A cerebellar-prepontine circuit for tonic immobility triggered by an inescapable threat.

Sudden changes in the environment are frequently perceived as threats and provoke defensive behavioral states. One such state is tonic immobility, a conserved defensive strategy characterized by powerful suppression of movement and motor reflexes. Tonic immobility has been associated with multiple brainstem regions, but the underlying circuit is unknown. Here, we demonstrate that a strong vibratory stimulus evokes tonic immobility in larval zebrafish defined by suppressed locomotion and sensorimotor responses. Using a circuit-breaking screen and targeted neuron ablations, we show that cerebellar granule cells and a cluster of glutamatergic ventral prepontine neurons (vPPNs) that express key stress-associated neuropeptides are critical components of the circuit that suppresses movement. The complete sensorimotor circuit transmits information from sensory ganglia through the cerebellum to vPPNs to regulate reticulospinal premotor neurons. These results show that cerebellar regulation of a neuropeptide-rich prepontine structure governs a conserved and ancestral defensive behavior that is triggered by an inescapable threat.

Store-Operated Ca2+ Entry Contributes to Piezo1-Induced Ca2+ Increase in Human Endometrial Stem Cells.

Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment. Mechanosensitive channels are part of the cellular toolkit that feels the mechanical environment and can transduce mechanical stimuli to intracellular signaling pathways. Here, we identify previously recorded, mechanosensitive (MS), stretch-activated channels as Piezo1 proteins in the plasma membrane of eMSCs. Piezo1 activity triggered by the channel agonist Yoda1 elicits influx of Ca2+, a known modulator of cytoskeleton reorganization and cell motility. We found that store-operated Ca2+ entry (SOCE) formed by Ca2+-selective channel ORAI1 and Ca2+ sensors STIM1/STIM2 contributes to Piezo1-induced Ca2+ influx in eMSCs. Particularly, the Yoda1-induced increase in intracellular Ca2+ ([Ca2+]i) is partially abolished by 2-APB, a well-known inhibitor of SOCE. Flow cytometry analysis and wound healing assay showed that long-term activation of Piezo1 or SOCE does not have a cytotoxic effect on eMSCs but suppresses their migratory capacity and the rate of cell proliferation. We propose that the Piezo1 and SOCE are both important determinants in [Ca2+]i regulation, which critically affects the migratory activity of eMSCs and, therefore, could influence the regenerative potential of these cells.

Impact of lipid rafts on transient receptor potential channel activities.

Members of the transient receptor potential (TRP) superfamily are cation channels that are expressed in nearly every mammalian cell type and respond as cellular sensors to various environmental stimuli. Light, pressure, osmolarity, temperature, and other stimuli can induce TRP calcium conductivity and correspondingly trigger many signaling processes in cells. Disruption of TRP channel activity, as a rule, harms cellular function. Despite numerous studies, the mechanisms of TRP channel regulation are not yet sufficiently clear, in part, because TRP channels are regulated by a broad set of ligands having diverse physical and chemical features. It is now known that some TRP members are located in membrane microdomains termed lipid rafts. Moreover, interaction between specific raft-associated lipids with channels may be a key regulation mechanism. This review examines recent findings related to the roles of lipid rafts in regulation of TRP channel activity. The mechanistic events of channel interactions with the main lipid raft constituent, cholesterol, are being clarified. Better understanding of mechanisms behind such interactions would help establish the key elements of TRP channel regulation and hence allow control of cellular responses to environmental stimuli.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish.

AIM: We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. METHODS: A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. RESULTS: The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. CONCLUSION: Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

The bullies are the leaders of the next generation: Inherited aminergic neurotransmitter system changes in socially dominant zebrafish, Danio rerio.

We studied the social hierarchy in zebrafish and assessed differences in neurotransmitters and behavior in the F1 generation offspring of dominant and subordinate zebrafish (Danio rerio). We used behavioral assays to study locomotion, ability to complete cognitive tasks, social interaction and aggression. To study the neurochemical changes, we applied quantitative polymerase chain reaction, high pressure liquid chromatography and immunohistochemistry. Social hierarchies were formed both by males and females when animals were kept in same sex pairs in the dyadic dominant-subordinate hierarchy test. The offspring of dominant animals were the leaders in social interactions, however aggression in the mirror-test was not altered in any group. Serotonin and noradrenaline levels were lower in the F1 generation subordinate animals when compared with dominant animals, but not compared with animals that were naïve to social hierarchy. The mRNA level of the rate-limiting enzyme in histamine synthesis, histidine decarboxylase, was significantly lower in dominant and subordinate larval zebrafish when compared with control animals. In the dominant adult zebrafish tyrosine hydroxylase 1 mRNA level was lower compared with control animals, whereas tyrosine hydroxylase 2 mRNA was not different. The result was verified with immunohistochemistry. There were gender specific differences between the dominant and subordinate animals, where the dominant females performed better in cognitive tasks such as the T-maze than subordinate females. This was not observed in males, as the behavior of the dominant and subordinate males did not differ. These results add to the understanding of the plastic nature of the central nervous system and show that neurochemical features in aminergic neurotransmitter systems are associated with social leadership and dominance.

Lipid raft integrity is required for human leukemia Jurkat T-cell migratory activity.

Lipid rafts are membrane microdomains featuring high cholesterol, sphingolipid, and protein content. These microdomains recruit various receptors, ion channels, and signaling molecules for coordination of various cellular functions, including synaptic transmission, immune response, cytoskeletal organization, adhesion, and migration. Many of these processes also depend on Ca2+ intake. We have previously shown in Jurkat cells that activity of transient receptor potential vanilloid, type 6 (TRPV6) calcium channel, and TRPV6-mediated Ca2+ influx, depend on lipid raft integrity. In this study, using the transwell cell migration assay and time-lapse video microscopy with Jurkat cells, we found that lipid raft destruction was associated with: inhibited cell adhesion and migration; and decreased mean speed, maximum speed, and trajectory length. Using String Server, we constructed a Protein Interaction Network (PIN). The network indicated that TRPV6 proteins interact with the highest probability (0.9) with Src family kinase members (SFKs) involved in processes related to cell migration. Analysis of detergent-resistant membrane fractions and immunoelectron microscopy data confirmed an association in lipid rafts between TRPV6 and Lck kinase, an SFKs member. Destruction of lipid rafts led to uncoupling of TRPV6 clusters with Lck and their departure from the plasma membrane into the cytosol of the cells. Src family kinases are generally associated with their roles in tumor invasion and progression, epithelial-mesenchymal transitions, angiogenesis, and metastatic development. We suggest that a functional interaction between TRPV6 calcium channels and SFKs members in lipid rafts is one of necessary elements of migration and oncogenic signaling in leukemia cells.

Sex differences and Tat expression affect dopaminergic receptor expression and response to antioxidant treatment in methamphetamine-sensitized HIV Tat transgenic mice.

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders.

Cerebral Dopamine Neurotrophic Factor Regulates Multiple Neuronal Subtypes and Behavior.

Cerebral dopamine neurotrophic factor (CDNF) protects dopaminergic neurons against toxic damage in the rodent brain and is in clinical trials to treat Parkinson's disease patients. Yet the underlying mechanism is poorly understood. To examine its significance for neural circuits and behavior, we examined the development of neurotransmitter systems from larval to male adult mutant zebrafish lacking cdnf Although a lack of cdnf did not affect overall brain dopamine levels, dopaminergic neuronal clusters showed significant abnormalities. The number of histamine neurons that surround the dopaminergic neurons was significantly reduced. Expression of tyrosine hydroxylase 2 in the brain was elevated in cdnf mutants throughout their lifespan. There were abnormally few GABA neurons in the hypothalamus in the mutant larvae, and expression of glutamate decarboxylase was reduced throughout the brain. cdnf mutant adults showed a range of behavioral phenotypes, including increased sensitivity to pentylenetetrazole-induced seizures. Shoaling behavior of mutant adults was abnormal, and they did not display social attraction to conspecifics. CDNF plays a profound role in shaping the neurotransmitter circuit structure, seizure susceptibility, and complex behaviors in zebrafish. These findings are informative for dissecting the diverse functions of this poorly understood factor in human conditions related to Parkinson's disease and complex behaviors.SIGNIFICANCE STATEMENT A zebrafish lacking cdnf grows normally and shows no overt morphologic phenotype throughout the life span. Remarkably, impaired social cohesion and increased seizure susceptibility were found in adult cdnf KO fish conceivably associated with significant changes of dopaminergic, GABAergic, and histaminergic systems in selective brain areas. These findings suggest that cdnf has broad effects on regulating neurogenesis and maturation of transmitter-specific neuronal types during development and throughout adulthood, rather than ones restricted to the dopaminergic systems.

Systems Biology Analysis of the Antagonizing Effects of HIV-1 Tat Expression in the Brain over Transcriptional Changes Caused by Methamphetamine Sensitization.

Methamphetamine (Meth) abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein, trans-activator of transcription (Tat), has been described to induce changes in brain gene transcription that can result in impaired reward circuitry, as well as in inflammatory processes. In transgenic mice with doxycycline-induced Tat protein expression in the brain, i.e., a mouse model of neuroHIV, we tested global gene expression patterns induced by Meth sensitization. Meth-induced locomotor sensitization included repeated daily Meth or saline injections for seven days and Meth challenge after a seven-day abstinence period. Brain samples were collected 30 min after the Meth challenge. We investigated global gene expression changes in the caudate putamen, an area with relevance in behavior and HIV pathogenesis, and performed pathway and transcriptional factor usage predictions using systems biology strategies. We found that Tat expression alone had a very limited impact in gene transcription after the Meth challenge. In contrast, Meth-induced sensitization in the absence of Tat induced a global suppression of gene transcription. Interestingly, the interaction between Tat and Meth broadly prevented the Meth-induced global transcriptional suppression, by maintaining regulation pathways, and resulting in gene expression profiles that were more similar to the controls. Pathways associated with mitochondrial health, initiation of transcription and translation, as well as with epigenetic control, were heavily affected by Meth, and by its interaction with Tat in anti-directional ways. A series of systems strategies have predicted several components impacted by these interactions, including mitochondrial pathways, mTOR/RICTOR, AP-1 transcription factor, and eukaryotic initiation factors involved in transcription and translation. In spite of the antagonizing effects of Tat, a few genes identified in relevant gene networks remained downregulated, such as sirtuin 1, and the amyloid precursor protein (APP). In conclusion, Tat expression in the brain had a low acute transcriptional impact but strongly interacted with Meth sensitization, to modify effects in the global transcriptome.

Adenosine-5'-triphosphate suppresses proliferation and migration capacity of human endometrial stem cells.

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP-induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X7 receptor in eMSCs. In spite of this, cell activation with specific P2X7 receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X7 receptor inhibitor, AZ10606120 also did not prevent ATP-induced inhibition of cell migration, confirming that inhibition occurs without P2X7 receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.

Brain Reward Function after Chronic and Binge Methamphetamine Regimens in Mice Expressing the HIV-1 TAT Protein.

BACKGROUND: Methamphetamine abuse and human immunodeficiency virus (HIV) are common comorbidities. HIV-associated proteins, such as the regulatory protein TAT, may contribute to brain reward dysfunction, inducing an altered sensitivity to methamphetamine reward and/or withdrawal in this population. OBJECTIVE: These studies examined the combined effects of TAT protein expression and, chronic and binge methamphetamine regimens on brain reward function. METHODS: Transgenic mice with inducible brain expression of the TAT protein were exposed to either saline, a chronic, or a binge methamphetamine regimen. TAT expression was induced via doxycycline treatment during the last week of methamphetamine exposure. Brain reward function was assessed daily throughout the regimens, using the intracranial self-stimulation procedure, and after a subsequent acute methamphetamine challenge. RESULTS: Both methamphetamine regimens induced withdrawal-related decreases in reward function. TAT expression substantially, but not significantly increased the withdrawal associated with exposure to the binge regimen compared to the chronic regimen, but did not alter the response to acute methamphetamine challenge. TAT expression also led to persistent changes in adenosine 2B receptor expression in the caudate putamen, regardless of methamphetamine exposure. These results suggest that TAT expression may differentially affect brain reward function, dependent on the pattern of methamphetamine exposure. CONCLUSION: The subtle effects observed in these studies highlight that longer-term TAT expression, or its induction at earlier stages of methamphetamine exposure, may be more consequential at inducing behavioral and neurochemical effects.

Abnormal behavior, striatal dopamine turnover and opioid peptide gene expression in histamine-deficient mice.

Hypothalamic histaminergic neurons regulate a variety of homeostatic, metabolic and cognitive functions. Recent data have suggested a modulatory role of histamine and histamine receptors in shaping striatal activity and connected the histaminergic system to neuropsychiatric disorders. We characterized exploratory behavior and striatal neurotransmission in mice lacking the histamine producing enzyme histidine decarboxylase (Hdc). The mutant mice showed a distinct behavioral pattern during exploration of novel environment, specifically, increased frequency of rearing seated against the wall, jumping and head/body shakes. This behavioral phenotype was associated with decreased levels of striatal dopamine and serotonin and increased level of dopamine metabolite DOPAC. Gene expression levels of dynorphin and enkephalin, opioids released by medium spiny neurons of striatal direct and indirect pathways respectively, were lower in Hdc mutant mice than in control animals. A low dose of amphetamine led to similar behavioral and biochemical outcomes in both genotypes. Increased striatal dopamine turnover was observed in Hdc KO mice after treatment with dopamine precursor l-Dopa. Overall, our study suggests a role for striatal dopamine and opioid peptides in formation of distinct behavioral phenotype of Hdc KO mice.

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line.

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca2+ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca 2+ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch-clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl-ß-cyclodextrin (MßCD). Continuous measurement of intracellular Ca2+ revealed a reduction of Ca2+ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MßCD-treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6-mediated Ca2+ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.

Regeneration of Dopaminergic Neurons in Adult Zebrafish Depends on Immune System Activation and Differs for Distinct Populations.

Adult zebrafish, in contrast to mammals, regenerate neurons in their brain, but the extent and variability of this capacity is unclear. Here we ask whether the loss of various dopaminergic neuron populations is sufficient to trigger their functional regeneration. Both sexes of zebrafish were analyzed. Genetic lineage tracing shows that specific diencephalic ependymo-radial glial (ERG) progenitor cells give rise to new dopaminergic [tyrosine hydroxylase-positive (TH+)] neurons. Ablation elicits an immune response, increased proliferation of ERG progenitor cells, and increased addition of new TH+ neurons in populations that constitutively add new neurons (e.g., diencephalic population 5/6). Inhibiting the immune response attenuates neurogenesis to control levels. Boosting the immune response enhances ERG proliferation, but not addition of TH+ neurons. In contrast, in populations in which constitutive neurogenesis is undetectable (e.g., the posterior tuberculum and locus ceruleus), cell replacement and tissue integration are incomplete and transient. This is associated with a loss of spinal TH+ axons, as well as permanent deficits in shoaling and reproductive behavior. Hence, dopaminergic neuron populations in the adult zebrafish brain show vast differences in regenerative capacity that correlate with constitutive addition of neurons and depend on immune system activation.SIGNIFICANCE STATEMENT Despite the fact that zebrafish show a high propensity to regenerate neurons in the brain, this study reveals that not all types of dopaminergic neurons are functionally regenerated after specific ablation. Hence, in the same adult vertebrate brain, mechanisms of successful and incomplete regeneration can be studied. We identify progenitor cells for dopaminergic neurons and show that activating the immune system promotes the proliferation of these cells. However, in some areas of the brain this only leads to insufficient replacement of functionally important dopaminergic neurons that later disappear. Understanding the mechanisms of regeneration in zebrafish may inform interventions targeting the regeneration of functionally important neurons, such as dopaminergic neurons, from endogenous progenitor cells in nonregenerating mammals.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

Differential effects of withdrawal from intermittent and continuous nicotine exposure on reward deficit and somatic aspects of nicotine withdrawal and expression of α4ß2* nAChRs in Wistar male rats.

BACKGROUND: Chronic nicotine exposure produces neuroadaptations in brain reward systems and α4ß2 nicotinic acetylcholine receptors (nAChRs) in the corticolimbic brain areas. We previously demonstrated opposite effects of nicotine exposure delivered by self-administration or pumps on brain reward thresholds that can be attributed to the different temporal pattern and contingency of nicotine exposure. We investigated the effects of these two factors on reward thresholds and somatic signs during nicotine withdrawal, and on nAChRs binding in corticolimbic brain areas. METHODS: The intracranial self-stimulation procedure was used to assess reward thresholds in rats prepared with pumps delivering various doses of nicotine continuously or intermittently. Separate group of rats were randomly exposed to nicotine via pumps (non-contingent) or nicotine self-administration (contingent) to determine [125I]-epibatidine binding at α4ß2* nAChRs. RESULTS: Withdrawal from continuous non-contingent nicotine exposure led to significant elevations in thresholds and increases in somatic signs in rats, while there was no significant effect of withdrawal from intermittent non-contingent nicotine exposure at the same doses. nAChRs were upregulated during withdrawal from continuous non-contingent nicotine exposure. α4ß2* nAChRs were upregulated in the ventral tegmental area and prelimbic cortex during withdrawal from non-contingent intermittent exposure and in the nucleus accumbens during withdrawal from contingent intermittent nicotine exposure to the same dose. CONCLUSIONS: During non-contingent nicotine exposure, the temporal pattern of nicotine delivery differentially affected thresholds and somatic signs of withdrawal. Upregulation of α4ß2* nAChRs was brain site-specific and depended on both temporal pattern and contingency of nicotine exposure.

Effects of HIV-1 TAT protein and methamphetamine exposure on visual discrimination and executive function in mice.

Mild neurocognitive impairments are common in people with human immunodeficiency virus (HIV) infection. HIV-encoded proteins, such as trans-activator of transcription (TAT), contribute to neuropathology and cognitive function in medicated subjects. The combination of TAT and comorbid methamphetamine use may further impair neurocognitive function in HIV-positive individuals by affecting dopaminergic systems in the brain. The current study examined the effects of TAT protein expression and methamphetamine exposure on cognitive function and dopamine systems in mice. Transgenic mice with inducible brain expression of the TAT protein were exposed to a binge methamphetamine regimen. TAT expression was induced via a doxycycline-containing diet during the final stage of the regimen and maintained throughout cognitive testing. Learning and executive function were assessed using an operant visual discrimination protocol, with a strategy switch and reversal. TAT expression and methamphetamine exposure improved visual discrimination learning. Combined TAT expression and methamphetamine exposure increased perseverative errors during reversal learning. TAT expression altered reversal learning by improving early stage, but impairing late stage, learning. TAT expression was also associated with an increase in dopamine transporter expression in the caudate putamen. These results highlight that TAT expression and methamphetamine exposure likely affect a range of selective cognitive processes, with some potentially improving function under certain conditions.

Metabotropic Glutamate Receptor 5 as a Target for the Treatment of Depression and Smoking: Robust Preclinical Data but Inconclusive Clinical Efficacy.

The ability of novel pharmacological compounds to improve outcomes in preclinical models is often not translated into clinical efficacy. Psychiatric disorders do not have biological boundaries, and identifying mechanisms to improve the translational bottleneck between preclinical and clinical research domains is an important and challenging task. Glutamate transmission is disrupted in several neuropsychiatric disorders. Metabotropic glutamate (mGlu) receptors represent a diverse class of receptors that contribute to excitatory neurotransmission. Given the wide, yet region-specific manner of expression, developing pharmacological compounds to modulate mGlu receptor activity provides an opportunity to subtly and selectively modulate excitatory neurotransmission. This review focuses on the potential involvement of mGlu5 receptor disruption in major depressive disorder and substance and/or alcohol use disorders. We provide an overview of the justification of targeting mGlu5 receptors in the treatment of these disorders, summarize the preclinical evidence for negatively modulating mGlu5 receptors as a therapeutic target for major depressive disorders and nicotine dependence, and highlight the outcomes of recent clinical trials. While the evidence of mGlu5 receptor negative allosteric modulation has been promising in preclinical investigations, these beneficial effects have not translated into clinical efficacy. In this review, we identify key challenges that may contribute to poor clinical translation and provide suggested approaches moving forward to potentially improve the translation from preclinical to clinical domains. Such approaches may increase the success of clinical trials and may reduce the translational bottleneck that exists in drug discovery for psychiatric disorders.