Kappa Opioid Receptor Mediated Differential Regulation of Serotonin and Dopamine Transporters in Mood and Substance Use Disorder.

Dynorphin (DYN) is an endogenous neurosecretory peptide which exerts its activity by binding to the family of G protein-coupled receptors, namely the kappa opioid receptor (KOR). Opioids are associated with pain, analgesia, and drug abuse, which play a central role in mood disorders with monoamine neurotransmitter interactions. Growing evidence demonstrates the cellular signaling cascades linked to KOR-mediated monoamine transporters regulation in cell models and native brain tissues. This chapter will review DYN/KOR role in mood and addiction in relevance to dopaminergic and serotonergic neurotransmissions. Also, we discuss the recent findings on KOR-mediated differential regulation of serotonin and dopamine transporters (SERT and DAT). These findings led to a better understanding of the role of DYN/KOR system in aminergic neurotransmission via its modulatory effect on both amine release and clearance. Detailed knowledge of these processes at the molecular level enables designing novel pharmacological reagents to target transporter motifs to treat mood and addiction and reduce unwanted side effects such as aversion, dysphoria, sedation, and psychomimesis.

A Mutation in Hnrnph1 That Decreases Methamphetamine-Induced Reinforcement, Reward, and Dopamine Release and Increases Synaptosomal hnRNP H and Mitochondrial Proteins.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

Glycogen synthase kinase-3ß supports serotonin transporter function and trafficking in a phosphorylation-dependent manner.

Serotonin (5-HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase-3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax , surface expression with a reduction in 5-HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK-293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild-type hSERT (hSERT-WT) resulted in a time and dose-dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT-S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, over-expression of constitutively active GSK3ß with hSERT-WT, but not with hSERT-S48A, reduced SERT function, Vmax , surface density, and enhanced transporter phosphorylation. Both hSERT-WT and hSERT-S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine-48 phosphorylation.

Neurokinin-1 Antagonism Distinguishes the Role of Norepinephrine Transporter from Dopamine Transporter in Mediating Amphetamine Behaviors.

BACKGROUND: Amphetamine (AMPH) and other psychostimulants act on the norepinephrine (NE) transporter (NET) and the dopamine (DA) transporter (DAT) and enhance NE and DA signaling. Both NET and DAT share anatomical and functional characteristics and are regulated similarly by psychostimulants and receptor-linked signaling pathways. We and others have demonstrated that NET and DAT are downregulated by AMPH and substance P/neurokinin-1 receptor (NK1R)-mediated protein kinase C pathway. OBJECTIVES: Since both NET and DAT are downregulated by AMPH and NK1R activation and share high sequence homology, the objective of the study was to determine the catecholamine transporter specificity in NK1R modulation of AMPH-induced behaviors. METHODS: The effect of NK1R antagonism on AMPH-induced conditioned place preference (CPP) as well as AMPH-induced NET and DAT downregulation was examined using NET and DAT knockout mice (NET-KO and DAT-KO) along with their wild-type littermates. RESULTS: Aprepitant (5 mg/kg i.p.) significantly attenuated AMPH (2 mg/kg i.p.)-induced CPP in the wild-type and DAT-KO but not in the NET-KO. Locomotor activity measured during the post-conditioning test (in the absence of AMPH) showed higher locomotor activity in DAT-KO compared to wild-type or NET-KO. However, the locomotor activity of all 3 genotypes remained unchanged following aprepitant. Additionally, in the ventral striatum of wild-type, the AMPH-induced downregulation of NET function and surface expression but not that of DAT was attenuated by aprepitant. CONCLUSIONS: The results from the current study demonstrate that aprepitant attenuates the expression of AMPH-induced CPP in DAT-KO mice but not in NET-KO mice suggesting a role for NK1R-mediated NET regulation in AMPH-induced behaviors.

Activity-dependent expression of miR-132 regulates immediate-early gene induction during olfactory learning in the greater short-nosed fruit bat, Cynopterus sphinx.

The activity-dependent expression of immediate-early genes (IEGs) and microRNA (miR)-132 has been implicated in synaptic plasticity and the formation of long-term memory (LTM). In the present study, we show that olfactory training induces the expression of IEGs (EGR-1, C-fos, C-jun) and miR-132 at similar time scale in olfactory bulb (OB) of Cynopterus sphinx. We examined the role of miR-132 in the OB using antisense oligodeoxynucleotide (AS-ODN) and demonstrated that a local infusion of AS-ODN in the OB 2h prior to training impaired olfactory memory formation in C. sphinx. However, the infusion of AS-ODN post-training did not cause a deficit in memory formation. Furthermore, the inhibition of miR-132 reduced the olfactory training-induced expression of IEGs and post synaptic density protein-95 (PSD-95) in the OB. Additionally, we show that miR-132 regulates the activation of calcium/calmodulin-dependent protein kinase-II (CaMKII) and cAMP response element binding protein (CREB), possibly through miR-148a. These data suggest that olfactory training induces the expression of miR-132 and IEGs, which in turn activates post-synaptic proteins that regulate olfactory memory formation.

Prevalence of Cardiovascular Diseases in South Asians: Scrutinizing Traditional Risk Factors and Newly Recognized Risk Factors Sarcopenia and Osteopenia/Osteoporosis.

One of the primary reasons for complications and death worldwide are cardiovascular diseases (CVDs), with a death toll of approximately 18 million per year. CVDs include cardiomyopathy, hypertension, ischemic heart disease, coronary heart disease, myocardial infarction, heart attack, hearth failure, etc. Over 80% of the CVD mortality is recorded from lower and middle-income countries. Records from the past decade have highlighted the increase of CVDs among the South Asian populations, and the prime purpose of the review is to jot down the reasons for the steep spike in CVDs. Studies analyzing the causative factors for the increase of CVDs in South Asians are still to be verified. Apart from known predisposing and lifestyle factors, other emerging risk factors associated with CVDs, namely the musculoskeletal diseases sarcopenia and osteopenia, should be tracked to tackle research gaps in upcoming analyses. This requires loads of scientific efforts. With proper monitoring, the raising alarm that the CVD burden generates can be reduced. This review discusses the already established signs and recognizes important clues to the emerging etiology of CVDs in the Asian population and prevention measures to keep it at bay.

Environmental enrichment upregulates micro-RNA-183 and alters acetylcholinesterase splice variants to reduce anxiety-like behavior in the little Indian field mouse (Mus booduga).

Environmental enrichment (EE) has an influential role in reducing behavioral reactivity to stress. We previously observed that EE reduces the anxiety-like behavior in the field mouse Mus booduga accompanied by a reduction in the expression of molecules involved in the stress pathway. In this study, we demonstrate the effect of different housing condition on regulation of micro-RNA-183-SC35-mediated splicing of acetylcholinesterase (AChE). Adult male M. booduga were captured from an agricultural field and housed under nonenriched standard conditions (SC) for 7 days and considered as directly from the wild (DW). On day 8, individuals were randomly assigned to three groups; DW, SC, and EE. The DW group's anxiety-like behavior was assessed in the elevated plus maze (EPM) and open field test (OFT). The SC and EE groups were transferred to their respective conditions and housed for another 30 days. The mice housed in EE showed less anxiety-like behavior on EPM and in OFT compared with DW and SC mice. Interestingly, miR-183 expression was increased following exposure to EPM in EE mice but not in SC mice. Subsequently, the upregulated miR-183 expression suppresses the SC35 expression and shifting of splicing from AChE-S (synaptic) to AChE-R (read-through) form, whereas standard housing condition downregulate miR-183 and induces the splicing of AChE. The upregulated AChE-R form possibly terminates ACh transmission, which is reflected in the level of anxiety-like behavior. Overall, the present study suggests that EE effectively regulates the miR-183 pathway to reduce anxiety-like behavior.

Blunted Amphetamine-induced Reinforcing Behaviors and Transporter Downregulation in Knock-in Mice Carrying Alanine Mutations at Threonine-258 and Serine-259 of Norepinephrine Transporter.

Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.

Inhibition of monoamine oxidase attenuates social defeat-induced memory impairment in goldfish, (Carassius auratus): A possible involvement of synaptic proteins and BDNF.

Social defeat (SD) has been implicated in different modulatory effects of physiology and behaviour including learning and memory. We designed an experiment to test the functional role of monoamine oxidase (MAO) in regulation of synaptic transmission, synaptic plasticity and memory in goldfish Carassius auratus. To test this, individuals were divided into three groups: (i) control; (ii) social defeat (SD) group (individuals were subjected to social defeat for 10 min by Pseudotropheus demasoni) and (iii) SD + MAO inhibitor pre-treated group. All experimental groups were subjected to spatial learning and then memory. Our results suggest that SD affects a spatial learning and memory, whereas SD exerts no influence on MAOI pre-treated group. In addition, we noted that the expression of monoamine oxidase-A (MAO-A) was up-regulated and level of serotonin (5-hydroxytryptamine; 5-HT), expression of serotonin transporter (SERT), synaptophysin (SYP), synaptotagmin -1 (SYT-1), N-methyl-D-asparate (NMDA) receptors subunits (NR2A and NR2B), postsynaptic density-95 (PSD-95) and brain-derived neurotrophic factor (BDNF) were reduced by SD, while MAOIs pretreatment protects the effect of SD. Taken together, our results suggest that MAO is an essential component in the serotonergic system that finely tunes the level of 5-HT, which further regulates the molecules involving in synaptic transmission, synaptic plasticity and memory.

Novelty-induced hyperactivity and suppressed cocaine induced locomotor activation in mice lacking threonine 53 phosphorylation of dopamine transporter.

Dopamine (DA) transporter (DAT) is dynamically regulated by several protein kinases and the Thr53 phosphorylation of DAT (pT53-DAT) is documented in heterologous cell models and in rat brain. However, the role of endogenous pT53-DAT in living animals has never been addressed. Here we generated and studied the pT53-lacking DAT mouse model (DAT-Ala53) by CRISPR/Cas9 technology. DAT-Ala53 mice showed normal growth, body weight, body temperature, grip strength, and sucrose preference while pT53-DAT was completely absent. However, DAT-Ala53 mice showed hyperlocomotion, pronounced vertical exploratory behavior, and stereotypy in a novel environment compared to wild-type littermates (WT). DAT-Ala53 mice displayed unaltered levels of monoamines, glutamate, and GABA in the striatum compared to WT. There were also no significant differences between DAT-Ala53 mice and WT in tyrosine hydroxylase (TH) and phospho-TH levels, or in total and surface DAT levels, or in DA-transport kinetic parameters Vmax and Km. Immunohistochemical and colocalization analyses of TH and DAT in caudate-putamen and nucleus accumbens revealed no significant differences between DAT-Ala53 and WT mice. Interestingly, cocaine's potency to inhibit striatal DA transport and cocaine-induced locomotor activation were significantly reduced in the DAT-Ala53 mice. Also, ERK1/2 inhibitors completely failed to inhibit striatal DA uptake in DAT-Ala53 mice. Collectively, our findings reveal that the mice lacking pT53-DAT display novelty-induced hyperactive phenotype despite having normal transporter protein expression, DA-transport kinetics and DA-linked markers. The results also reveal that the lack of endogenous pT53-DAT renders DAT resistant to ERK1/2 inhibition and also less susceptible to cocaine inhibition and cocaine-evoked locomotor stimulation.

Anorexia induces a microglial associated pro-inflammatory environment and correlates with neurodegeneration in the prefrontal cortex of young female rats.

Dehydration-Induced Anorexia (DIA) is a murine model that reproduces weight loss and avoidance of food, despite its availability. The prefrontal cortex (PFC) integrates sensory inputs and updates associative learning to promote (hunger) or inhibit (satiety) food-seeking behavior. In this study we tested if anorexia induces a pro-inflammatory environment associated with microglia in the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC), specific subregions of the PFC involved in appetite. Our results showed that anorexia increased microglial density, promoted a de-ramified morphology and augmented the de-ramified/ramified ratio in the mPFC and OFC but not in the motor cortex. Anorexia also increased the expression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß. This pro-inflammatory environment associated with microglia activation correlates with neuronal damage as revealed by Fluoro Jade C (FJC) and NeuN immunolabeling. We conclude that anorexia triggers a pro-inflammatory environment associated with microglia that correlates with neurodegeneration in the mPFC and OFC.

Histamine Receptors Regulate the Activity, Surface Expression, and Phosphorylation of Serotonin Transporters.

Reuptake and clearance of released serotonin (5-HT) are critical in serotonergic neurotransmission. Serotonin transporter (SERT) is mainly responsible for clearing the extracellular 5-HT. Controlled trafficking, phosphorylation, and protein stability have been attributed to robust SERT activity. H3 histamine receptors (H3Rs) act in conjunction and regulate 5-HT release. H3Rs are expressed in the nervous system and located at the serotonergic terminals, where they act as heteroreceptors. Although histaminergic and serotonergic neurotransmissions are thought to be two separate events, whether H3Rs influence SERT in the CNS to control 5-HT reuptake has never been addressed. With a priori knowledge gained from our studies, we explored the possibility of using rat hippocampal synaptosomal preparations. We found that treatment with H3R/H4R-agonists immepip and (R)-(-)-α-methyl-histamine indeed resulted in a time- and concentration-dependent decrease in 5-HT transport. On the other hand, treatment with H3R/H4R-inverse agonist thioperamide caused a moderate increase in 5-HT uptake while blocking the inhibitory effect of H3R/H4R agonists. When investigated further, immepip treatment reduced the level of SERT on the plasma membrane and its phosphorylation. Likewise, CaMKII inhibitor KN93 or calcineurin inhibitor cyclosporine A also inhibited SERT function; however, an additive effect with immepip was not seen. High-speed in vivo chronoamperometry demonstrated that immepip delayed 5-HT clearance while thioperamide accelerated 5-HT clearance from the extracellular space. Immepip selectively inhibited SERT activity in the hippocampus and cortex but not in the striatum, midbrain, and brain stem. Thus, we report here a novel mechanism of regulating SERT activity by H3R-mediated CaMKII/calcineurin pathway in a brain-region-specific manner and perhaps synaptic 5-HT in the CNS that controls 5-HT clearance.

Anorexia increases microglial density and cytokine expression in the hippocampus of young female rats.

Anorexia by osmotic dehydration is an adaptive response to hypernatremia and hyperosmolaemia induced by ingestion of a hypertonic solution. Dehydration-induced anorexia (DIA) reproduces weight loss and avoidance of food, despite its availability. By using this model, we previously showed increased reactive astrocyte density in the rat dorsal hippocampus, suggesting a pro-inflammatory environment where microglia may play an important role. However, whether such anorexic condition increases a pro-inflammatory response is unknown. The aim of this study was to test if DIA increases microglial density in the dorsal hippocampus, as well as the expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1ß) in the hippocampus of young female rats. Our results showed that DIA significantly increased microglial density in CA2-CA3 and dentate gyrus (DG) but not in CA1. However, forced food restriction (FFR) only increased microglial density in the DG. Accordingly, the activated/resting microglia ratio was significantly increased in CA2-CA3 and DG, in DIA and FFR groups. Finally, western blot analysis showed increased expression of IBA1, TNF-α, IL-6 and IL-1ß in the hippocampus of both experimental groups. We conclude that anorexia triggers increased reactive microglial density and expression of TNF-α, IL-6 and IL-1ß; this environment may result in hippocampal neuroinflammation.

Environmental Enrichment Reduces Anxiety by Differentially Activating Serotonergic and Neuropeptide Y (NPY)-Ergic System in Indian Field Mouse (Mus booduga): An Animal Model of Post-Traumatic Stress Disorder.

Exposure to a predator elicits an innate fear response and mimics several behavioral disorders related to post-traumatic stress disorder (PTSD). The protective role of an enriched condition (EC) against psychogenic stressors in various animal models has been well documented. However, this condition has not been tested in field mice in the context of PTSD. In this study, we show that field mice (Mus booduga) housed under EC exhibit predominantly proactive and less reactive behavior compared with mice housed under standard conditions (SC) during exposure to their natural predator (field rat Rattus rattus). Furthermore, we observed that EC mice displayed less anxiety-like behavior in an elevated plus maze (EPM) and light/dark-box after exposure to the predator (7 hrs/7 days). In EC mice, predator exposure elevated the level of serotonin (5-Hydroxytrypamine, [5-HT]) in the amygdala as part of the coping response. Subsequently, the serotonin transporter (SERT) and 5-HT1A receptor were up-regulated significantly, but the same did not occur in the 5-HT2C receptor, which is associated with the activation of calmodulin-dependent protein kinase-II (CaMKII) and a transcription factor cAMP response element binding protein (CREB). Our results show that predator exposure induced the activation of CaMKII/CREB, which is accompanied with increased levels of histone acetylation (H3, H4) and decreased histone deacetylases (HDAC1, 2). Subsequently, in the amygdala, the transcription of brain-derived neurotrophic factor (BDNF), neuropeptide Y (NPY) and its Y1 receptor were up-regulated, whereas the Y2 receptor was down-regulated. Therefore, EC facilitated a coping response against a fear associated cue in a PTSD animal model and reduced anxiety by differentially activating serotonergic and NPY-ergic systems.

Egr-1 antisense oligodeoxynucleotide administration into the olfactory bulb impairs olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx.

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We examined two important examples of these, calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95, in regard to the functional role of early growth response gene-1 (egr-1) in regulation of olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx (family Pteropodidae). To test whether activation of egr-1 in the olfactory bulb (OB) is required for olfactory memory of these bats, bilaterally canulated individuals were infused with antisense (AS) or non-sense (NS)-oligodeoxynucleotides (ODN) of egr-1, or with phosphate buffer saline (PBS), 2h before the olfactory training. Our results showed that behavioral training significantly up-regulates immediate early gene (IEG) EGR-1 and key synaptic proteins Synaptotagmin-1(SYT-1), CaMKII and PSD-95, and phosphorylation of CaMKII in the OB at the protein level per se. Subsequently, we observed that egr-1 antisense-ODN infusion in the OB impaired olfactory memory and down regulates the expression of CaMKII and PSD-95, and the phosphorylation of CaMKII but not SYT-1. In contrast, NS-ODN or PBS had no effect on the expression of the PSDs CaMKII or PSD-95, or on the phosphorylation of CaMKII. When the egr-1 NS-ODN was infused in the OB after training for the novel odor there was no effect on olfactory memory. These findings suggest that egr-1 control the activation of CaMKII and PSD-95 during the process of olfactory memory formation.