Vesicular glutamate transporter 2 expression in the ventral tegmental area of outbred male rats following exposure to nicotine and alcohol.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Expression and regulation of CYP17A1 and 3ß-hydroxysteroid dehydrogenase in cells of the nervous system: Potential effects of vitamin D on brain steroidogenesis.

Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). 3ß-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3ß-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3ß-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3ß-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3ß-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3ß-HSD, particularly on the transcriptional level, may play a role in the nervous system.

Reversal of opiate-induced apoptosis by human recombinant growth hormone in murine foetus primary hippocampal neuronal cell cultures.

Previous studies have shown that chronic opiates may inhibit cell growth and trigger apoptosis leading to impaired cognitive capabilities in both humans and other mammals. In contrast, growth hormone (GH) has been demonstrated to stimulate cell growth and counteract apoptosis. GH has also been shown to improve learning and memory in both human and rodents. In this work, we demonstrate that GH may reverse opiate-induced apoptosis in cells derived from prenatal mouse hippocampus. Primary hippocampal cell cultures derived from 16-day-old fetal mouse neurons were treated with morphine for 7 days during growth in the absence or presence of recombinant human GH (rhGH). The release of lactate dehydrogenase (LDH) into the culture media and the level of cleaved caspase-3 were measured. Results indicate that morphine (15 microM) decreased the cell content in a concentration-dependent manner and increased LDH release and caspase-3 activity. Thus, fetal mouse neurons treated with morphine showed less viability compared with controls. Interestingly, the addition of rhGH (1 microM) counteracted the morphine-induced effect on the cell density. Furthermore, the hormone attenuated the effects on LHD release and caspase-3 activity elicited by morphine. These results suggest that the hormone is capable of preventing or even repairing morphine-induced damage to hippocampal cells.

Dopamine melanin-loaded PC12 cells: a model for studies on pigmented neurons.

The most conspicuous feature in idiopathic parkinsonism is the degeneration of pigmented neurons in the substantia nigra. A major problem for the study of the significance of neuromelanin for the development of parkinsonism is that common experimental animals lack neuromelanin in substantia nigra. The aim of this study was to develop an in vitro model that could be used to study the role of neuromelanin in chemically induced toxicity in dopaminergic cells. Cultured neuron-like PC12 cells were exposed to synthetic dopamine melanin (0-1.0 mg/ml) for 48 h, resulting in uptake of dopamine melanin particles into the cells. The intracellular distribution of dopamine melanin granules was similar to that found in neuromelanin-containing neurons. Dopamine melanin, up to 0.5 mg/ml, had negligible effects on ultrastructure, induction of the endoplasmic reticulum-stress protein glucose regulating protein 78, activation of caspase-3 and cell viability. The decreased cell viability in response to the cytotoxic peptide amyloid-beta25-35 was similar in melanin-loaded cells and in control cells without melanin. The results of the studies suggest that melanin-loaded PC12 cells can serve as an in vitro model for studies on the role of neuromelanin for the toxicity of chemicals, in particular neurotoxicants with melanin affinity, in pigmented neurons.

Neuronal nicotinic and muscarinic receptor subtypes at different ages of transgenic mice overexpressing human acetylcholinesterase.

Subtypes of nicotinic (alpha4 and alpha7) as well as muscarinic (M1 and M2) receptor binding sites were quantified in the brain of transgenic mice overexpressing human acetylcholinesterase (AChE) at different ages using selective radioligands. A significant increase in [(3)H]cytisine (alpha4) binding was found in the cortex and striatum of AChE transgenic (hAChE-Tg) mice from 3 days to 12 months of age in comparison to non-transgenic mice. In addition a significant increase in [(3)H]AF-DX-384 (M2) binding was found in the striatum of hAChE-Tg mice at 3 months of age compared to controls. No major alteration was observed in the [(125)I]alpha-bungarotoxin (alpha7) or the [(3)H]pirenzepine (M1) binding sites. The persistent increase in alpha4 and M2 receptor binding sites in hAChE-Tg mice suggests that these receptor subtypes may play an important role in compensatory mechanisms facilitating the impaired cholinergic neurotransmission in hAChE-Tg.

Upregulation of neuronal nicotinic receptor subunits alpha4, beta2, and alpha7 in transgenic mice overexpressing human acetylcholinesterase.

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits alpha4, beta2, and alpha7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [3H]epibatidine and [125I]alphabungarotoxin binding in the cortex and the caudate putamen of these mice. Quantitativein situ hybridization showed significant upregulation of mRNA corresponding to the nicotinic receptor subunits alpha4, beta2, and alpha7 in various brain regions in the transgenic mice compared to nontransgenic controls. Our results suggest that disruption of balanced cholinergic transmission by constitutive overexpression of acetylcholinesterase is accompanied by variable upregulation of several nicotinic receptor subtypes, in particular these associated with cholinergic terminals participating in compensatory response.