Enhanced Novel Object Recognition and Spatial Memory in Rats Selectively Bred for High Nicotine Preference.

This study examined the influence of genetic background on cognitive performance in a selectively bred high nicotine-preferring (NP) rat line. Using the novel object recognition (NOR), novel location recognition (NLR), and Morris water maze (MWM) tests, we evaluated object memory, spatial memory, and spatial navigation in nicotine-naive NP rats compared to controls. Our results demonstrate that in the NOR test, both male and female NP rats spent more time exploring the novel object (higher discrimination index) compared to sex-matched controls. In the NLR, the discrimination index differed significantly from zero chance (no preference) in both NP males and females but not in controls, indicating enhanced spatial memory in the NP line. During MWM acquisition, the NP groups and control males took a shorter path to reach the platform compared to control females. On the probe trial, the distance traveled in the target quadrant was longer for NP males and females compared to their respective controls, suggesting enhanced spatial navigation and learning in the NP rats. The interesting preference for novel objects and locations displayed by NP rats may indicate a potential novelty-seeking phenotype in this line. These results highlight the complex interplay between genetic factors, cognitive function, and nicotine preference.

Chronic Nicotine Consumption and Withdrawal Regulate Melanocortin Receptor, CRF, and CRF Receptor mRNA Levels in the Rat Brain.

Alterations in the various neuropeptide systems in the mesocorticolimbic circuitry have been implicated in negative effects associated with drug withdrawal. The corticotropin-releasing factor (CRF) and α-melanocyte-stimulating hormone are two peptides that may be involved. This study investigated the regulatory effects of chronic nicotine exposure and withdrawal on the mRNA levels of melanocortin receptors (MC3R, MC4R), CRF, and CRF receptors (CRFR1 and CRFR2) expressed in the mesocorticolimbic system. Rats were given drinking water with nicotine or without nicotine (control group) for 12 weeks, after which they continued receiving nicotine (chronic exposure) or were withdrawn from nicotine for 24 or 48 h. The animals were decapitated following behavioral testing for withdrawal signs. Quantitative real-time PCR analysis demonstrated that nicotine exposure (with or without withdrawal) increased levels of CRF and CRFR1 mRNA in the amygdala, CRF mRNA in the medial prefrontal cortex, and CRFR1 mRNA in the septum. Nicotine withdrawal also enhanced MC3R and MC4R mRNA levels in different brain regions, while chronic nicotine exposure was associated with increased MC4R mRNA levels in the nucleus accumbens. These results suggest that chronic nicotine exposure and withdrawal regulate CRF and melanocortin signaling in the mesocorticolimbic system, possibly contributing to negative affective state and nicotine addiction.

Decreased anxiety-like behavior in a selectively bred high nicotine-preferring rat line.

Genetic vulnerability contributes significantly to the individual variability observed in nicotine dependence. Selective breeding for sensitivity to a particular effect of abused drugs has produced rodent lines useful for studying genetic vulnerability to drug addiction. Previous research showed that anxiety-related personality traits are associated with nicotine dependence. Therefore, we examined the differences in anxiety-like behavior between a high nicotine-preferring rat line and their controls. At the beginning of the study, all rats, naïve to any drug, were exposed sequentially to open field arena, marble-burying and elevated plus-maze paradigms. In the second step, all rats received nicotine in drinking water for 7 weeks. Behavioral tests were rerun on the final 2 weeks of chronic nicotine treatment. Elevated plus-maze testings under basal condition and during chronic nicotine treatment showed that the time spent on the open arms, preference for being in the open arms, and the latency to enter the closed arms were higher, whereas open arm avoidance index was lower in nicotine-preferring rats compared to the controls. In the open field test, nicotine-preferring rats spent longer time in the central zone and excreted less fecal pellets; they buried less marbles in the marble-burying test. These findings indicate a lower level of anxiety-like behavior in nicotine-preferring rat line under basal conditions and during chronic nicotine treatment. We conclude that lower anxiety level in nicotine-preferring rat line is consistent with novelty-seeking personality type and may increase vulnerability to nicotine dependence in this rat line.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein.

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Chronic oral nicotine administration and withdrawal regulate the expression of neuropeptide Y and its receptors in the mesocorticolimbic system.

Neuropeptide Y (NPY) and its receptors are involved in the regulation of mood, stress, and anxiety. In parallel, NPY signaling may play a vital role in the negative affective state induced by drug withdrawal. This study examined the changes in the transcript levels of NPY, Y1, Y2, and Y5 receptors in the mesocorticolimbic system during chronic nicotine exposure and withdrawal. Rats were administered with nicotine (initial dose: 25 µg/ml, maintenance dose: 50 µg/ml, free base) in drinking water for 12 weeks. Control group received only tap water. In the final week of the study, some of the nicotine-treated animals continued to receive nicotine (0-W), whereas some were withdrawn for either 24 (24-W) or 48 (48-W) h. All animals were decapitated after the evaluation of somatic signs (frequency of gasps, eye blinks, ptosis, shakes, teeth chatter) and the duration of locomotor activity and immobility. mRNA levels of NPY, Y1, Y2, and Y5 receptors in the mesocorticolimbic system were measured by quantitative real-time PCR (qRT-PCR). Results showed that nicotine withdrawal increased overall somatic signs. Moreover, chronic nicotine treatment increased the duration of locomotor activity, whereas withdrawal increased the duration of immobility. qRT-PCR analysis revealed that chronic nicotine treatment increased NPY mRNA levels in the hippocampus. On the other hand, 24- and 48-h withdrawals increased NPY mRNA levels in the amygdala and medial prefrontal cortex (mPFC), Y1 and Y2 mRNA levels in the nucleus accumbens and mPFC, and Y5 mRNA levels in the mPFC. These findings suggest that nicotine withdrawal enhances NPY signaling in the mesocorticolimbic system, which could be an important mechanism involved in regulating the negative affective state triggered during nicotine withdrawal.

Divergent Modulation of Proteostasis in Prostate Cancer.

Proteostasis regulates key cellular processes such as cell proliferation, differentiation, transcription, and apoptosis. The mechanisms by which proteostasis is regulated are crucial and the deterioration of cellular proteostasis has been significantly associated with tumorigenesis since it specifically targets key oncoproteins and tumor suppressors. Prostate cancer (PCa) is the second most common cause of cancer death in men worldwide. Androgens mediate one of the most central signaling pathways in all stages of PCa via the androgen receptor (AR). In addition to their regulation by hormones, PCa cells are also known to be highly secretory and are particularly prone to ER stress as proper ER function is essential. Alterations in various complex signaling pathways and cellular processes including cell cycle control, transcription, DNA repair, apoptosis, cell adhesion, epithelial-mesenchymal transition (EMT), and angiogenesis are critical factors influencing PCa development through key molecular changes mainly by posttranslational modifications in PCa-related proteins, including AR, NKX3.1, PTEN, p53, cyclin D1, and p27. Several ubiquitin ligases like MDM2, Siah2, RNF6, CHIP, and substrate-binding adaptor SPOP; deubiquitinases such as USP7, USP10, USP26, and USP12 are just some of the modifiers involved in the regulation of these key proteins via ubiquitin-proteasome system (UPS). Some ubiquitin-like modifiers, especially SUMOs, have been also closely associated with PCa. On the other hand, the proteotoxicity resulting from misfolded proteins and failure of ER adaptive capacity induce unfolded protein response (UPR) that is an indispensable signaling mechanism for PCa development. Lastly, ER-associated degradation (ERAD) also plays a crucial role in prostate tumorigenesis. In this section, the relationship between prostate cancer and proteostasis will be discussed in terms of UPS, UPR, SUMOylation, ERAD, and autophagy.

Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied.

Gene expression of pro-opiomelanocortin and melanocortin receptors is regulated in the hypothalamus and mesocorticolimbic system following nicotine administration.

Pro-opiomelanocortin (POMC)-derived peptides and their receptors have been shown to play important roles in natural and drug-induced reward and reinforcement. Reward process may involve the regulation of POMC gene expression and the gene expression of POMC-derived peptide receptors. The present study investigated the alterations observed in the transcript levels of POMC, melanocortin 3 (MC3R), melanocortin 4 (MC4R) and mu-opioid receptors (MOR) in the hypothalamus and mesocorticolimbic system during nicotine exposure. Rats were injected subcutaneously for 5days with one of the three doses (0.2, 0.4 or 0.6mg/kg/day, free base) of nicotine and were decapitated one hour after a challenge dose on the sixth day. mRNA levels of POMC in the hypothalamus, MC3R in the ventral tegmental area (VTA), MC4R and MOR in the medial prefrontal cortex (mPFC), nucleus accumbens, dorsal striatum, amygdala, lateral hypothalamic area and VTA were measured by quantitative real-time PCR. Our results showed that treatment with 0.6mg/kg/day nicotine upregulated POMC mRNA in the hypothalamus and MC4R mRNA in the mPFC. Additionally, all three nicotine doses increased MC3R mRNA expression in the VTA. On the other hand, none of the nicotine doses altered MOR mRNA levels in the mesocorticolimbic system and associated limbic structures. These results suggest that nicotine may enhance melanocortin signaling in the mesocorticolimbic system and this alteration may be an important mechanism mediating nicotine reward.

Nicotinic cholinergic and dopaminergic receptor mRNA expression in male and female rats with high or low preference for nicotine.

BACKGROUND: Nicotine exerts its central actions through nicotinic acetylcholine receptors (nAChRs), which in turn regulate major neurotransmitter systems including dopamine. Nicotinic and dopaminergic systems play significant roles in physiological functions, neuropsychiatric disorders, and addiction. OBJECTIVES: To evaluate possible differences in the expression of nAChR subunit and dopamine receptor (DR) mRNAs following voluntary nicotine intake. METHODS: Male and female rats (n = 67) were exposed to long-term free-choice oral nicotine (24 hours/day, 6 weeks); rats with maximum and minimum nicotine preference/intake were selected. The mRNA levels of genes encoding α4,ß2,α5, and α7 nAChR subunits and DR Drd1and Drd2 subtypes were evaluated in the striatum (STR), prefrontal cortex (PFC), and hippocampus using quantitative real-time polymerase chain reaction in selected rats (n = 30) and their control groups (n = 15). RESULTS: In addition to baseline differences, expression changes were observed in the mRNA levels of evaluated genes in rats exposed to voluntary oral nicotine in a brain region-, sex-, and preference-related manner. Nicotine intake is correlated negatively with Chrnb2, Chrna7 and positively with Drd1 expression. In the cholinergic system, regional differences in Chnrb2 and Chrna5, sex differences in Chrna4 and Chrna5, and nicotine preference effects in the expression of all subunits except α4 were observed. Chrna5 was lower in maximum than in minimum preferring, and in male than female rats, supporting the inhibitory role of the α5 subunit in nicotine dependence. Nicotine increased Drd2 mRNA expression only in minimum preferring female rats in STR and PFC. CONCLUSION: Modulation of nAChR and DR gene expression by nicotine may have clinical implications and aid drug development. Pharmaceuticals targeting the nicotinic cholinergic and dopaminergic systems might be expected to have differential efficacy that varies with the patient's sex or smoking status.

Nicotine regulates cocaine-amphetamine-Regulated Transcript (Cart) in the mesocorticolimbic system.

Cocaine-and-Amphetamine Regulated Transcript (CART) mRNA and peptides are intensely expressed in the brain regions comprising mesocorticolimbic system. Studies suggest that CART peptides may have a role in the regulation of reward circuitry. The present study aimed to examine the effect of nicotine on CART expression in the mesocorticolimbic system. Three different doses of nicotine (0.2, 0.4, 0.6 mg/kg free base) were injected subcutaneously for 5 days, and on day 6, rats were decapitated following a challenge dose. CART mRNA and peptide levels in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum (DST), amygdala (AMG), lateral hypothalamic area (LHA), and ventral tegmental area (VTA) were measured by quantitative real-time PCR (qPCR) and Western Blot analysis, respectively. In the mPFC, 0.4 and 0.6 mg/kg nicotine, decreased CART peptide levels whereas there was no effect on CART mRNA levels. In the VTA, a down-regulation of CART peptide expression was observed with 0.2 and 0.6 mg/kg nicotine. Conversely, 0.4 and 0.6 mg/kg nicotine increased CART mRNA levels in the AMG without affecting the CART peptide expression. Nicotine did not regulate CART mRNA or CART peptide expression in the NAc, DST, and LHA. We conclude that nicotine regulates CART expression in the mesocorticolimbic system and this regulation may play an important role in nicotine reward. Synapse 70:283-292, 2016. © 2016 Wiley Periodicals, Inc.

The epigenetic effect of nicotine on dopamine D1 receptor expression in rat prefrontal cortex.

Nicotine is a highly addictive drug and exerts its effect partially through causing dopamine release, thereby increasing intrasynaptic dopamine levels in the brain reward systems. Dopaine D1 receptor (DRD1) mRNAs and receptors are localized in reward-related brain regions, which receive cholinergic input. The aim of this study is to evaluate whether nicotine administration affects the expression of DRD1s, and if so, whether epigenetic mechanisms, such as histone acetylation, are involved. Twenty Male Sprague Dawley rats received nicotine (0.4 mg/kg/day, s.c.) or saline injections for 15 days. After nicotine/saline treatment, rats were perfused with saline; prefrontal cortex (PFC), corpus striatum (STR), and ventral tegmental area (VTA) were dissected. Homogenates were divided into two parts for total RNA isolation and histone H4 acetylation studies. DRD1 mRNA expression was significantly higher in the PFC of the nicotine-treated group compared with controls; similar trends were observed in the VTA and STR. To study epigenetic regulation, the 2kb upstream region of the DRD1 gene promoter was investigated for histone H4 acetylation in PFC samples. After chromatin immunoprecipitation with anti-acetyl histone H4 antibody, we found an increase in histone acetylation by two different primer pairs which amplified the -1365 to -1202 (P < 0.005) and -170 to +12 (P < 0.05) upstream regions of the DRD1 promoter. Our results suggest that intermittent subcutaneous nicotine administration increases the expression of DRD1 mRNA in the PFC of rats, and this increase may be due to changes in histone H4 acetylation of the 2kb promoter of the DRD1 gene.

A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.

p97/VCP is a hexameric AAA type ATPase that functions in a variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, autophagy and cell-cycle regulation. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder which has been attributed to mutations in p97/VCP. Several missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Here, we analyzed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Intriguingly, although almost all mutants exhibit enhanced p47 and Ufd1-Npl4 binding, the P137L mutation completely abolishes p97/VCP interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding. While recombinant R155C mutant protein consistently interacts with both Ufd1 and VIM of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. The differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD.

Region- and sex-specific changes in CART mRNA in rat hypothalamic nuclei induced by forced swim stress.

Cocaine and amphetamine regulated transcript (CART) mRNA and peptides are highly expressed in the paraventricular (PVN), dorsomedial (DMH) and arcuate (ARC) nuclei of the hypothalamus. It has been suggested that these nuclei regulate the hypothalamic-pituitary-adrenal (HPA) axis, autonomic nervous system activity, and feeding behavior. Our previous studies showed that forced swim stress augmented CART peptide expression significantly in whole hypothalamus of male rats. In another study, forced swim stress increased the number of CART-immunoreactive cells in female PVN, whereas no effect was observed in male PVN or in the ARC nucleus of either sex. In the present study, we evaluated the effect of forced swim stress on CART mRNA expression in PVN, DMH and ARC nuclei in both male and female rats. Twelve male (stressed and controls, n=6 each) and 12 female (stressed and controls, n=6 each) Sprague-Dawley rats were used. Control animals were only handled, whereas forced swim stress procedure was applied to the stressed groups. Brains were dissected and brain sections containing PVN, DMH and ARC nuclei were prepared. CART mRNA levels were determined by in situ hybridization. In male rats, forced swim stress upregulated CART mRNA expression in DMH and downregulated it in the ARC. In female rats, forced swim stress increased CART mRNA expression in PVN and DMH, whereas a decrease was observed in the ARC nucleus. Our results show that forced swim stress elicits region- and sex-specific changes in CART mRNA expression in rat hypothalamus that may help in explaining some of the effects of stress.

Efects of hormone replacement and tamoxifen on depression in ovariectomized rats.

OBJECTIVES: To determine the effects of tamoxifen and hormone replacement therapy in order to assess their role in depressive behavior: MATERIAL AND METHODS: Different protocols of hormone replacement therapies were administered to surgically ovariectomized rats. Intact rats were used for tamoxifen experiments. Properly assigned control groups were used and cognitive processes were studied on animal models of surgical menopause using the Porsolt forced swim test and locomotor activity experiments. RESULTS: In the tamoxifen experiments, an interaction between treatment and days did not reach statistical significance, but indicated a trend in this direction [F(1,26)=3.557, p = 0.071]. The number of repeated movements significantly decreased after the Porsolt test (F(1,44) = 8.483, P < 0.006) in the hormone replacement experiments. In the tamoxifen experiments, the number of repeated movements significantly decreased after the Porsolt test (F(1,26) = 74.410, P < 0.001). CONCLUSIONS: While sequential hormone replacement is found to be protective against depression, tamoxifen seems to augment behavioral despair

Electrochemical based detection of microRNA, mir21 in breast cancer cells.

In this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3' end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin-avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (α-NAP, the product). α-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from α-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6 pmol for cell lysates.

Forced swim stress elicits region-specific changes in CART expression in the stress axis and stress regulatory brain areas.

CART mRNA and peptides are highly expressed in the anatomical structures composing the hypothalamo-pituitary-adrenal (HPA) axis and sympatho-adrenal system. Anatomical and functional studies suggest that CART peptides may have a role in the regulation of the neuroendocrine and autonomic responses during stress. Our previous study showed that CART peptides increased significantly in the male hypothalamus and amygdala 10min after the forced swim stress. The present study aimed to examine the effect of forced swim stress on CART peptide expression in selected brain regions, including those where CART peptide expression has not been reported before (frontal cortex, pons, medulla oblongata), as well as in endocrine glands related to stress in male Sprague Dawley rats. A total of 16 (n=8) animals were used, including control groups. Rats were subjected to forced swim on two consecutive days, and sacrificed on the second day, 2h after the termination of the stress procedure. Frontal cortex, pons, medulla oblongata, hypothalamus, pituitary and adrenal glands were dissected and homogenized. CART peptide expression in these tissues was measured by Western Blotting and six different CART peptide fragments were identified. Our results showed that forced swim stress elicited region-specific changes in CART peptide expression. CART was upregulated in the frontal cortex, hypothalamus, medulla oblongata and adrenal gland while there was no change in the pons and pituitary. Enhanced CART peptide fragments in these brain regions and adrenal glands may have a role in the regulation of the HPA and sympatho-adrenal axis activity during stress response.

Hippocampal neuronal nitric oxide synthase (nNOS) is regulated by nicotine and stress in female but not in male rats.

NO (nitric oxide) produced in limbic brain regions has important roles in the regulation of autonomic nervous system and HPA axis activity, anxiety, fear learning, long-term memory formation, and depression. NO is synthesized from l-arginine in a reaction catalyzed by nitric oxide synthase (NOS). Neuronal nitric oxide synthase (nNOS), one of the three isoforms of NOS, is synthesized constitutively in nerve cells. Increasing evidence indicates that nNOS expression in the nervous system may be regulated by stress and nicotinic receptors. Furthermore, data obtained from several studies suggest that signaling pathways induced by stress and nicotinic receptors may converge on various signal transduction molecules to regulate nNOS expression in brain. In the present study, we used Western Blot analysis to test the effect of forced swim stress, chronic nicotine administration, and the combined effect of both procedures on nNOS expression in the hippocampus, amygdala and frontal cortex of the male and female rat brain. Basal nNOS levels of the three brain regions examined did not show sex differences. However, forced swim stress and chronic nicotine administration increased nNOS expression in the hippocampus of female rats. When stress and nicotine were applied together, no additional increment was observed. Stress and nicotine did not regulate nNOS expression in the amygdala and the frontal cortex of either sex. Data obtained from the present study indicate that the regulation of stress and nicotine induced-nNOS expression in rat hippocampus shows sexual dimorphism and nNOS expression in the female rat hippocampus increases by nicotine and stress.

Epigenetic regulation of neuron-dependent induction of astroglial synaptic protein GLT1.

Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.

Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.

The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia regulate synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. Astroglial mechanisms underlying this essential neuron-glial communication are not known. We now show that presynaptic terminals regulate astroglial synaptic functions, GLT1/EAAT2, via kappa B-motif binding phosphoprotein (KBBP), the mouse homolog of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the GLT1/EAAT2 promoter. Neuron-stimulated KBBP is required for GLT1/EAAT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling by corticospinal tract transection, ricin-induced motor neuron death, or neurodegeneration in amyotrophic lateral sclerosis all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.