Transcriptomic Analysis Reveals Priming of The Host Antiviral Interferon Signaling Pathway by Bronchobini® Resulting in Balanced Immune Response to Rhinovirus Infection in Mouse Lung Tissue Slices.

Rhinovirus (RV) is the predominant virus causing respiratory tract infections. Bronchobini® is a low dose multi component, multi target preparation used to treat inflammatory respiratory diseases such as the common cold, described to ease severity of symptoms such as cough and viscous mucus production. The aim of the study was to assess the efficacy of Bronchobini® in RV infection and to elucidate its mode of action. Therefore, Bronchobini®'s ingredients (BRO) were assessed in an ex vivo model of RV infection using mouse precision-cut lung slices, an organotypic tissue capable to reflect the host immune response to RV infection. Cytokine profiles were assessed using enzyme-linked immunosorbent assay (ELISA) and mesoscale discovery (MSD). Gene expression analysis was performed using Affymetrix microarrays and ingenuity pathway analysis. BRO treatment resulted in the significant suppression of RV-induced antiviral and pro-inflammatory cytokine release. Transcriptome analysis revealed a multifactorial mode of action of BRO, with a strong inhibition of the RV-induced pro-inflammatory and antiviral host response mediated by nuclear factor kappa B (NFkB) and interferon signaling pathways. Interestingly, this was due to priming of these pathways in the absence of virus. Overall, BRO exerted its beneficial anti-inflammatory effect by priming the antiviral host response resulting in a reduced inflammatory response to RV infection, thereby balancing an otherwise excessive inflammatory response.

Dopamine and opioid systems adaptation in alcoholism revisited: Convergent evidence from positron emission tomography and postmortem studies.

A major hypothesis in the addiction field suggests deficits in dopamine signaling during abstinence as a driving mechanism for the relapsing course of the disorder. Paradoxically, blockade of mu-opioid receptors (MORs) intended to suppress dopamine release and alcohol reward is a widely used treatment for preventing relapse in alcohol use disorder (AUD). To elucidate this apparent discrepancy, we systematically survey the literature on experimental studies in AUD subjects and animal models, which assessed striatal dopamine levels and D1, D2-like receptor, dopamine transporter and MOR via positron emission tomography (PET) and ex vivo receptor binding assays. The reported evidence indicates a changing dopaminergic signaling over time, which is associated with concomitant alterations in MOR, thus suggesting a highly dynamic regulation of the reward system during abstinence. Such a view can reconcile the various evidences from in vivo and postmortem studies, but makes developing an effective pharmacological intervention that specifically targets either dopamine receptors or the transporter system a daunting task.

Dissociable Role of Corticotropin Releasing Hormone Receptor Subtype 1 on Dopaminergic and D1 Dopaminoceptive Neurons in Cocaine Seeking Behavior.

The ability of many drugs of abuse, including cocaine, to mediate reinforcement and drug-seeking behaviors is in part mediated by the corticotropin-releasing hormone (CRH) system, in which CRH exerts its effects partly via the CRH receptor subtype 1 (CRHR1) in extra-hypothalamic areas. In fact, CRHR1 expressed in regions of the mesolimbic dopamine (DA) system have been demonstrated to modify cocaine-induced DA release and alter cocaine-mediated behaviors. Here we examined the role of neuronal selectivity of CRHR1 within the mesolimbic system on cocaine-induced behaviors. First we used a transgenic mouse line expressing GFP under the control of the Crhr1 promoter for double fluorescence immunohistochemistry to demonstrate the cellular location of CRHR1 in both dopaminergic and D1 dopaminoceptive neurons. We then studied cocaine sensitization, self-administration, and reinstatement in inducible CRHR1 knockouts using the CreERT2/loxP in either dopamine transporter (DAT)-containing neurons (DAT-Crhr1) or dopamine receptor 1 (D1)-containing neurons (D1-Crhr1). For sensitization testing, mice received five daily injections of cocaine (15 mg/kg IP). For self-administration, mice received eight daily 2 h cocaine (0.5 mg/kg per infusion) self-administration sessions followed by extinction and reinstatement testing. There were no differences in the acute or sensitized locomotor response to cocaine in DAT-Crhr1 or D1-Crhr1 mice and their respective controls. Furthermore, both DAT-Crhr1 and D1-Crhr1 mice reliably self-administered cocaine at the level of controls. However, DAT-Crhr1 mice demonstrated a significant increase in cue-induced reinstatement relative to controls, whereas D1-Crhr1 mice demonstrated a significant decrease in cue-induced reinstatement relative to controls. These data demonstrate the involvement of CRHR1 in cue-induced reinstatement following cocaine self-administration, and implicate a bi-directional role of CRHR1 for cocaine craving.

Differential Roles for L-Type Calcium Channel Subtypes in Alcohol Dependence.

It has previously been shown that the inhibition of L-type calcium channels (LTCCs) decreases alcohol consumption, although the contribution of the central LTCC subtypes Cav1.2 and Cav1.3 remains unknown. Here, we determined changes in Cav1.2 (Cacna1c) and Cav1.3 (Cacna1d) mRNA and protein expression in alcohol-dependent rats during protracted abstinence and naive controls using in situ hybridization and western blot analysis. Functional validation was obtained by electrophysiological recordings of calcium currents in dissociated hippocampal pyramidal neurons. We then measured alcohol self-administration and cue-induced reinstatement of alcohol seeking in dependent and nondependent rats after intracerebroventricular (i.c.v.) injection of the LTCC antagonist verapamil, as well as in mice with an inducible knockout (KO) of Cav1.2 in Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-expressing neurons. Our results show that Cacna1c mRNA concentration was increased in the amygdala and hippocampus of alcohol-dependent rats after 21 days of abstinence, with no changes in Cacna1d mRNA. This was associated with increased Cav1.2 protein concentration and L-type calcium current amplitudes. Further analysis of Cacna1c mRNA in the CA1, basolateral amygdala (BLA), and central amygdala (CeA) revealed a dynamic regulation over time during the development of alcohol dependence. The inhibition of central LTCCs via i.c.v. administration of verapamil prevented cue-induced reinstatement of alcohol seeking in alcohol-dependent rats. Further studies in conditional Cav1.2-KO mice showed a lack of dependence-induced increase of alcohol-seeking behavior. Together, our data indicate that central Cav1.2 channels, rather than Cav1.3, mediate alcohol-seeking behavior. This finding may be of interest for the development of new antirelapse medications.

Low µ-Opioid Receptor Status in Alcohol Dependence Identified by Combined Positron Emission Tomography and Post-Mortem Brain Analysis.

Blockade of the µ-opioid receptor (MOR) by naltrexone reduces relapse risk in a subpopulation of alcohol-dependent patients. Previous positron-emission-tomography (PET) studies using the MOR ligand [11C]carfentanil have found increased MOR availability in abstinent alcoholics, which may reflect either increased MOR expression or lower endogenous ligand concentration. To differentiate between both effects, we investigated two cohorts of alcoholic subjects using either post-mortem or clinical PET analysis. Post-mortem brain tissue of alcohol-dependent subjects and controls (N=43/group) was quantitatively analyzed for MOR ([3H]DAMGO)-binding sites and OPRM1 mRNA in striatal regions. [11C]carfentanil PET was performed in detoxified, medication free alcohol-dependent patients (N=38), followed by a randomized controlled study of naltrexone versus placebo and follow-up for 1 year (clinical trial number: NCT00317031). Because the functional OPRM1 variant rs1799971:A>G affects the ligand binding, allele carrier status was considered in the analyses. MOR-binding sites were reduced by 23-51% in post-mortem striatal tissue of alcoholics. In the PET study, a significant interaction of OPRM1 genotype, binding potential (BPND) for [11C]carfentanil in the ventral striatum, and relapse risk was found. Particularly in G-allele carriers, lower striatal BPND was associated with a higher relapse risk. Interestingly, this effect was more pronounced in the naltrexone treatment group. Reduced MOR is interpreted as a neuroadaptation to an alcohol-induced release of endogenous ligands in patients with severe alcoholism. Low MOR availability may explain the ineffectiveness of naltrexone treatment in this subpopulation. Finally, low MOR-binding sites are proposed as a molecular marker for a negative disease course.

Reduced oxytocin receptor gene expression and binding sites in different brain regions in schizophrenia: A post-mortem study.

Schizophrenia is a severe neuropsychiatric disorder with impairments in social cognition. Several brain regions have been implicated in social cognition, including the nucleus caudatus, prefrontal and temporal cortex, and cerebellum. Oxytocin is a critical modulator of social cognition and the formation and maintenance of social relationships and was shown to improve symptoms and social cognition in schizophrenia patients. However, it is unknown whether the oxytocin receptor is altered in the brain. Therefore, we used qRT-PCR and Ornithine Vasotocin Analog ([125I]OVTA)-based receptor autoradiography to investigate oxytocin receptor expression at both the mRNA and protein level in the left prefrontal and middle temporal cortex, left nucleus caudatus, and right posterior superior vermis in 10 schizophrenia patients and 6 healthy controls. Furthermore, to investigate confounding effects of long-term antipsychotic medication we treated rats with clozapine or haloperidol for 12weeks and assessed expression of the oxytocin receptor in cortical and subcortical brain regions. In schizophrenia patients, we found a downregulation of oxytocin receptor mRNA in the temporal cortex and a decrease in receptor binding in the vermis. In the other regions, the results showed trends in the same direction, without reaching statistical significance. We found no differences between antipsychotic-treated rats and controls. Downregulated expression and binding of the oxytocin receptor in brain regions involved in social cognition may lead to a dysfunction of oxytocin signaling. Our results support a dysfunction of the oxytocin receptor in schizophrenia, which may contribute to deficits of social cognition.

Convergent evidence from alcohol-dependent humans and rats for a hyperdopaminergic state in protracted abstinence.

A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Adaptive changes in serotonin metabolism preserve normal behavior in mice with reduced TPH2 activity.

Polymorphisms in the TPH2 gene coding for the serotonin synthesizing enzyme in the brain are considered as risk factors associated with depression and anxiety in humans. However, whether a certain variation in the TPH2 gene leads to decreased brain serotonin production and development of psychological abnormalities remains unresolved. We generated a new mouse model, carrying one Tph2-null allele and one Tph21473G-allele, coding for a hypoactive form of the enzyme. We tested these mice along with C57BL/6 mice (Tph2C/C), congenic C57BL/6 mice homozygous for the Tph21473G-allele (Tph2G/G), and heterozygous Tph2-deficient mice (Tph2C/-) for anxiety- and depression-like behavior, and evaluated brain serotonin metabolism and 5-HT1AR signaling by high-performance liquid chromatography and quantitative autoradiography, respectively. Progressive reduction in TPH2 activity had no effect on emotional behavior, and only slightly affected brain serotonin levels. However, serotonin degradation rate was drastically decreased in mice with reduced TPH2 activity, thereby compensating for the lowered rate of serotonin production in these mice. In addition, the hypothermic response to the 5-HT1AR agonist, 8-OH-DPAT, was attenuated in mice with reduced serotonin production. In contrast, 5-HT1A autoreceptor density and G-protein coupling were not changed in mice with gradual decrease in central serotonin. Taken together, these data suggest that in conditions of reduced serotonin production lowered serotonin degradation rate contributes to the maintenance of brain serotonin at levels sufficient for adequate behavior responses. These findings reveal that decreased TPH2 activity cannot be considered a reliable predisposition factor for impaired emotional behavior.

ß-Arrestin 2 knockout mice exhibit sensitized dopamine release and increased reward in response to a low dose of alcohol.

RATIONALE: The rewarding effects of alcohol have been attributed to interactions between opioid and dopaminergic system within the mesolimbic reward pathway. We have previously shown that ablation of ß-arrestin 2 (Arrb2), a crucial regulator of µ-opioid receptor function, attenuates alcohol-induced hyperlocomotion and c-fos activation in the nucleus accumbens. OBJECTIVES: Here, we further investigated the role of Arrb2 in modulating alcohol-induced dopamine (DA) release and conditioned place preference (CPP). We also assessed the functional importance of Arrb2 for µ-opioid receptor surface expression and signaling following an acute alcohol challenge. METHODS: Alcohol-evoked (0.375, 0.75, and 1.5 g/kg intraperitoneally) DA release was measured by in vivo microdialysis in the shell of nucleus accumbens. Reward was assessed by the CPP paradigm. Receptor function was assessed by µ-receptor binding and [(35)S]GTP-γ-S autoradiography. RESULTS: In Arrb2 knockout mice accumbal DA levels reach maximum response at a lower dose compared to wild-type (wt) animals. In line with these results, Arrb2 knockout mice display increased CPP for alcohol as compared to wt mice. Finally, Arrb2 mutant mice display increased µ-opioid receptor signaling in the ventral and dorsal striatum and amygdala in response to a low dose of alcohol, indicating impaired desensitization mechanisms in these mice. CONCLUSIONS: Our results show that Arrb2 modulates the response to low doses of alcohol on various levels including µ-opioid receptor signaling, DA release, and reward. They also reveal a clear dissociation between the effects of Arrb2 on psychomotor and reward behaviors.