Burcu Balkan

Cocaine and amphetamine regulated transcript (CART) and the stress response

CART is expressed abundantly in the hypothalamic paraventricular nucleus and locus coeruleus, major corticotropin releasing factor (CRF) and noradrenaline sources, respectively. There is a bidirectional relation between CART and hypothalamo-pituitary-adrenal axis activity. CART stimulates CRF, adrenocorticotropic hormone and glucocorticoid secretion, whereas CRF and glucocorticoids increase the transcriptional activity of the CART gene; adrenalectomy declines CART expression in the hypothalamus. Stress exposure modulates CART expression in hypothalamus and amygdala in rat brain in a region and sex specific manner. CART may be a mediator peptide in the interaction between stress, drug abuse, and feeding. The review discusses the established role of CART as it relates to the stress response.

Sexually dimorphic cognitive style in rats emerges after puberty.

In a water maze (WM), rats employ different and sexually dimorphic behavioral strategies to solve a place-learning task, a test of cognitive/propositional ability. Puberty is the last step in brain development and marks an important phase with regard to sexually dimorphic cognitive performance and behavior. The present study assessed possible sex differences in cognitive style before and after puberty in a WM place-learning task. Since nitric oxide (NO) is implicated in spatial learning and hippocampal function, and since brain NO(-)(2) + NO(-)(3) levels (stable metabolites of NO) display region-specific sex differences in rat brain, NO(-)(2) + NO(-)(3) levels were determined after behavioral testing. The sex-related style difference emerged very clearly but only in the adult rats, which suggests that the female behavioral strategy in the WM place-learning task requires the presence of female sex hormones at puberty. Although NO(-)(2) + NO(-)(3) levels were higher in the adult rats and males compared to prepubertal and female rats, respectively, no significant correlations emerged between brain NO and behavior. The fact that the behavioral sexually dimorphic cognitive-style effect observed here and in previous studies appears to emerge only after puberty suggests that awareness of such postpubertal sex differences may also be important in human educational and therapeutic contexts.

The effect of adrenalectomy on cocaine and amphetamine-regulated transcript (CART) expression in the hypothalamic nuclei of the rat.

CART peptide is a neurotransmitter involved in various physiological processes including feeding, sensory processing, development, addiction, and stress. Substantial amounts of CART mRNA and CART peptide expression have been demonstrated in the hypothalamic periventricular area, the paraventricular nucleus of hypothalamus, the anterior lobe of the pituitary gland and the adrenal gland in addition to many other brain areas. This localization defines the HPA axis, responsible for the stress response. The aim of the present study was to assess the possible mediation of the CART peptides in the stress response by testing for changes in CART in adrenalectomized animals. Three groups of male Sprague-Dawley rats were used for the study: sham operated, adrenalectomized (ADX), and ADX+hormone replacement (corticosterone, 30 microg/ml in drinking water/5 days). All rats were perfused 7 days after the surgery, brains were removed and serial coronal sections were prepared. Immunohistochemistry was used to assess CART peptide expression in paraventricular and supraoptic cells. ADX lowered both the number and percentage of CART-positive cells compared to the sham-operated group, and hormone replacement partially restored the decrease in the CART cell numbers in ADX animals. There were no significant changes in the supraoptic nucleus. Our results suggest a role for CART peptides in the stress response.

Nicotine modulates nitric oxide in rat brain

Nicotine exerts its central actions by regulating cationic fluxes through nicotinic acetylcholine receptors (nAChRs). By this effect, the drug likely also modifies events occurring beyond the nAChR, including the regulation of nitric oxide (NO) synthesis. The present study was undertaken to assess the effects of acute and chronic nicotine administration (0.4 mg/kg, s.c.) on levels of NO(-)(2)+NO(-)(3), stable metabolites of NO, in brain regions of male and female rats. Nicotine increased levels of the metabolites, and therefore presumably of NO, with sex differences in the degree of stimulation, the brain regions affected, and the variance between the effects of acute and chronic administration. Prior inhibition of NO synthase eliminated the effect of nicotine in all regions studied. While nicotine appeared to increase NO indirectly via glutamate receptors in the cortex and hippocampus, this was not true of the corpus striatum, where blocking NMDA-type glutamate receptors with MK-801 had no effect. The findings support the view that NO is likely involved in some of the central effects of nicotine.

Effects of nitric oxide synthase inhibition on spatial discrimination learning and central DA2 and mACh receptors.

Cholinergic and dopaminergic systems are involved in spatial memory and are modulated by nitric oxide (NO); NO has well documented effects on place learning in rodents. The aim of the present study was to investigate the effect of NOS inhibition on place learning in the water maze and to evaluate the relationships between NOS inhibition, learning performance, dopamine (DA) D2 and muscarinic acetylcholine (mACh) receptors. Male Sprague-Dawley rats received the NOS inhibitor Nomega-Nitro-l-Arginine (l-NA), or saline and were trained in the water maze. Rats that were not trained, but received the same treatments were also included. Following treatments with or without water maze training, [3H]-QNB and [3H]-spiperone binding in cortex, striatum and hippocampus were determined to assess the effects of NOS inhibition and/or learning on DA D2 and mACh receptor regulation. The overall results of the present study showed that: (1) NOS inhibition impairs performance in the MWM; (2) NOS inhibition does not affect specific binding to DA D2 (striatum and hippocampus) and mACh (cortex and hippocampus) receptors; (3) MWM training lowers D2 and mACh receptor binding in cortical regions.

CART expression in limbic regions of rat brain following forced swim stress : Sex differences

Our previous studies showed the modulation of cocaine and amphetamine regulated transcript (CART) positive neurons and CART mRNA by adrenalectomy and corticosterone replacement in hypothalamic nuclei of male rat brain. More recently, we have shown by CART immunohistochemistry that restraint and forced swim (FS) stress have sexually dimorphic and regionally specific effects on CART expression in the hypothalamic nuclei of male and female Sprague-Dawley rats. This study aimed to evaluate the effects of FS stress on CART peptide expression in hypothalamus, amygdala and hippocampus of male and female (in or near estrus) Sprague-Dawley rats. Initially basal CART levels in regions of interest were determined in male and female rats; no sex differences were observed. In FS test, rats were forced to swim on two consecutive days, in a Plexiglas cylinder for 15 and 6 min, respectively. Rats were decapitated on the second day, 10 min after the stress procedure. Hypothalami, amygdalae and hippocampi were dissected and homogenized. CART peptide expression in these regions was measured by Western blotting. In males, FS increased CART expression in hypothalamus and amygdala. On the other hand, in females, FS lowered CART expression in amygdala. CART expression in hippocampus was not affected by the stress procedure in either sex. Our results suggest sexually dimorphic modulation of CART expression in hypothalamus and amygdala by FS procedure. Although modulation of the CART peptide by glucocorticoids and gonadal hormones appears likely, future studies are needed to elucidate the underlying mechanisms in the involvement of CART peptide in stress response.

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.

Co-localization of cart peptide immunoreactivity and nitric oxide synthase activity in rat hypothalamus

Because of the reported presence of both CART peptide and NOS activity in the same hypothalamic nuclei, their colocalization was examined. Eighteen percent of the neurons in the supraoptic nuclei, and 16% of the neurons in the paraventricular nucleus contained both CART immunoreactivity and NOS activity. Many other neurons in these regions stained for only one marker although they were often close by. Thus, CART peptides and NO may interact in these regions.

Effects of chronic nicotine administration on nitric oxide synthase expression and activity in rat brain

Although there is substantial evidence concerning the influence of nicotine on nitric oxide (NO) synthesis in the vascular system, there are fewer studies concerning the central nervous system. Although NO metabolites (nitrates/nitrites) increase in several rat brain regions after chronic injection of nicotine, the cellular origin of this rise in NO levels is not known. The aim of the present work was to assess the effects of repetitive nicotine administration on nitric oxide synthase (NOS) expression and activity in male and female rat brains. To determine levels of nitrate/nitrite, the Griess reaction was carried out in tissue micropunched from the frontal cortex, striatum, and accumbens of both male and female rats untreated (naïve) or injected with saline or nicotine (0.4 mg/kg for 15 days). In parallel, coronal sections of fixed brains from equally treated animals were immunostained for neuronal NOS or histochemically labelled for NADPH‐diaphorase activity. Nicotine treatment increased NO metabolites significantly in all brain regions compared with naïve or saline‐treated rats. By contrast, analysis of the planimetric counting of NOS/NADPH‐diaphorase‐positive neurons failed to demonstrate any significant effect of the nicotine treatment. A significant decrease was observed with both techniques employed in saline‐injected female rats compared with naïve animals, suggesting a stress response. The mismatch between the biochemical and the histological data after chronic nicotine treatment is discussed. The up‐regulation of NO sources other than neurons is proposed.

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. Synapse 67:545–552, 2013.