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WOS: 000310414300006, PubMed ID: 22960117, 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. (C) 2012 Elsevier B.V. All rights reserved., NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [3 R01 DA010732-05S1]; National Center for Research ResourcesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR) [P51RR165]; Office of Research Infrastructure Programs/OD [P51OD11132], This study was supported by NIH Grant no. 3 R01 DA010732-05S1 and by the National Center for Research Resources (P51RR165) and is currently supported by the Office of Research Infrastructure Programs/OD (P51OD11132).

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 10 min 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, 2 h 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.

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 μg/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.

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.

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.

Sodium nitroprusside (SNP), a generator of nitric oxide (NO), decreased [3H]dopamine uptake into rat striatal synaptosomal preparations in a dose-, time- and temperature-dependent fashion. Reduced hemoglobin, a substance that binds NO, prevented the SNP-induced decrease in uptake. Potassium ferri- and ferrocyanide, compounds similar to SNP that do not produce NO, were without effect on uptake. SNP inhibited [3H]dopamine uptake in synaptosomes from nucleus accumbens and olfactory tubercle as well but with a lower potency. SNP inhibited [3H]serotonin and [3H]glutamate uptake but had no effect on [3H]norepinephrine uptake. S-Nitroso-N-acetylpenicillamine (SNAP), another generator of NO, had effects similar to those of SNP. The SNP-induced decrease in [3H]dopamine uptake was due to a Vmax decrease at 100 μM SNP and to both a Vmax and Km change at 300 μM SNP. Depletion of calcium by omission of calcium from buffers and addition of EGTA increased the potency of SNP in inhibiting uptake. There was no change in [3H]WIN 35428 binding to the dopamine transporter with doses of SNP that inhibited uptake. These data indicate that NO can decrease [3H]dopamine transporter function.