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.

Possible Correlation between Cholinergic System Alterations and Neuro/Inflammation in Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune and demyelinating disease of the central nervous system. Although the etiology of MS is still unknown, both genetic and environmental factors contribute to the pathogenesis of the disease. Acetylcholine participates in the modulation of central and peripheral inflammation. The cells of the immune system, as well as microglia, astrocytes and oligodendrocytes express cholinergic markers and receptors of muscarinic and nicotinic type. The role played by acetylcholine in MS has been recently investigated. In the present review, we summarize the evidence indicating the cholinergic dysfunction in serum and cerebrospinal fluid of relapsing-remitting (RR)-MS patients and in the brains of the MS animal model experimental autoimmune encephalomyelitis (EAE). The correlation between the increased activity of the cholinergic hydrolyzing enzymes acetylcholinesterase and butyrylcholinesterase, the reduced levels of acetylcholine and the increase of pro-inflammatory cytokines production were recently described in immune cells of MS patients. Moreover, the genetic polymorphisms for both hydrolyzing enzymes and the possible correlation with the altered levels of their enzymatic activity have been also reported. Finally, the changes in cholinergic markers expression in the central nervous system of EAE mice in peak and chronic phases suggest the involvement of the acetylcholine also in neuro-inflammatory processes.

Comparative study of the expression of cholinergic system components in the CNS of experimental autoimmune encephalomyelitis mice: Acute vs remitting phase.

Acetylcholine (ACh) is involved in the modulation of the inflammatory response. ACh levels are regulated by its synthesizing enzyme, choline acetyltransferase (ChAT), and by its hydrolyzing enzymes, mainly acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A more comprehensive understanding of the cholinergic system in experimental autoimmune encephalomyelitis (EAE) disease progression could pave the path for the development of therapies to ameliorate multiple sclerosis (MS). In this work, we analyzed possible alterations of the CNS cholinergic system in the neuroinflammation process by using a MOG-induced EAE mice model. MOG- and vehicle-treated animals were studied at acute and remitting phases. We examined neuropathology and analyzed mRNA expression of ChAT, AChE and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR), as well as AChE and BuChE enzyme activities, in brain and spinal cord sections during disease progression. The mRNA expression and enzyme activities of these cholinergic markers were up- or down-regulated in many cholinergic areas and other brain areas of EAE mice in the acute and remitting phases of the disease. BuChE was present in a higher proportion of astroglia and microglia/macrophage cells in the EAE remitting group. The observed changes in cholinergic markers expression and cellular localization in the CNS during EAE disease progression suggests their potential involvement in the development of the neuroinflammatory process and may lay the ground to consider cholinergic system components as putative anti-inflammatory therapeutic targets for MS.

Galanin (1-15) enhancement of the behavioral effects of Fluoxetine in the forced swimming test gives a new therapeutic strategy against depression.

The pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5-HT) activity. Specifically, selective 5-HT reuptake inhibitors, including Fluoxetine (FLX), are the most commonly used for treatment of major depression. However, the understanding of the mechanism of action of FLX beyond its effect of elevating 5-HT is limited. The interaction between serotoninergic system and neuropeptides signaling could be a key aspect. We examined the ability of the neuropeptide Galanin(1-15) [GAL(1-15)] to modulate the behavioral effects of FLX in the forced swimming test (FST) and studied feasible molecular mechanisms. The data show that GAL(1-15) enhances the antidepressant-like effects induced by FLX in the FST, and we demonstrate the involvement of GALR1/GALR2 heteroreceptor complex in the GAL(1-15)-mediated effect using in vivo rat models for siRNA GALR1 or GALR2 knockdown. Importantly, 5-HT1A receptors (5HT1A-R) also participate in the GAL(1-15)/FLX interactions since the 5HT1AR antagonist WAY100635 blocked the behavioral effects in the FST induced by the coadministration of GAL(1-15) and FLX. The mechanism underlying GAL(1-15)/FLX interactions affected the binding characteristics as well as the mRNA levels of 5-HT1A-R specifically in the dorsal hippocampus while leaving unaffected mRNA levels and affinity and binding sites of this receptor in the dorsal raphe. The results open up the possibility to use GAL(1-15) as for a combination therapy with FLX as a novel strategy for treatment of depression.

Cholinergic System and Neuroinflammation: Implication in Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS) characterized by leucocytes infiltration, demyelination, axonal degeneration and neuronal death. Although the etiology of MS is still unkwon, inflammation and autoimmunity are considered to be key players of the disease. Nervous System: The severe alterations affecting the nervous system contribute to the motor and cognitive disabilities and are in large part dependent on severe inflammatory processes active in both central nervous system and immune system. Acetylcholine (ACh) appears to be involved in the modulation of central and peripheral inflammation. Immune cells as well as astrocytes and microglia respond to ACh stimuli by activation of cholinergic receptors. Muscarinic and nicotinic receptors differently contribute to the modulation of immunological and inflammatory processes stimulating pro- and anti-inflammatory cytokines respectively. The role played by ACh in MS is not yet fully understood, although some results point to its involvement in different neurological disorders such as Alzheimer's disease and schizophrenia. CONCLUSION: In the present review we summarize the evidence indicating the correlation between nervous system dysfunction in MS, with inflammation and cholinergic system alterations. Experiments performed in MS animal models and analyses on biological fluids from MS patients such as blood, serum and cerebrospinal fluid suggest that cholinergic alterations may contribute to the dysregulated inflammatory processes of MS. Many current therapeutic approaches in MS are based on anti-inflammatory drugs. We also discuss how the use of cholinesterase inhibitors or ACh mimetics may represent a new interesting therapeutic approach in MS.

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.

Mct8 and trh co-expression throughout the hypothalamic paraventricular nucleus is modified by dehydration-induced anorexia in rats.

Thyrotropin-releasing hormone (TRH) is a neuropeptide with endocrine and neuromodulatory effects. TRH from the paraventricular hypothalamic nucleus (PVN) participates in the control of energy homeostasis; as a neuromodulator TRH has anorexigenic effects. Negative energy balance decreases PVN TRH expression and TSH concentration; in contrast, a particular model of anorexia (dehydration) induces in rats a paradoxical increase in TRH expression in hypophysiotropic cells from caudal PVN and high TSH serum levels, despite their apparent hypothalamic hyperthyroidism and low body weight. We compared here the mRNA co-expression pattern of one of the brain thyroid hormones' transporters, the monocarboxylate transporter-8 (MCT8) with that of TRH in PVN subdivisions of dehydration-induced anorexic (DIA) and control rats. Our aim was to identify whether a low MCT8 expression in anorexic rats could contribute to their high TRH mRNA content.We registered daily food intake and body weight of 7-day DIA and control rats and analyzed TRH and MCT8 mRNA co-expression throughout the PVN by double in situ hybridization assays. We found that DIA rats showed increased number of TRHergic cells in caudal PVN, as well as a decreased percentage of TRH-expressing neurons that co-expressed MCT8 mRNA signal. Results suggest that the reduced proportion of double TRH/MCT8 expressing cells may be limiting the entry of hypothalamic triiodothyronine to the greater number of TRH-expressing neurons from caudal PVN and be in part responsible for the high TRH expression in anorexia rats and for the lack of adaptation of their hypothalamic-pituitary-thyroid axis to their low food intake.

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.

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.

Critical role for PDE4 subfamilies in the development of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis exhibiting neuroinflammation, axonal damage and demyelination, further characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein. Pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses through modulation of cAMP levels. The PDE4B subfamily has been related to the inflammatory immune response in mice and PDE4 inhibition produces amelioration of the clinical signs and delayed onset in the EAE model. Analyses of the expression of the mRNA coding for PDE4B splice variants revealed an upregulation of PDE4B2 in the brainstem and spinal cord of EAE mice which correlated with forkhead box P3 (FoxP3) and transforming growth factor beta (TGF-ß) mRNAs expression in a score-dependent manner. The increase observed for the PDE4B protein was mainly found in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages, in areas with high cellular infiltration. Unexpectedly, PDE4B(-/-) mice showed an earlier onset of the disease compared to wildtype mice. The results point to a possible role of the PDE4B enzyme and in particular the PDE4B2 splice variant during EAE pathogenesis, probably by modulating cAMP levels in APCs, consequently influencing the cytokine environment important for T-cell differentiation.

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.

Expression of α(1)-adrenergic receptors in rat prefrontal cortex: cellular co-localization with 5-HT(2A) receptors.

The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellular expression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dorsoventral gradient, with a lower percentage of α(1)AR-positive neurons in infralimbic cortex compared to anterior cingulate and prelimbic cortex. The expression of α(1A), α(1B) and α(1D) adrenergic receptors was segregated in different layers and subdivisions. In all them there is a high co-expression with 5-HT(2A)Rs (∼80%). These observations indicate that NE controls the activity of most PFC pyramidal neurons via α(1)ARs, either directly or indirectly, via GABAergic interneurons. Antipsychotic drugs can thus modulate the activity of PFC via α(1)AR blockade. The high co-expression with 5-HT(2A)Rs indicates a convergence of excitatory serotonergic and noradrenergic inputs onto the same neuronal populations. Moreover, atypical antipsychotics may exert a more powerful control of PFC function through the simultaneous blockade of α(1)ARs and 5-HT(2A)Rs.

Identification in silico and experimental validation of novel phosphodiesterase 7 inhibitors with efficacy in experimental autoimmune encephalomyelitis mice.

A neural network model has been developed to predict the inhibitory capacity of any chemical structure to be a phosphodiesterase 7 (PDE7) inhibitor, a new promising kind of drugs for the treatment of neurological disorders. The numerical definition of the structures was achieved using CODES program. Through the validation of this neural network model, a novel family of 5-imino-1,2,4-thiadiazoles (ITDZs) has been identified as inhibitors of PDE7. Experimental extensive biological studies have demonstrated the ability of ITDZs to inhibit PDE7 and to increase intracellular levels of cAMP. Among them, the derivative 15 showed a high in vitro potency with desirable pharmacokinetic profile (safe genotoxicity and blood brain barrier penetration). Administration of ITDZ 15 in an experimental autoimmune encephalomyelitis (EAE) mouse model results in a significant attenuation of clinical symptoms, showing the potential of ITDZs, especially compound 15, for the effective treatment of multiple sclerosis.

Sex-related differences of cAMP-specific PDE4B3 mRNA in oligodendrocytes following systemic inflammation.

Sex-related differences have been observed in the incidence and severity of several neurological diseases and in sepsis in humans. Cyclic adenosine monophosphate (cAMP) has been shown to play an important role in modulating the inflammatory environment during neuroinflammation and importantly in protecting myelin from excitotoxic cell death. Considering the sexual dimorphism in the functional properties of oligodendrocytes and the importance of a systemic inflammation in the progression of multiple sclerosis, we focused on identifying possible sex-related differences in the alterations previously reported for the two phosphodiesterase4B (PDE4B) splice-variants (PDE4B2 and PDE4B3) mRNA expression during innate neuroinflammation. PDE4A, PDE4B, and PDE4D are present in oligodendrocytes and we have previously reported that PDE4B3 mRNA is readily expressed in both oligodendrocytes and neurons. In this study, we analyzed the influence of an intraperitoneal lipopolysaccharide injection on the distribution pattern and expression levels of the PDE4B mRNA splicing variants in both male and female mice brains. Clear differences were observed in PDE4B2 and PDE4B3 mRNA expression levels in males compared with females in a time-dependent manner. Furthermore, we observed that the clear downregulation of PDE4B3 mRNA was reflected in a lower percentage of oligodendrocytes positive for this transcript which correlated with a decrease in inducible cAMP early repressor expression in female corpus callosum.

Comparison of cAMP-specific phosphodiesterase mRNAs distribution in mouse and rat brain.

There are eleven families of phosphodiesterases that regulate cellular levels of cyclic nucleotides by degradation of cAMP or cGMP. Knowledge of the expression sites of different PDE genes in brain is of special importance for studies on development of specific inhibitors considering that, for example, PDE4 inhibitor treatments exhibit profound anti-inflammatory effects. To address possible species differences we examined the expression of mRNAs coding for the cAMP specific PDE4 and PDE7 families since inhibitors have been used in clinic for schizophrenia, mood disorders, cognition and inflammatory diseases treatment. We have compared the expression of these PDEs in mouse brain by in situ hybridization histochemistry in comparison with rat brain and found that their neuroanatomical distribution differs in a few areas.

Effect of phosphodiesterase 7 (PDE7) inhibitors in experimental autoimmune encephalomyelitis mice. Discovery of a new chemically diverse family of compounds.

Phosphodiesterase (PDE) 7 is involved in proinflammatory processes, being widely expressed both on lymphocytes and on certain brain regions. Specific inhibitors of PDE7 have been recently reported as potential new drugs for the treatment of neurological disorders because of their ability to increase intracellular levels of cAMP and thus to modulate the inflammatory process, as a neuroprotective well-established strategy. Multiple sclerosis is an unmet disease in which pathologies on the immune system, T-cells, and specific neural cells are involved simultaneously. Therefore, PDE7 inhibitors able to interfere with all these targets may represent an innovative therapy for this pathology. Here, we report a new chemically diverse family of heterocyclic PDE7 inhibitors, discovered and optimized by using molecular modeling studies, able to increase cAMP levels in cells, decrease inflammatory activation on primary neural cultures, and also attenuate the clinical symptoms in the experimental autoimmune encephalomyelitis (EAE) mouse model. These results led us to propose the use of PDE7 inhibitors as innovative therapeutic agents for the treatment of multiple sclerosis.

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.

Expression of parvalbumin and glutamic acid decarboxylase-67 after acute administration of MK-801. Implications for the NMDA hypofunction model of schizophrenia.

RATIONALE: A reduction of GABAergic markers in postmortem tissue is consistently found in schizophrenia. This is generally mediated by a decreased expression of the calcium-binding protein, parvalbumin (PV), and the 67-kDa isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD(67)). Similar reductions of PV or GAD(67) are observed after repeated exposure to N-methyl-D-aspartate (NMDA) receptor antagonists but less attention has been paid to what occurs after their acute administration. OBJECTIVES: Here, we have used in situ hybridization to examine the expression of PV and GAD(67) mRNAs at 4 h and 24 h after an acute administration of MK-801 (1 mg/kg). RESULTS: Four hours after MK-801, the expression of PV mRNA decreased only in dentate gyrus of the hippocampus. Twenty four hours after this treatment, a reduction of the levels of PV mRNA was found in the medial prefrontal, orbitofrontal and entorhinal cortices, hippocampus and the basolateral nucleus of the amygdala. In contrast, no changes in the expression of GAD(67) were observed in any of the brain regions examined. Interestingly, the reduction in PV mRNA expression is observed in discrete corticolimbic subregions that have been implicated in schizophrenia, which is coincident with changes observed in postmortem tissue of schizophrenia brain. CONCLUSIONS: These findings indicate that acute administration of a NMDA antagonist delineate a pattern of changes in GABAergic markers different from those observed in postmortem tissue in schizophrenia inasmuch as only deficits in parvalbumin (but not GAD(67)) were seen.

Activation of thalamocortical networks by the N-methyl-D-aspartate receptor antagonist phencyclidine: reversal by clozapine.

BACKGROUND: Noncompetitive N-methyl-D-aspartate receptor antagonists are widely used as pharmacological models of schizophrenia. Their neurobiological actions are still poorly understood, although the prefrontal cortex (PFC) appears as a key target area. METHODS: We examined the effect of phencyclidine (PCP) on neuronal activity of the mediodorsal (MD) and centromedial (CM) thalamic nuclei, reciprocally connected with the PFC, using extracellular recordings (n = 50 neurons from 35 Wistar rats) and c-fos expression. RESULTS: Phencyclidine (.25 mg/kg intravenous [IV]) markedly disorganized the activity of MD/CM neurons, increasing (424%) and decreasing (41%) the activity of 57% and 20% of the recorded neurons, respectively (23% remained unaffected). Phencyclidine reduced delta oscillations (.15-4 Hz) as assessed by recording local field potentials. The subsequent clozapine administration (1 mg/kg IV) reversed PCP effects on neuronal discharge and delta oscillations. Double in situ hybridization experiments revealed that PCP (10 mg/kg intraperitoneal [IP]) markedly increased c-fos expression in glutamatergic neurons of several cortical areas (prefrontal, somatosensory, retrosplenial, entorhinal) and in thalamic nuclei, including MD/CM. Phencyclidine also increased c-fos expression in the amygdala; yet, it had a small effect in the hippocampus. Phencyclidine did not increase c-fos expression in gamma-aminobutyric acidergic cells except in hippocampus, amygdala, somatosensory, and retrosplenial cortices. Clozapine (5 mg/kg IP) had no effect by itself but significantly prevented PCP-induced c-fos expression. CONCLUSIONS: Phencyclidine likely exerts its psychotomimetic action by increasing excitatory neurotransmission in thalamo-cortico-thalamic networks involving, among others, PFC, retrosplenial, and somatosensory cortices. The antipsychotic action of clozapine includes, among other actions, an attenuation of the neuronal hyperactivity in thalamocortical networks.

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.