Cocaine- and amphetamine-regulated transcript promoter regulated by nicotine in nerve growth factor-treated PC12 cells.

Nicotine and cocaine- and amphetamine-regulated transcripts (CART) have several overlapping functions, such as the regulation of reward, feeding behavior, stress response, and anxiety. Previous studies showed that nicotine regulates CART expression in various brain regions. However, the molecular mechanisms underlying this regulation are not known. This study investigated the regulatory effect of nicotine on promoter activity of the CART gene in PC12 cells, which were differentiated into a neuronal phenotype by nerve growth factor (NGF) treatment. Two vectors containing reporter genes (Gaussia luciferase or mCherry) and the 1,140-bp upstream of the transcriptional start site of the mouse CART gene are used to analyze the CART promoter activity. Transient transfection of PC12 cells with either vector displayed strong promoter activity in both undifferentiated and differentiated PC12 cells. CART promoter activity in the PC12 cell line is increased by forskolin or NGF treatment. In differentiated PC12 cells, exposure to 50 nM nicotine for 6 h increased CART promoter activity. However, treatment with higher nicotine doses for 6 h and treatment with all nicotine doses for 24 h showed no effect. A nicotine concentration of 50 nM is comparable to brain nicotine levels experienced by chronic smokers over long periods of time. Taken together, these data indicate that nicotine may exert some of its actions through the regulation of CART transcription in the brain.

Effects of laterality and sex on cognitive strategy in a water maze place learning task and modification by nicotine and nitric oxide synthase inhibition in rats.

The aim of the present study was to investigate sex differences in learning strategies and to elucidate the mechanisms, which may underlie these differences. In two separate experiments, rats were presented with different strategies that could be employed to learn the position of a platform in a water maze (WM); furthermore, rats received treatments that could influence these strategies. In the first experiment, we demonstrated that the response-learning paradigm can be applied to the WM and can be compared with visually cued learning and reversal learning. Naïve rats of either sex could acquire this protocol relatively easily. On the probe trial, where the rats are presented with a choice between using response versus visually cued learning, initially response learning was preferred, however, during these experiments, laterality emerged as a significant factor and rats trained to turn right had difficulty in reversing the learned pattern to find the platform. The second part of our study evaluated the effects of nicotine and nitric oxide synthase (NOS) inhibition on the aforementioned parameters. Drug treatments impaired acquisition compared to saline treatments and the effect was more pronounced with NOS inhibition. During the probe trial, while NOS inhibition enhanced the right-side bias in both sexes, nicotine treatment had the same effect only in males. In conclusion, naïve rats can acquire place learning using visible cues or response learning; however, there is a right side bias in both sexes and the laterality effect is more pronounced in male rats. In drug-treated animals, while NOS inhibition enhances laterality (right bias) in both sexes similarly, nicotine modifies the cognitive strategy in a sexually dimorphic manner by augmenting the right bias only in male rats.

The effect of nitric oxide synthase inhibition on cognitive ability and strategies employed for place learning in the water maze: sex differences.

Male and female rats use different cognitive strategies in the solution of place-learning problems in the water maze despite similar abilities. The female-type strategy has been negatively correlated with cortical nitric oxide (NO) metabolites. The present study aimed to examine the effect of NO synthase (NOS) inhibition (N(omega)-nitro-L-arginine, L-NA) on cognitive ability and strategy in the water maze, and to evaluate possible sex differences. In a 2 (male versus female) x2 (L-NA versus saline) factorial design, rats were trained to find the platform (visible or hidden), always in the same position, for 12 days. L-NA impaired acquisition, during the earlier phases and more prominently in females. This impairment was quite dramatic and unique to females during the first day that the platform was hidden following 3 days of visible-platform conditions. After acquisition, the visible platform's position was shifted, thereby presenting the rats with a choice (searching for the hidden platform in the previous location, i.e. adopting a conceptual cognitive style, or escaping to the visible platform in a new position, i.e. adopting a perceptual style). On the first of the four shift trials (where the newly positioned platform was proximal to the rat's starting position), female rats showed the previously found tendency to adopt a perceptual style escape directly in clear contrast to saline-treated males. The L-NA-treated males tended to manifest female-like perceptual style, suggesting that inhibition of NO synthesis in males weakened the tendency to choose a conceptual style in this shifted-platform task. The role of NO in both cognitive and non-cognitive psychological functions is discussed.

The effect of octreotide on kainate-induced wet dog shakes and seizure activity in male and female rats.

Systemic kainic acid (KA) administration to rats triggers wet dog shakes (WDS) followed by epileptic seizures. Although WDS are often associated with the occurrence of seizures, we have recently shown that following nitric oxide (NO) synthesis inhibition, the number of WDS decreased; subsequently the onset of seizure activity was shortened, and the number of convulsions was increased. Somatostatin (SS), whose release appears to be controlled by NO, inhibits seizure activity. There are sex differences in seizure susceptibility as well as in SS and NO activities in brain. The present study was undertaken to assess the effect of octreotide (OC), a stable SS analogue, on KA-induced WDS and seizures in rats, with emphasis on possible sex differences. WDS and seizures were induced by KA in male and female (proestrus) Sprague Dawley rats; OC or saline was injected 30 min before KA and the behavior was monitored for 120 min after KA. Octreotide increased the number of WDS and decreased the number of convulsions; this effect was more pronounced in males. Onset of KA-induced seizure activity was earlier in females than males; however, there was no effect of OC on seizure latency. Seizure activity started after the termination of WDS. These results show OC has opposite effects on WDS and convulsions, in that it stimulates the former and inhibits the latter. These results support our previous findings that WDS and seizure activity involve separate mechanisms and suggest that WDS may have an inhibitory effect on limbic seizures.

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.

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.

Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain.

Cocaine- and amphetamine-regulated transcript (CART) is a brain-enriched mRNA with a protein product(s) that is a candidate brain neurotransmitter. We have developed antisera to CART peptide fragment 106-129 and have demonstrated specific immunoreactivity (IR) at the light microscopic level throughout the brain, spinal cord, and retina. All brain nuclei previously shown to express CART mRNA are now shown to contain CART peptide IR. Although it is premature to define CART peptide(s) as a neurotransmitter(s), the localization found here suggests an involvement of CART in many processes. CART peptide staining in the nucleus accumbens and basolateral amygdala continue to suggest a role in drug-induced reward and reinforcement. Staining in the olfactory bulbs, the cortical barrels, the retina and its projection areas, the thalamic nuclei, the lateral and dorsal horns of the spinal cord, and the nuclei of the solitary tract are compatible with a major role for CART in sensory processing and autonomic regulation. CART peptides appear to colocalize with some classical neurotransmitters and appear to occur in peripheral neurons as well.

Ultrastructural localization of CART (cocaine- and amphetamine-regulated transcript) peptides in the nucleus accumbens of monkeys.

CART (cocaine- and amphetamine-regulated transcript) peptides are proposed to play a role in the action of psychostimulants as neurotransmitters/neuromodulators. In the present study, we demonstrate that the shell of the nucleus accumbens, a brain structure involved in drug reinforcement, is densely innervated by a dense plexus of CART peptide-immunoreactive varicose fibers in register with immunoreactive perikarya in monkeys. At the electron microscopic level, varicosities appeared as immunoreactive axon terminals packed with round electron-lucent vesicles and a variable number of darkly stained dense-core vesicles that formed symmetric synapses with dendrites. These findings suggest that CART peptides may be a cotransmitter with gamma aminobutyric acid (GABA) in intrinsic axon collaterals of striatal projection neurons or interneurons in the primate nucleus accumbens.

Further studies on the anatomical distribution of CART by in situ hybridization.

CART (cocaine and amphetamine regulated transcript) is a novel, brain-enriched mRNA which predicts a novel protein without homology to any known protein or peptide. In situ hybridization studies have identified many expression sites in the brain and periphery as well as clarify its expression in three known areas. CART mRNA has been localized to ganglion cells of the retina, lamina X of the spinal cord, mitral and tufted cells of the olfactory bulb, barrel field neurons of the somatosensory cortex, the anterior pituitary, and the medulla of the adrenal cortex. The two alternatively spliced CART variants present in the rat brain were found to have identical and overlapping distributions in the rat forebrain. This central nervous system expression pattern suggests a role for CART in processing of peripheral sensory information. Its localization within the pituitary completes its identification within the three levels of the hypothalamic-pituitary-adrenal axis and perhaps suggests a role in mediating stress responses. CART's distribution and predicted protein sequence is reminiscent of characteristics shared by many brain neuropeptides such as proopiomelanocortin; CART may encode a new peptide transmitter or signaling molecule.

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland.

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.