice were locally infused with vehicle, nonsense siRNA, and 1A-siRNA into dorsal raphe nucleus (DR). 5-HT(1A)R knockout mice (1A-KO) were also used. Histological approaches, in vivo microdialysis, and stress-related behaviors were performed to assess the effects of 5-HT(1A) autoreceptor knockdown. RESULTS: Intra-DR 1A-siRNA infusion selectively reduced 5-HT(1A)R mRNA and binding levels and canceled 8-OH-DPAT-induced hypothermia. Basal extracellular 5-HT in medial prefrontal cortex (mPFC) did not differ among treatments. However, 1A-siRNA-treated mice displayed less immobility in the tail suspension and forced swim tests, as did 1A-KO mice. This was accompanied by a greater increase in prefrontal 5-HT release during tail suspension test. Moreover, intra-DR 1A-siRNA infusion augmented the increase of extracellular 5-HT in mPFC evoked by fluoxetine, up to the level in 1A-KO mice. CONCLUSION: Together with our previous report, the present results indicate that acute suppression of 5-HT(1A) autoreceptor expression evokes robust antidepressant-like effects, likely mediated by an increased capacity of serotonergic neurons to release 5-HT in stressful conditions.

Compliance with guidelines in the treatment of asthma exacerbations in primary care.

OBJECTIVE: To describe the approach to asthma exacerbations in a primary care centre in comparison with current guidelines and to ascertain the resolution of the episodes. METHODS: A cross-sectional, descriptive study was performed in asthmatic patients over 14 years old who were consulting for asthma exacerbations in a primary care centre during a 6-month period. The treatment given and the resolution obtained were evaluated. RESULTS: One hundred and twenty-three asthma exacerbations were registered, corresponding to 96 patients. A total of 74% were mild exacerbations, 24.4% moderate and 1.6% severe. The severity of asthma correlated directly with the severity of exacerbations. The frequency of resolution was 98.4%, with an average duration of medical attention of 30 min (SD 16.5). According to guidelines, 60.2% of the mild exacerbations were well treated, as were 26.7% of the moderate exacerbations and none of the severe episodes. Peak expiratory flow was measured in 54.5% of patients. In 82 cases (66.6%) salbutamol was given with a large-volume spacer chamber. Treatment after discharge was correct in 27.3% of the mild and 23.3% of the moderate exacerbations. A total of 23 (23.9%) patients presented more than one exacerbation during the study period. CONCLUSIONS: Most exacerbations seen in primary care are mild. Administration of salbutamol was sufficient for the resolution of these exacerbations. However, treatment after discharge was not compliant with guidelines in most cases. The primary care team was able to resolve most of the asthma exacerbations.

Lipopolysaccharide administration in vivo induces differential expression of cAMP-specific phosphodiesterase 4B mRNA splice variants in the mouse brain.

Many inflammatory processes involve cAMP. Pharmacological manipulation of cAMP levels using specific phosphodiesterase (PDE) inhibitors provokes an antiinflammatory response. The aim of this study was to investigate changes in the pattern and levels of expression of mRNAs coding for the cAMP-specific PDE4 family and subfamilies in mouse brain during the immediate acute immune response provoked by an intraperitoneal injection of lipopolysaccharide (LPS). PDE4B, and furthermore the splice variants PDE4B2 and PDE4B3, were the only mRNAs that showed altered expression. Whereas PDE4B2 presented increased expression at both 3 and 8 hr postinjection, PDE4B3 mRNA showed decreased expression that reached a minimum 8 hr postinjection. PDE4B2 mRNA upregulation was observed mainly in endothelial and macrophage/neutrophil cell populations in the leptomeninges, and the downregulation of PDE4B3 was observed mainly in oligodendrocytes throughout the brain. Our results clearly illustrate the distinctive anatomical distribution and cellular localization of the PDE4Bs during neuroinflammation and emphasize the importance of PDE4B splice-variant-specific inhibitors as therapeutic tools.

Simultaneous projections from prefrontal cortex to dopaminergic and serotonergic nuclei.

Derangements of the prefrontal cortex (PFC) and of brainstem monoaminergic systems occur in depression and schizophrenia. Anatomical and functional evidence supports a PFC control of the brainstem monoaminergic systems. Similarly, the PFC contains a high density of monoamine receptors for which antipsychotic drugs exhibit high affinity. This raises the possibility that pathological or drug-induced changes in PFC may subsequently alter monoaminergic activity. Recent data indicate that a substantial proportion of PFC pyramidal neurons projecting to the ventral tegmental area (VTA) or the dorsal raphe nucleus (DR) express the 5-HT2A receptor mRNA, which suggests that atypical antipsychotic drugs affect serotonergic and dopaminergic function by targeting PFC 5-HT2A receptors. Using electrophysiological and tract-tracing techniques we examined whether PFC pyramidal neurons projecting to DR are segregated from those projecting to the VTA. Sequential electrical stimulation of these nuclei in anaesthetized rats evoked antidromic potentials from both areas in the same pyramidal neurons of the medial PFC (60%, n=30). A similar percentage of dual DR+VTA projection neurons (50%) was obtained using the reciprocal collision test (n=85). Similarly, tracer application (Fluoro-Gold in VTA and cholera toxin B in DR, or vice versa) retrogradely labelled pyramidal neurons in PFC projecting to VTA (81±18), to DR (52±9) and to both nuclei (31±4, n=5 rats). Overall, these results indicate that the PFC may simultaneously coordinate the activity of dopaminergic and serotonergic systems within a short temporal domain, supporting a concerted modulation of the ascending serotonergic and dopaminergic activity during antipsychotic drug treatment.

NMDA receptors in frontal cortex and hippocampus of alcohol consumers.

Specific binding of [³H]MK801 to N-methyl-D-aspartate (NMDA) receptors in the frontal cortex and hippocampus (CA1 and gyrus dentatus) was measured by receptor autoradiography in 16 Caucasian chronic alcohol consumers free of clinical manifestations of alcoholism, and compared with 16 Caucasian control subjects. Binding densities were not significantly different between heavy and moderate drinkers, neither between alcohol consumers that were abstinent or non-abstinent before death, nor between ethanol drinkers and controls. Continued alcohol consumption, in the absence of hepatic, neurologic or psychiatric disorders related to alcoholism, does not alter the binding properties of NMDA receptors in the brain areas studied.

Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT(1A) receptors but not 5-HT(2A) receptors.

Atypical antipsychotic drugs (APDs) increase dopamine (DA) release in prefrontal cortex (PFC), an effect probably mediated by the direct or indirect activation of the 5-HT(1A) receptor (5-HT(1A)R). Given the very low in-vitro affinity of most APDs for 5-HT(1A)Rs and the large co-expression of 5-HT(1A)Rs and 5-HT(2A) receptors (5-HT(2A)Rs) in the PFC, this effect might result from the imbalance of 5-HT(1A)R and 5-HT(2A)R activation after blockade of these receptors by APDs, for which they show high affinity. Here we tested this hypothesis by examining the dependence of the APD-induced DA release in medial PFC (mPFC) on each receptor by using in-vivo microdialysis in wild-type (WT) and 5-HT(1A)R and 5-HT(2A)R knockout (KO) mice. Local APDs (clozapine, olanzapine, risperidone) administered by reverse dialysis induced a dose-dependent increase in mPFC DA output equally in WT and 5-HT(2A)R KO mice whereas the DA increase was absent in 5-HT(1A)R KO mice. To examine the relative contribution of both receptors to the clozapine-induced DA release in rat mPFC, we silenced G-protein-coupled receptors (GPCRs) in vivo with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) while 5-HT(1A)Rs or 5-HT(2A)/2CRs in the mPFC were selectively protected with the respective antagonists WAY-100635 or ritanserin. The inactivation of GPCRs while preserving ∼70% of 5-HT(2A)/(2C)Rs prevented the clozapine-induced DA rise in mPFC. In contrast, clozapine increased DA in mPFC of EEDQ-treated rats whose 5-HT(1A)Rs were protected (∼50% of control rats). These results indicate that (1) 5-HT(1A)Rs are necessary for the APDs-induced elevation in cortical DA transmission, and (2) this effect does not require 5-HT(2A)R blockade by APDs.

Pyramidal neurons in rat prefrontal cortex projecting to ventral tegmental area and dorsal raphe nucleus express 5-HT2A receptors.

The prefrontal cortex (PFC) is involved in higher brain functions altered in schizophrenia. Classical antipsychotics modulate cortico-limbic circuits mainly through subcortical D2 receptor blockade, whereas second generation (atypical) antipsychotics preferentially target cortical 5-HT receptors. Anatomical and functional evidence supports a PFC-based control of the brainstem monoaminergic nuclei. Using a combination of retrograde tracing experiments and in situ hybridization we report that a substantial proportion of PFC pyramidal neurons projecting to the dorsal raphe (DR) and/or ventral tegmental area (VTA) express 5-HT(2A) receptors. Cholera-toxin B application into the DR and the VTA retrogradely labeled projection neurons in the medial PFC (mPFC) and in orbitofrontal cortex (OFC). In situ hybridization of 5-HT(2A) receptor mRNA in the same tissue sections labeled a large neuronal population in mPFC and OFC. The percentage of DR-projecting neurons expressing 5-HT(2A) receptor mRNA was approximately 60% in mPFC and approximately 75% in OFC (n = 3). Equivalent values for VTA-projecting neurons were approximately 55% in both mPFC and ventral OFC. Thus, 5-HT(2A) receptor activation/blockade in PFC may have downstream effects on dopaminergic and serotonergic systems via direct descending pathways. Atypical antipsychotics may distally modulate monoaminergic cells through PFC 5-HT(2A) receptor blockade, presumably decreasing the activity of neurons receiving direct cortical inputs.

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain.

G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.

Neuroprotection induced by the adenosine A2A antagonist CSC in the 6-OHDA rat model of parkinsonism: effect on the activity of striatal output pathways.

In Parkinson's disease (PD), the striatal dopamine depletion and the following overactivation of the indirect pathway of the basal ganglia leads to very early disinhibition of the subthalamic nucleus (STN) that may contribute to the progression of PD by glutamatergic overstimulation of the dopaminergic neurons in the substantia nigra. Adenosine A2A antagonism has been demonstrated to attenuate the overactivity of the striatopallidal pathway. To investigate whether neuroprotection exerted by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC) correlates with a diminution of the striatopallidal pathway activity, we have examined the changes in the mRNA encoding for enkephalin, dynorphin, and adenosine A2A receptors by in situ hybridization induced by subacute systemic pretreatment with CSC in rats with striatal 6-hydroxydopamine(6-OHDA) administration. Animals received CSC for 7 days until 30 min before 6-OHDA intrastriatal administration. Vehicle-treated group received a solution of dimethyl sulfoxide. CSC pretreatment partially attenuated the decrease in nigral tyrosine hydroxylase immunoreactivity induced by 6-OHDA, whereas no modification of the increase in preproenkephalin mRNA expression in the dorsolateral striatum was observed. The neuroprotective effect of the adenosine A2A antagonist CSC in striatal 6-OHDA-lesioned rats does not result from a normalization of the increase in striatal PPE mRNA expression in the DL striatum, suggesting that other different mechanisms may be involved.

Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions.

Serotonin 5-HT4 receptors are widely distributed in the periphery and in brain, where they modulate the release of various neurotransmitters and have been implicated in learning and memory. Nine C-terminal splice variants of this receptor have been cloned in mammalian species. In the rat, three such variants have been described: 5-HT4(a), 5-HT4(b), and 5-HT4(e). In the present study, we have examined several aspects of the distribution of these receptors in brain. First, we provide, in rat and guinea pig, a detailed comparison of the distribution of 5-HT4 receptors labeled by the antagonist [125I]-SB 207710 with the distribution of their encoding mRNA visualized by in situ hybridization histochemistry (ISHH). The results suggest that, in several projection systems (striato-nigral and striato-pallidal pathways, projection from dentate granule cells to field CA3, habenulo-interpeduncular pathway), 5-HT4 receptors are located both somatodendritically and axonally. Second, we have analyzed the distribution of mRNA for the three known rat splice variants by reverse transcription-polymerase chain reaction (RT-PCR) and by ISHH. RT-PCR indicates that all three variants are widely distributed, with 5-HT4(b) mRNA being present in all regions examined (olfactory tubercle, striatum, hippocampus, inferior colliculus, substantia nigra, parietal cortex) and 5-HT4(a) and 5-HT4(e) showing a somewhat more restricted distribution. In other regions (periaqueductal gray, reticular formation, medial septum, diagonal band), faint ISHH signals are observed for 5-HT4(a)+4(e) mRNAs, whereas 5-HT4(b) mRNA signals are almost undetectable. Finally, neurotoxic lesions of basal ganglia components in guinea pig also indicate a location of these receptors on terminals of striatal projection neurons.

Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei.

We have used double in situ hybridization to examine the cellular localization of 5-HT2C receptor mRNA in relation to serotonergic and GABAergic neurons in the anterior raphe nuclei of the rat. In the dorsal and median raphe nuclei 5-HT2C receptor mRNA was not detected in serotonergic cells identified as those expressing serotonin (5-HT) transporter mRNA. In contrast, 5-HT2C receptor mRNA was found in most GABAergic cells, recognized by the presence of glutamic acid decarboxylase mRNA. Such 5-HT2C receptor-positive GABAergic neurons were mainly located in the intermediolateral and lateral portions of the dorsal raphe and lateral part of the median raphe. The present data give anatomical support to a previous hypothesis that proposed a negative-feedback loop involving reciprocal connections between GABAergic interneurons bearing 5-HT2A/2C receptors and 5-HT neurons in the dorsal raphe and surrounding areas. According to this model, the excitation of GABAergic interneurons through these 5-HT2C (and also 5-HT2A) receptors would result in the suppression of 5-HT cell firing.

An autoradiographic study of the influence of pindolol upon [35S]GTPgammaS binding in rat, guinea pig and human brain.

The 5-HT1A/beta-adrenoceptor ligand (+/-)pindolol has been used in clinical trials to enhance the antidepressant effect of selective serotonin (5-HT) reuptake inhibitors (SSRIs). The accelerating effect of (+/-)pindolol is thought to derive from its blockade of the SSRI-induced, 5-HT1A autoreceptor-mediated inhibition of serotonergic cell firing and 5-HT release. However, controversial results have been reported in regard to its ability to antagonize the effect of 5-HT at such receptors. In the present study, we have analysed the effect of (+/-)pindolol on receptor-mediated G-protein activation by measuring guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) binding onto tissue sections from the hippocampus and dorsal raphe nucleus from rat, guinea pig and human brain. In these regions, enriched in 5-HT1A receptors, (+/-)pindolol antagonized the stimulation of [35S]GTPgammaS binding induced by 5-HT in a concentration-dependent manner. We found that in both rat and human brain the calculated pEC50 values were higher in the dorsal raphe nucleus than in hippocampus. This suggests a higher potency of (+/-)pindolol at somatodendritic 5-HT1A receptors compared to post-synaptic 5-HT1A sites. In the absence of 5-HT, (+/-)pindolol (up to 10(-3) M) did not modify [35S]GTPgammaS binding, which remained at basal levels, indicating that, in this assay, (+/-)pindolol acts as a neutral antagonist rather than a partial agonist as it has been observed in other experimental models. The present data are relevant for the understanding of the neurobiological basis of pindolol acceleration of the action of SSRI antidepressants.

5-ht5B receptor mRNA in the raphe nuclei: coexpression with serotonin transporter.

We used double-label in situ hybridization to examine the cellular localization of 5-ht(5B) receptor mRNA in relation to serotonin transporter mRNA in the rat dorsal raphe (DR) and central superior nucleus (CS, median raphe nucleus). 5-ht(5B) receptor mRNA hybridization signal was often found on serotonin transporter mRNA-positive neuron profiles. The degree of cellular colocalization of these mRNAs notably varied among the different regions of the raphe nuclei. In the DR, cell bodies showing 5-ht(5B) receptor mRNA expression were abundant in the medial portions of the nucleus, all of them being also labeled for serotonin transporter mRNA. In contrast, in the ventrolateral regions (lateral wings) of the DR, we observed serotonin transporter mRNA-positive cells, but they were devoid of 5-ht(5B) receptor mRNA signal. In the CS, the level of coexpression of 5-ht(5B) receptor mRNA with serotonin transporter mRNA was high in the intermediate portions of the nucleus; however, we were unable to detect specific 5-ht(5B) receptor mRNA hybridization signal in its caudal extent. Our results support the presence of 5-ht(5B) receptor in serotonergic neurons in the DR and CS, suggesting an autoreceptor role for this receptor subtype.

GABAB receptor mRNA in the raphe nuclei: co-expression with serotonin transporter and glutamic acid decarboxylase.

We have used double-label in situ hybridization techniques to examine the cellular localization of GABAB receptor mRNA in relation to serotonin transporter mRNA and glutamic acid decarboxylase mRNA in the rat dorsal raphe, median raphe and raphe magnus nuclei. The degree of cellular co-localization of these markers notably varied among the different nuclei. In the dorsal raphe, cell bodies showing GABAB receptor mRNA were very abundant, the 85% being also labelled for serotonin transporter mRNA, and a low proportion (5%) showing glutamic acid decarboxylase mRNA. In the median raphe, the level of co-expression of GABAB receptor mRNA with serotonin transporter mRNA was significantly lower. Some cells were also identified that contained GABAB receptor mRNA in the absence of either one of the other mRNA species studied. Our results support the presence of GABAB receptors in serotonergic as well as GABAergic neurones in the dorsal and median raphe, providing the anatomical basis for the reported dual inhibitory/disinhibitory effect of the GABAB agonist baclofen on serotonergic function.

Flip and flop splice variants of AMPA receptor subunits in the spinal cord of amyotrophic lateral sclerosis.

Excitotoxicity mediated by AMPA receptors has been suggested to be implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). To investigate the relevance of AMPA receptors to motor neuron degeneration in ALS, we evaluated the expression of mRNAs coding for flip and flop splice variants of AMPA receptor subunits (GluR-A to GluR-D) in the cervical segment of the spinal cord from control individuals and patients with ALS using in situ hybridization histochemistry. Transcript mRNAs coding for flop variants were significantly decreased in the ventral horn of the spinal cord from patients with ALS, whereas the mRNAs for flip variants were preserved. These findings suggest that the relative abundance of flip variants vs. flop variants is increased in spinal motor neurons of ALS patients when compared to that of control individuals. Flip variants promote assemblies of slowly desensitizing AMPA receptors. These results imply that spinal motor neurons of ALS patients possess more slowly desensitizing AMPA receptors than those of control individuals. This expression change of AMPA receptors in ALS may account for vulnerability of motor neurons in this disease.