Ersin O. Koylu

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. J. Comp. Neurol. 391:115–132, 1998.

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.

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. CARTs 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.

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. Synapse 27:90–94, 1997.

Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase

Cocaine and amphetamine regulated transcript (CART) encodes a novel brain‐enriched protein whose features are reminiscent of a neurotransmitter propeptide. We have now localized CART peptide(s) in the gastrointestinal tract by immunohistochemical methods. Polyclonal antisera raised to CART peptide 106–129 stained neuronal cell bodies and fibers in rat ileum myenteric plexus‐longitudinal muscle tissue preparations. Electron microscopic analysis of thin sections showed immunopositive axon terminals in close apposition to CART‐labelled and unlabelled neuronal cell bodies as well as to the longitudinal muscle. CART peptide‐immunoreactive terminals contained numerous ovoid electron‐lucent vesicles and a few dark‐stained dense‐core vesicles. Light microscopic double labelling studies revealed CART peptide immunoreactivity in a subpopulation of choline acetyltransferase (ChAT)‐immunoreactive neurons. This combined light microscopic and ultrastructural examination of CART peptides in the gastrointestinal tract suggests a role of CART peptides as transmitters or neuromodulators in the peripheral nervous system. Synapse 30:1–8, 1998.

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.

SEX DIFFERENCE IN UP-REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTORS IN RAT BRAIN

This study tested for sex differences in the effects of chronic nicotine administration and withdrawal on nicotinic acetylcholine receptor binding in brain. Rats received nicotine (0.6 mg/kg, s.c.) or saline once daily for 15 days, and were sacrificed 1 or 20 days after termination of treatment. Saturation studies of nAChR binding were performed using [3H]cytisine as the radioligand in whole brain minus cerebellum taken from animals in the chronic treatment groups and from naive rats. Male but not female rats that received chronic nicotine had higher receptor densities than corresponding control groups; up-regulation of nAChR was not seen 20 days after withdrawal. Furthermore, in groups that showed no up-regulation (controls and rats withdrawn for 20 days), nAChR densities were higher in female rats than males. The findings underscore the importance of sex differences in pharmacological responses as well as in basal neurochemical parameters.

Nicotinamide treatment reduces the levels of oxidative stress, apoptosis, and PARP-1 activity in Aβ(1-42)-induced rat model of Alzheimer's disease.

Abstract The underlying mechanisms of Alzheimers Disease (AD) are still unclear. It is suggested that poly(ADP-ribose) polymerase-1 (PARP-1) overactivation can cause neuroinflammation and cell death. In this study we searched the effects of nicotinamide (NA), endogenous PARP-1 inhibitor, on oxidative stress, apoptosis, and the regulation of PARP-1 and nuclear factor kappa B (NF-κB) in amyloid beta peptide (1–42) (Aβ(1–42))-induced neurodegeneration. Sprague–Dawley rats were divided into four groups as control, Aβ(1–42), Aβ(1–42) + NA(100 and 500 mg/kg). All groups were stereotaxically injected bilaterally into the hippocampus with Aβ(1–42) or saline. After surgery NA administrations were made intraperitoneally (ip) for 7 days. In order to investigate the effects of Aβ(1–42) and NA, protein carbonyls, lipid peroxidation, reactive oxygen species (ROS) production, glutathione (GSH) levels, activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), mitochondrial function, mRNA and protein levels of PARP-1, NF-κB, p53, Bax, and Bcl-2 were measured in specific brain regions such as cortex and hippocampus. Aβ(1–42) treatment only increased the oxidative stress parameters and caused decline in antioxidant enzyme activities, mitochondrial function, and GSH levels. Also, overexpression of PARP-1, NF-κB, p53, Bax, and the decreased levels of Bcl-2 were observed in Aβ(1–42)-treated group. NA treatments against Aβ(1–42)-upregulated Bcl-2 and downregulated PARP-1, NF-κB, p53, and Bax levels. NA treatments also decreased the oxidative stress parameters and elevated antioxidant enzyme activities, GSH levels, and mitochondrial function against Aβ(1–42) treatment. These data suggest that NA may have a therapeutic potential in neurodegenerative processes due to the decreased levels of oxidative stress, apoptosis, and PARP-1 activity.

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.