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.

Psychological impact of multigene cancer panel testing in patients with a clinical suspicion of hereditary cancer across Spain.

OBJECTIVE: Patients' psychological reactions to multigene cancer panel testing might differ compared with the single-gene testing reactions because of the complexity and uncertainty associated with the different possible results. Understanding patients' preferences and psychological impact of multigene panel testing is important to adapt the genetic counselling model. METHODS: One hundred eighty-seven unrelated patients with clinical suspicion of hereditary cancer undergoing a 25-gene panel test completed questionnaires after pretest genetic counselling and at 1 week, 3 months, and 12 months after results to elicit their preferences regarding results disclosure and to measure their cancer worry and testing-specific distress and uncertainty. RESULTS: A pathogenic variant was identified in 38 patients (34 high penetrance and 4 moderate penetrance variants), and 54 patients had at least one variant of uncertain significance. Overall, cancer panel testing was not associated with an increase in cancer worry after results disclosure (P value = .87). Twelve months after results, carriers of a moderate penetrance variant had higher distress and uncertainty scores compared with carriers of high penetrance variants. Cancer worry prior to genetic testing predicted genetic testing specific distress after results, especially at long term (P value <.001). Most of the patients reported the wish to know all genetic results. CONCLUSIONS: Our results suggest that patients can psychologically cope with cancer panel testing, but distress and uncertainty observed in carriers of moderate penetrance cancer variants in this cohort warrant further research.

Weekday and weekend food advertising varies on children's television in the USA but persuasive techniques and unhealthy items still dominate.

OBJECTIVE: In 2006, food industry self-regulatory efforts aimed to balance the mix of food advertisements to limit children's exposure to unhealthy food products. An update to these efforts proposed to eliminate all unhealthy advertisements during peak child viewing times and implement uniform nutrition criteria by December, 2013. Marketing techniques are not currently addressed in self-regulatory efforts. The food industry's pledge prompted researchers to conduct a content analysis to assess nutritional quality and presence of persuasive marketing techniques in child-directed food and beverage advertisements. STUDY DESIGN: Content analysis. METHODS: 32 h of children's television programming were recorded in February, 2013. Three independent coders assessed the nutritional content of food and beverage advertisements using the UK Nutrition Profiling System and assessed presence of persuasive techniques (PTs) using a rating form developed for this study. RESULTS: Overall, 13.75% of advertisements promoted a food or beverage product. Most food advertisements, 54.6%, represented unhealthy products and 95.48% of food advertisements contained at least one PT. The number of PTs was not significantly different for healthy (M = 4.98, SD = 2.07) and unhealthy food advertisements (M = 4.66, SD = 1.82) however food advertisements aimed at children used significantly more PTs (M = 5.5, SD = 1.43) than those targeting adults (M = 1.52, SD = 1.54), t (153) = 11.738, P < 0.0001. Saturday morning children's programming showed significantly fewer food advertisements compared to weekday morning children's programming. CONCLUSIONS: While a majority of food-related advertisements represented unhealthy items, advertisements airing during Saturday morning programming featured fewer food advertisements overall and were more frequently for healthier items compared to weekdays. Industry self-regulation may not be effective for reducing overall unhealthy ad exposure but may play a role in reduced exposure on weekends. Despite policy efforts, additional changes are needed to improve ad exposure experienced by children with a focus on addressing the persistent use of persuasive marketing techniques in food advertising intended for children.

Multiple conformations of 5-HT2A and 5-HT 2C receptors in rat brain: an autoradiographic study with [125I](±)DOI.

Earlier autoradiographic studies with the 5-HT2 receptor agonist [(125)I](±)DOI in human brain showed unexpected biphasic competition curves for various 5-HT2A antagonists. We have performed similar studies in rat brain regions with selective 5-HT2A (M100907) and 5-HT2C (SB242084) antagonists together with ketanserin and mesulergine. The effect of GTP analogues on antagonist competition was also studied. Increasing concentrations of Gpp(NH)p or GTPγS resulted in a maximal inhibition of [(125)I](±)DOI-specific binding of approximately 50 %. M100907 competed biphasically in all regions. In the presence of 100 µM Gpp(NH)p, M100907 still displaced biphasically the remaining [(125)I](±)DOI binding. Ketanserin showed biphasic curves in some regions and monophasic curves in others. In the latter, Gpp(NH)p evidenced an additional high-affinity site. SB242084 competed biphasically in brainstem nuclei and monophasically in the other regions. In most areas, SB242084 affinities were not notably altered by Gpp(NH)p. Mesulergine competed monophasically in all regions without alteration by Gpp(NH)p. These results conform with the extended ternary complex model of receptor action: receptor exists as an equilibrium of multiple conformations, i.e. ground (R), partly activated (R*) and activated G-protein-coupled (R*G) conformation/s. Thus, [(125)I](±)DOI would label multiple conformations of both 5-HT2A and 5-HT2C receptors in rat brain, and M100907 and ketanserin would recognise these conformations with different affinities.

Discrimination of motor and sensorimotor effects of phencyclidine and MK-801: Involvement of GluN2C-containing NMDA receptors in psychosis-like models.

Non-competitive NMDA receptor (NMDA-R) antagonists like ketamine, phencyclidine (PCP) and MK-801 are routinely used as pharmacological models of schizophrenia. However, the NMDA-R subtypes, neuronal types (e.g., GABA vs. glutamatergic neurons) and brain regions involved in psychotomimetic actions are not fully understood. PCP activates thalamo-cortical circuits after NMDA-R blockade in reticular thalamic GABAergic neurons. GluN2C subunits are densely expressed in thalamus and cerebellum. Therefore, we examined their involvement in the behavioral and functional effects elicited by PCP and MK-801 using GluN2C knockout (GluN2CKO) and wild-type mice, under the working hypothesis that psychotomimetic effects should be attenuated in mutant mice. PCP and MK-801 induced a disorganized and meandered hyperlocomotion in both genotypes. Interestingly, stereotyped behaviors like circling/rotation, rearings and ataxia signs were dramatically reduced in GluN2CKO mice, indicating a better motor coordination in absence of GluN2C subunits. In contrast, other motor or sensorimotor (pre-pulse inhibition of the startle response) aspects of the behavioral syndrome remained unaltered by GluN2C deletion. PCP and MK-801 evoked a general pattern of c-fos activation in mouse brain (including thalamo-cortical networks) but not in the cerebellum, where they markedly reduced c-fos expression, with significant genotype differences paralleling those in motor coordination. Finally, resting-state fMRI showed an enhanced cortico-thalamic-cerebellar connectivity in GluN2CKO mice, less affected by MK-801 than controls. Hence, the GluN2C subunit allows the dissection of the behavioral alterations induced by PCP and MK-801, showing that some motor effects (in particular, motor incoordination), but not deficits in sensorimotor gating, likely depend on GluN2C-containing NMDA-R blockade in cerebellar circuits.

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.

Morphine desensitization, internalization, and down-regulation of the mu opioid receptor is facilitated by serotonin 5-hydroxytryptamine2A receptor coactivation.

Analysis of the distribution of mRNA encoding the serotonin (5-hydroxytryptamine) 5-HT(2A) receptor and the mu opioid peptide receptor in rat brain demonstrated their coexpression in neurons in several distinct regions. These regions included the periaqueductal gray, an area that plays an important role in morphine-induced analgesia but also in the development of tolerance to morphine. To explore potential cross-regulation between these G protein-coupled receptors, the human mu opioid peptide receptor was expressed stably and constitutively in Flp-In T-REx human embryonic kidney 293 cells that harbored the human 5-HT(2A) receptor at the inducible Flp-In locus. In the absence of the 5-HT(2A) receptor, pretreatment with the enkephalin agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin but not with the alkaloid agonist morphine produced desensitization, internalization, and down-regulation of the mu opioid peptide receptor. Induction of 5-HT(2A) receptor expression in these cells resulted in up-regulation of mu opioid peptide receptor levels that was blocked by both a 5-HT(2A) receptor inverse agonist and selective inhibition of signaling via Galpha(q)/Galpha(11) G proteins. After induction of the 5-HT(2A) receptor, coaddition of 5-HT with morphine now also resulted in desensitization, receptor internalization, and down-regulation of the mu opioid peptide receptor. It has been argued that enhancement of mu opioid peptide receptor internalization in response to morphine would limit the development of tolerance without limiting analgesia. These data suggest that selective activation of the 5-HT(2A) receptor in concert with treatment with morphine might achieve this aim.

[Systemic AA amyloidosis induced by benign neoplasms]. / Amiloidosis secundaria (AA) asociada a tumoraciones benignas.

Amyloidosis is a systemic disorder characterized by the extracellular tissue deposition of insoluble, toxic aggregates in bundles of beta-sheet fibrillar proteins. These deposits are typically identified on the bases of their apple-green birrefringence under a polarized light microscope after staining with Congo red, and by the presence of rigid, nonbranching fibrils 8 to 10 nm in diameter on electron microscopy. The type of amyloid fibril unit can be further defined by immunohistology or by immunoelectron microscopy. It has been described at least 25 different human protein precursors of amyloid fibrils, which will describe its corresponding amyloid disease. The most common types of amyloidosis are AL (primary) and AA (secondary) types; the former, is the most frequent and is due to deposition of proteins derived from immunoglobulin light chain fragments, occurring alone or in association with multiple myeloma. The later (AA), is caused by deposition of fibrils composed of fragments of the acute phase reactant serum amyloid A (SAA) and complicates chronic diseases with ongoing or recurring inflammation, namely; rheumatoid arthritis (RA), juvenile chronic polyarthritis, ankylosing spondylitis, familial periodic fever syndromes (Familial Mediterranean Fever), chronic infections and furthermore, some neoplasms (mainly renal cell carcinoma and Hodgkin's disease). Despite its less frequent association, some benign neoplasms can subsequently complicate to AA amyloidosis, therefore, an early diagnose and successful treatment may lead indeed, to regression of the amyloid disease. Herein, we present two cases of AA amyloidosis, both of them caused by 2 different benign neoplasms: 1. A 34 year-old woman, after chronic oral contraceptive use, developed an hepatic adenoma (fig. 1) which finally lead to AA amyloidosis with primary kidney presentation (pure nephrotic syndrome) (table 1). Post-surgical complications yield to acute renal failure from which unfortunately could not be recovered. After being on hemodialysis therapy during 10 months she received a first renal allograft without any complication. 2. A 20 year old woman, was diagnosed of AA amyloidosis after a renal biopsy (fig. 2) because of nephrotic syndrome (table 1). Further investigation lead to the finding of a hialyne-vascular type Castleman's disease located in the retroperitoneum (fig. 2). Despite surgical resection and medical treatment (colchicine) she developed progressive renal failure requiring initialization of hemodialysis therapy. After 6 years being on hemodialysis, she received a first renal allograft which is currently functioning after one year of follow- up. Although other chronic inflammatory diseases complicate more frequently to AA amyloidosis, benign tumors have to be taken into account as a potential ethiological cause for secondary amyloidosis.

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.

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.

Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

Mapping of 5-HT2A receptors and their mRNA in monkey brain: [3H]MDL100,907 autoradiography and in situ hybridization studies.

The anatomic distribution of serotonin 5-HT2A receptors visualized with [3H]MDL100,907 and of their mRNA detected by in situ hybridization were studied in monkey brain. Both autoradiographic patterns of signal showed heterogeneous distributions and were in general in good agreement in the majority of brain regions. In most neocortical areas, [3H]MDL100,907 presented a four-banded pattern with layers I and III-IV more intensely labeled and layers II and V-VI showing weaker labeling. 5-HT2A receptor mRNA was detected in layers III and IV, and in some cases also in layers II and V. In intra- and extra-calcarine areas of striate cortex a five-banded pattern was distinguished, with layers III and IVc-V showing the highest densities of [3H]MDL100,907 labeling. These two areas showed the highest neocortical hybridization signal. An unexpected finding was the presence of low densities of [3H]MDL100,907 labeling and 5-HT2A receptor mRNA in choroid plexus. Comparison of the distribution of [3H]MDL100,907 and [3H]ketanserin binding sites in monkey brain regions with high nonspecific [3H]ketanserin binding (caudate, putamen, substantia nigra, inferior olive) revealed specific binding of [3H]MDL100,907 with very low nonspecific binding. Some differences were noted between the distribution of [3H]MDL100,907-labeled 5-HT2A receptors in monkey brain and the previously reported distribution of these receptors in human brain: absence of striosome labeling in monkey striatum and different patterns of neocortical labeling. The present results provide the first detailed comparison of 5-HT2A receptor and mRNA distribution in primate brain. The observed species differences in 5-HT2A receptor distribution should be considered when extrapolating results among different species.

Desensitization of 5-HT(1A) autoreceptors by a low chronic fluoxetine dose effect of the concurrent administration of WAY-100635.

Using microdialysis, receptor autoradiography and in situ hybridization, we examined the effects of fluoxetine alone or with WAY-100635 on: (a) extracellular 5-HT in frontal cortex; and (b) density and sensitivity of 5-HT(1A) autoreceptors in rat brain. WAY-100635 (0.3 mg/kg, s.c.) doubled the increase in extracellular 5-HT produced by fluoxetine (3 mg/kg, i.p.) in frontal cortex. Two-week minipump treatments with these daily doses significantly raised extracellular 5-HT to 275 +/- 33% (fluoxetine) and 245 +/- 10% (fluoxetine plus WAY-100635) of controls. Fluoxetine 3 mg/kg.day desensitized dorsal raphe 5-HT(1A) autoreceptors, an effect prevented by the concurrent WAY-100635 administration. However, WAY-100635 (alone or with fluoxetine) did not change 5-HT(1A) autoreceptor sensitivity. The density of 5-HT(1A) receptors and its encoding mRNA, was unaffected by these treatments. These results suggest that prolonged blockade of 5-HT(1A) receptors in vivo prevents the autoreceptor desensitization induced by fluoxetine but does not result in receptor sensitization.

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.

[3H]MDL 100,907 labels 5-HT2A serotonin receptors selectively in primate brain.

The selective antagonist for the 5-HT2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [3H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (Kd = 0.14-0.19 nM in human brain; Kd= 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL 100,907-labeled receptors: MDL 100,907 > spiperone > ketanserin > mesulergine). The autoradiographical signal obtained with [3H]MDL 100,907 was compared to the signal obtained with [3H]ketanserin, [3H]RP62203 and [3H]mesulergine in both species, and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [3H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [3H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [3H]mesulergine confirmed the lack of labeling of the 5-HT2A receptor by this radioligand in primate brain. The similarity of the distribution of [3H]MDL 100,907-labeled receptors and 5-HT2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [3H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT2A receptors in mammalian brain including post-mortem human brain.

Muscarinic cholinergic receptors in the rat caudate-putamen and olfactory tubercle belong predominantly to the m4 class: in situ hybridization and receptor autoradiography evidence.

In order to establish the nature of the muscarinic cholinergic receptors present in the rat caudate-putamen and olfactory tubercle, we have combined in situ hybridization histochemistry with oligonucleotide probes and receptor autoradiography with N-[3H]methyl scopolamine and several subtype-selective antagonists: hexahydro-sila-difenidol, p-fluoro-hexahydro-sila-difenidol, 4-diphenyl-acetoxy-N-methylpiperidine methbromide, AF-DX 116, pirenzepine and methoctramine. In both brain regions, transcripts for the m4 muscarinic receptor subtype were the most abundant, followed by transcripts for the m1 subtype. m2 and m3 transcripts were much less abundant, whereas m5 mRNA was not detected under the present conditions. The binding profiles obtained in these areas were clearly distinct from those obtained in the CA1 layer of the hippocampus and in the pontine nuclei, regions enriched in M1 and M2 sites, respectively. In contrast, they were good agreement with the characteristics of atypical muscarinic receptors present in cell lines such as NG108-15, which contains mRNA for the m4 subtype, and PC12. The profiles displayed by some of the compounds used in the present study for cloned m4 receptors expressed in mammalian cells also agree with our results in rat caudate-putamen and olfactory tubercule. Taken together, these facts support the existence, in rat caudate-putamen and olfactory tubercle, of a major population of muscarinic cholinergic receptors belonging to the M4 type.

Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells in the rat brain: a correlative study using in situ hybridization histochemistry and receptor autoradiography.

The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of muscarinic cholinergic receptors in the rat brain. In situ hybridization histochemistry was used, with oligonucleotides as hybridization probes. The distribution of cholinergic cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of muscarinic cholinergic binding sites was examined with a non-selective ligand ([3H]N-methylscopolamine) and with ligands proposed to be M1-selective ([3H]pirenzepine) or M2-selective ([3H]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholinergic) cell groups (medial septum-diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habenula and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M2 preferential agonist [3H]oxotremorine-M, but not with [3H]pirenzepine binding sites. Regions where the presence of [3H]oxotremorine-M binding sites was not correlated with that of m2 mRNA are the caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. The present results strongly suggest that the M2 receptor is expressed by a majority of cholinergic cells, where it probably plays a role as autoreceptor. However, many non-cholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M2-selective ligand [3H]oxotremorine-M indicates that this ligand, in addition to M2 receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M4.

Muscarinic receptor subtypes expression in rat and chick dorsal root ganglia.

In the present work we have analyzed by Northern blot, RT-PCR and in situ hybridization the expression of muscarinic receptor subtype mRNAs in rat and chick dorsal root ganglia. Northern blot analysis performed on rat total RNA revealed a strong signal for M(2) while a faint band was observed for M(3) and M(4) subtypes; no signal was evident for M(1) and M(5), while in chick total RNA no signal was detected for any of the analyzed subtypes (M(2), M(3), M(4)). On the other hand, RT-PCR revealed that all muscarinic subtype mRNAs were present both in rat and chick DRG, although the level of their expression may be different. In chick DRG, the presence of various muscarinic subtypes was confirmed by competition binding experiments. In situ hybridization in rat DRG showed that M(3) and M(4) transcripts, similarly to what has been previously described for M(2) mRNA, were preferentially localized in medium-small neurons. Large neurons were usually negative or faintly labelled. No hybridization signal was detected in rat DRG with probes for M(1) and M(5) muscarinic subtypes. The presence of various muscarinic receptors in DRG and their preferential expression in the medium-small sensory neurons suggest their possible involvement in the modulation of nociceptive stimuli transduction.

Multiplicity of muscarinic autoreceptor subtypes? Comparison of the distribution of cholinergic cells and cells containing mRNA for five subtypes of muscarinic receptors in the rat brain.

In situ hybridization was used to compare the microscopic distribution in the rat brain of cells containing mRNA for choline acetyltransferase (ChAT) (i.e. cholinergic cells) with that of cells containing mRNA for the five subtypes of muscarinic receptors, in an attempt to establish the potential role as autoreceptors (i.e. muscarinic cholinoceptors present in cholinergic cells) of the different muscarinic receptor subtypes. [32P]alpha-dATP-labelled synthetic oligonucleotides were used as hybridization probes in serial sections. Transcripts for all five subtypes of muscarinic receptors were detected in cells co-distributing with ChAT mRNA-containing cells in one or more regions of the brain. Cells containing m2, m3, m4 or m5 mRNAs were observed in the regions of the basal forebrain where cholinergic cells are located (medial septum/diagonal band nuclei, ventral pallidum, basal nucleus of Meynert). m2, m3 and m5 mRNAs were abundant in the parabigeminal nucleus. m2, m3 and m4 transcripts were detected in the pedunculopontine and laterodorsal tegmental nuclei. m1, m2 and m3 mRNAs were present in several cranial nerve nuclei. The present results suggest that muscarinic autoreceptors belonging to the five subtypes cloned to date may exist.

Distribution of AMPA receptor subunit mRNAs in the human basal ganglia: an in situ hybridization study.

The distribution of AMPA receptor subunit mRNAs (spliced flip and flop variants of GluR-A to GluR-D) in the human post-mortem striatum, nucleus accumbens, globus pallidus and basal nucleus of Meynert was determined by in situ hybridization histochemistry. In the striatum and nucleus accumbens, for each subunit, the mRNA for the flop variant was more enriched than that for the corresponding flip variant. The GluR-C(flop) mRNA was most abundant, followed by the GluR-A(flop) mRNA. Transcripts for flop forms were evenly distributed in these regions, whereas those for flip forms showed a dorsoventral increasing gradient of the hybridization signals. The signals in these areas were found to originate mainly from medium-sized neurons. In the globus pallidus, mRNAs encoding GluR-A(flop) and GluR-C(flop) were also abundantly expressed. The basal nucleus of Meynert was enriched for mRNAs of flop forms. In conclusion, AMPA receptors in these areas of the human basal ganglia appeared to be mainly composed of flop variants, especially GluR-A(flop) and GluR-C(flop). However, the finding that flip transcripts were more abundant in the nucleus accumbens than in the striatum implies differences in functions of AMPA receptors between the two regions.