The Role of Ryanodine Receptor 2 in Drug-Associated Learning.

Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca 2+ from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.

NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress-induced anhedonia-like behavior and reductions in excitatory synapses.

Chronic stress can produce reward system deficits (i.e., anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and it is required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation in the mice. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reductions in pyramidal neuron dendritic spine density, excitatory synaptic transmission, and presynaptic function, revealing a relationship between perturbation in excitatory synaptic transmission and the expression of anhedonia-like behavior in the mice. Finally, analysis of the mice mPFC tissues revealed that NPAS4 regulates the expression of numerous genes linked to glutamatergic synapses and ribosomal function, the expression of upregulated genes in CSDS-susceptible animals, and differentially expressed genes in postmortem human brains of patients with common neuropsychiatric disorders, including depression. Together, our findings position NPAS4 as a key mediator of chronic stress-induced hypofrontal states and anhedonia-like behavior.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) regulates anxiety- and novelty-related behaviors.

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)-a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR-subunit surface levels in the adult NAc, and the Arc-deficient mice show reductions in anxiety-like behavior, deficits in social novelty preference, and antidepressive-like behavior. Viral-mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety-like behavior in the elevated plus maze (EPM). Consistent with this finding, viral-mediated reduction of Arc in the adult NAc reduces anxiety-like behavior in male, but not female, mice in the EPM. NAc-specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood- and anxiety-related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.

It is a complex issue: emerging connections between epigenetic regulators in drug addiction.

Drug use leads to addiction in some individuals, but the underlying brain mechanisms that control the transition from casual drug use to an intractable substance use disorder (SUD) are not well understood. Gene x environment interactions such as the frequency of drug use and the type of substance used likely to promote maladaptive plastic changes in brain regions that are critical for controlling addiction-related behavior. Epigenetics encompasses a broad spectrum of mechanisms important for regulating gene transcription that are not dependent on changes in DNA base pair sequences. This review focuses on the proteins and complexes contributing to epigenetic modifications in the nucleus accumbens (NAc) following drug experience. We discuss in detail the three major mechanisms: histone acetylation and deacetylation, histone methylation, and DNA methylation. We discuss how drug use alters the regulation of the associated proteins regulating these processes and highlight how experimental manipulations of these proteins in the NAc can alter drug-related behaviors. Finally, we discuss the ways that histone modifications and DNA methylation coordinate actions by recruiting large epigenetic enzyme complexes to aid in transcriptional repression. Targeting these multiprotein epigenetic enzyme complexes - and the individual proteins that comprise them - might lead to effective therapeutics to reverse or treat SUDs in patients.

Cellular Targets and Receptor of Sexual Transmission of Zika Virus.

STUDY QUESTION: What is the mechanism of sexual transmission of Zika virus (ZIKV)? SUMMARY ANSWER: By utilizing exquisite reverse transcriptase-initiated in situ polymerase chain reaction (RT-in situ PCR), which enables an improved visualization of spermatozoa's subcellular compartment, we precisely localized the mid-piece of sperm that carry receptors for ZIKV. WHAT IS ALREADY KNOWN: ZIKV is transmitted sexually and recent studies have verified ZIKV presence in semen of previously Zika-infected patients for >6-month postinfection when ZIKV had disappeared from blood, saliva, and urine. Strong serial analyses of various body fluids suggest that ZIKV can be transmitted between sexual partners. Currently, there is limited information on the association of the virus with human semen cell types that may carry the virus. STUDY DESIGN, SIZE, DURATION: Analyses were carried out to localize ZIKV for subcellular localization of ZIKV on cell types. The Tyro3 receptor for ZIKV was colocalized by dual immunocytochemistry with specific monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three semen specimens were purchased from a commercial sperm bank. Motile sperm was separated from nonmotile cells by the "swim-up" technique. Each of the semen fractions was infected with ZIKV at the multiplicity of infection of 0.1.0 and 1.0 and evaluated for the primary targets of ZIKV in the semen cells by RT-in situ PCR and confirmed by real-time RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: ZIKV was present primarily at the mid-piece of mature spermatozoa in about 30% of the sperm. In addition, we determined that Tyro3 receptors, primarily expressed on mid-piece of human spermatozoa, play a role in ZIKV-binding and entry into spermatozoa. Our data strongly suggest a potential sexual/horizontal route of transmission for ZIKV primarily via infected sperms; most likely ZIKV enters the sperm via the Tyro3 receptor found at the mid-piece of the mature spermatozoa. LIMITATIONS, REASONS FOR CAUTION: We are uncertain as to what phase of spermatogenesis, that in human takes about 120 days, sperms are permissive to ZIKV. If permissiveness was very early during spermatogenesis males could be infectious for ∼120 days after the disappearance of viremia in an infected man. WIDER IMPLICATIONS OF THE FINDINGS: Our findings bring a new focus on the current affords to develop ZIKV vaccine. Why in the presence of anti-ZIKV antibodies infected men are still able to transmit the virus sexually? We suggest that only certain subclass of immunoglobulin (Ig)G (ie, IgG4) can cross the blood-Sertoli barrier therefore, a successful vaccine must provoke a subclass of IgG can quell ZIKV inside the seminiferous tubules.

HDAC5 and Its Target Gene, Npas4, Function in the Nucleus Accumbens to Regulate Cocaine-Conditioned Behaviors.

Individuals suffering from substance-use disorders develop strong associations between the drug's rewarding effects and environmental cues, creating powerful, enduring triggers for relapse. We found that dephosphorylated, nuclear histone deacetylase 5 (HDAC5) in the nucleus accumbens (NAc) reduced cocaine reward-context associations and relapse-like behaviors in a cocaine self-administration model. We also discovered that HDAC5 associates with an activity-sensitive enhancer of the Npas4 gene and negatively regulates NPAS4 expression. Exposure to cocaine and the test chamber induced rapid and transient NPAS4 expression in a small subpopulation of FOS-positive neurons in the NAc. Conditional deletion of Npas4 in the NAc significantly reduced cocaine conditioned place preference and delayed learning of the drug-reinforced action during cocaine self-administration, without affecting cue-induced reinstatement of drug seeking. These data suggest that HDAC5 and NPAS4 in the NAc are critically involved in reward-relevant learning and memory processes and that nuclear HDAC5 limits reinstatement of drug seeking independent of NPAS4.

Computational identification of mutually homologous Zika virus miRNAs that target microcephaly genes.

Background Zika virus (ZIKV) has been associated with a variety of neuropathologies, including microcephaly. We hypothesize that ZIKV genes activate host microRNAs (miRNAs) causing dysfunctional development of human fetal brains. Objectives/methods A bioinformatics search for miRNA genome-wide binding sites in the prototypic ZIKV (strain MR766) was undertaken to hunt for miRNAs with significant similarities with MCPH genetic sequences responsible for inducing MCHP in human fetal brains. Results Six ZIKV miRNAs were found to share mutual homology with 12 MCPH genetic sequences responsible for inducing MCPH. Noteworthy was miR-1304, which expressed 100% identity to six different MCPH genes. Conclusions We suggest that following infection of fetal neurons ZIKV may modulate the action of various miRNAs, and miR-1304 in particular, resulting in microcephaly.

Infectivity of Immature Neurons to Zika Virus: A Link to Congenital Zika Syndrome.

BACKGROUND: Epidemiological data strongly suggest that microcephaly cases in Brazil are associated with the ongoing epidemic of Zika virus (ZIKV). In order to further solidify the possible link, we investigated the infectivity of ZIKV using various neuroblastoma (NB) cell lines. METHODS: Six undifferentiated, two terminally differentiated and two retinoic acid (RA) -induced, partially differentiated cell lines were exposed to ZIKV strain PRVABC59, which is genetically similar to the French Polynesia strain, with 97-100% genetic homology to the current ZIKV strain found in Brazil. All infections were confirmed by real-time PCR (RT-qPCR), immunofluorescence assay (IFA) probing with anti-flavivirus E antibody, and evaluation of cytopathic effects. FINDINGS: ZIKV infected all six undifferentiated NB cell lines. In five out of six NB cell lines, between 90 and 70% cells were positive by IFA whereas for one cell line, CCL-127, ~80% of cells were positive for ZIKV as determined by IFA but showed persistent infection. Two differentiated cell lines, JFEN and T-268, were highly resistant to ZIKV with <1% of the cells being susceptible, as determined by IFA and confirmed by qRT-PCR. Two retinoic acid (RA)-induced NB partially differentiated cell lines showed no difference in permissiveness as compared to their undifferentiated mother cell lines. INTERPRETATION: These findings strengthen the reported association between high incidences of microcephaly and ZIKV infection in newborns in Brazil. Our results suggest that the undifferentiated neurons are highly permissive to ZIKV infection, as one would expect during the early stages of neurogenesis in fetal brains; whereas differentiated neurons, representative of adult brain neurons, are relatively resistant to the virus, which explains the rare occurrence of neurological complications in adults infected with ZIKV. Our studies confirm the neurotropism of the ZIKV strain closely related to the current epidemic in Latin America.

Environmental factors may contribute to autism development and male bias: Effects of fragrances on developing neurons.

BACKGROUND: Autism spectrum disorders (ASDs) are developmental conditions characterized by deficits in social interaction, impairments in verbal and nonverbal communication, and stereotyped patterns of behavior. Previous studies have implicated environmental factors in the development of ASD. Although no reliable neurophysiological network is associated with ASD, low levels of plasma oxytocin (OXY) and arginine vasopressin (AVP) have been reported. The "twin" nonapeptides OXY and AVP are mainly produced in the brain of mammals, and dysregulation of these neuropeptides has been associated with changes in behavior, especially social interactions. METHODS: Previously, we analyzed 91 commonly used fragrances and reported significant mutagenic, neurocytotoxic, and stimulatory effects on fetal neuroblastoma cell lines (NBC). In this study, we analyzed the neuromodifications of three selected fragrances on male and female human fetal brain neurons, utilizing immunohistochemistry. RESULTS: We show that exposure to femtomolar concentrations of fragrances results in morphological changes by light microscopy in the NBC. Importantly, these fragrances significantly reduced the OXY- and AVP-receptor positive (OXYR+ and AVPR+) neurons in male NBC but not in female NBC, possibly contributing to the development of male bias in ASD. CONCLUSION: This study is the first to show a potential link between fragrance exposure, depletion of OXYR+ and AVPR+ neurons, and a male bias in autism.