Journal of Neurochemistry

Abstract: We have isolated and characterized the 5′‐flanking regulatory region of the murine serotonin 5‐HT transporter (5‐HTT) gene. A TATA‐like motif and several potential binding sites for transcription factors, including two AP1, several AP2 and AP4 binding sites, CCAAT and GC boxes (SP1 binding sites), a nuclear factor‐κB, and a cyclic AMP response element‐like motif, are present in the 5′‐flanking region. A ∼2.2‐kb fragment (−2,143 to +51 with respect to the transcription start site), which had been fused to the luciferase reporter gene and transiently expressed in a 5‐HTT‐expressing cell line and in serotonergic raphe neurons derived from embryonic rat brain‐stem, displayed both constitutive and inducible promoter activity. Functional promoter mapping revealed two clusters of activating elements from bp −82 to −527 and bp −1,001 to −1,937. A cell/neuron‐selective silencer element(s) is contained between bp −294 and −527. Our findings suggest that (1) the murine 5‐HTT gene promoter is active in serotonergic raphe neurons but significantly repressed in neuronal cells from frontal cortex that do not express 5‐HTT, (2) the information contained within ∼0.5 kb of the 5′‐flanking sequence is sufficient to confer its cell‐selective expression, (3) the promoter responds to cyclic AMP‐ and protein kinase C‐dependent induction, and (4) the expression of the 5‐HTT is regulated by a combination of positive and negative cis‐acting elements operating through a basal promoter unit defined by a TATA‐like motif. Fusion of the 5‐HTT gene promoter unit to a gene of choice may aid its cell‐selective expression in transgenic strategies.

Abstract: The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH₄, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, α‐ketoglutarate, and malate in either tissue. The changes of NH₄+, glutamine, and lactate levels as well as gutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.

Abstract: The sequence of molecular events linking depolarisation‐dependent calcium influx to the release of neurotransmitters from nerve terminals is unknown; however, calcium‐stimulated protein phosphorylation may play a role. In this study the incorporation of phosphate into proteins was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. The rate and relative incorporation of label into individual phosphoproteins depended on the prelabelling time and buffer concentrations of calcium and phosphate. After prelabelling for 45 min, depolarisation caused a >20% increase in the labelling of 10 phosphoproteins, and this initial increase was maximal with 41 mM K⁺ for 5 s, or 30 μM veratridine for 15 s, in the presence of 1 mM calcium. Both agents also led to an initial dephosphorylation of four phosphoproteins. Depolarisation for 5 min led to a significant decrease in the labelling of all phosphoproteins. All of the depolarisation‐stimulated changes in protein phosphorylation were calcium‐dependent. The depolarisation conditions found to optimally alter the phosphorylation of synaptosomal proteins find many parallels in studies on calcium uptake and neurotransmitter release. However, the uniform responses of such a large number of phosphoproteins to the multitude of depolarisation conditions studied suggest that the changes could equally well relate to recovery events such as biosynthesis of neurotransmitters and regulation of intraterminal metabolic activity.

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer’s disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have usedin vivomicrodialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE‐/‐ mice). Extracellular ACh levels in the hippocampus were 60‐fold elevated in AChE‐/‐ mice compared with wild‐type (AChE+/+) animals. In AChE‐/‐ mice, calcium‐free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose‐dependent, up to 16‐fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE‐/‐ mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE‐/‐ mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis‐norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.

Abstract: Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up‐regulated sites labeled by [³H]cytisine in cerebral cortex appear to be composed exclusively of α4 and β2 subunits. It is unknown whether receptor subtypes that do not bind [³H]‐cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [³H]cytisine with that of [³H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [³H]‐cytisine and [³H]epibatidine. In contrast, no change in [³H]‐epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high‐affinity [³H]cytisine binding. These data indicate that, under the conditions used here, nicotine up‐regulates the α4β2 nicotinic receptor subtype, which can be labeled by [³H]cytisine and [³H]epibatidine, but not non‐α4β2 subtypes, which can be labeled by [³H]epibatidine.

The export of glutamine from astrocytes, and the uptake of glutamine by neurons, are integral steps in the glutamate‐glutamine cycle, a major pathway for the replenishment of neuronal glutamate. We review here the functional and molecular identification of the transporters that mediate this transfer. The emerging picture of glutamine transfer in adult brain is of a dominant pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons. The participating glutamine transporters are functionally and structurally related, sharing the following properties: (a) unlike many neutral amino acid transporters which have proven to be obligate exchangers, these glutamine transporters mediate net substrate transfer energized by coupling to ionic gradients; (b) they are sensitive to small pH changes in the physiological range; (c) they are susceptible to adaptive and humoral regulation; (d) they are related structurally to the AAAP (amino acid and auxin permeases) family of transporters. A key difference between SN1 and the SAT/ATA transporters is the ready reversibility of glutamine fluxes via SN1 under physiological conditions, which allows SN1 both to sustain a glutamine concentration gradient in astrocytes and to mediate the net outward flux of glutamine. It is likely that the ASCT2 transporter, an obligate exchanger of neutral amino acids, displaces the SN1 transporter as the main carrier of glutamine export in proliferating astrocytes.

Choline acetyltransferase levels have been measured in specific nuclei of the diencephalon. On the whole, the thalamic nuclei contain higher concentrations of this enzyme than do the hypothalamic and preoptic nuclei. Those nuclei which seem to receive the most dense cholinergic innervation contain the highest levels of choline acetyltransferase.

Abstract—

The standardization of a radiochemical assay of choline acetyltransferase (acetyl‐CoA: choline‐O‐acetyltransferase EC 2.3.1.6. [ChAc]) is described. The method depends upon the use of [l‐C¹⁴]acetyl‐CoA as substrate and has been modified from the procedure originally published by MCCAMAN and HUNT (1965).