Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximate to -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na+ channels, low- and high-voltage-activated Ca2+ channels and delayed-rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Ethanol prevents NMDA receptor reduction by maternal separation in neonatal rat hippocampus

We measured the effects of ethanol on glutamate receptor levels in the hippocampus of neonatal Wistar rats using a vapor chamber model. Two control groups were used; a normal suckle group and a maternal separation group. Levels of NMDA receptors were not significantly altered in ethanol-treated animals compared to the normal suckle control group, as shown by [H-3]MK-801 binding and Western blot analysis. However, MK-801 binding and NR1 subunit immunoreactivity were greatly reduced in the hippocampus of separation control animals. Neither ethanol treatment nor maternal separation altered levels of GluR1 or GluR2(4). These results have serious implications for the importance of maternal contact for normal brain development.

Isolation and characterization at cholinergic nicotinic receptors of a neurotoxin from the venom of the Acanthophis sp Seram death adder

The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (naChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 muM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA(2) Of 8.36 +/- 0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type 11) neurotoxin, alpha-bungarotoxin. With a pK(i) value of 4.48, acantoxin IVa was approximately 25,000 times less potent than a-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [H-3]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [H-3]-epibatidine binding sites. While long-chain neurotoxin resistant [H-3]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type 1) neurotoxin is capable of fully inhibiting specific [H-3]-MLA binding. (C) 2004 Elsevier Inc. All rights reserved.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Cognitive disorders and neurogenesis deficits in Huntington's disease mice are rescued by fluoxetine

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat encoding an extended polyglutamine tract in the huntingtin protein. Affected individuals display progressive motor, cognitive and psychiatric symptoms (including depression), leading to terminal decline. Given that transgenic HD mice have decreased hippocampal cell proliferation and that a deficit in neurogenesis has been postulated as an underlying cause of depression, we hypothesized that decreased hippocampal neurogenesis contributes to depressive symptoms and cognitive decline in HD. Fluoxetine, a serotonin-reuptake inhibitor commonly prescribed for the treatment of depression, is known to increase neurogenesis in the dentate gyrus of wild-type mouse hippocampus. Here we show that hippocampal-dependent cognitive and depressive-like behavioural symptoms occur in HD mice, and that the administration of fluoxetine produces a marked improvement in these deficits. Furthermore, fluoxetine was found to rescue deficits of neurogenesis and volume loss in the dentate gyrus of HD mice.

Changes in neuronal protein 22 expression and cytoskeletal association in the alcohol-dependent and withdrawn rat brain

The action of alcohol on neuronal pathways has been an issue of increasing research focus, with numerous findings contradicting the previously accepted idea that its effect is nonspecific. The human NP22 (hNP22) gene was revealed by its elevated expression in the frontal cortex of the human alcoholic. The sequences of hNP22 and the rat orthologue rNP22 contain a number of domains consistent with those of cytoskeletal-interacting proteins. Localization of rNP22 is restricted to the cytoplasm and processes of neurons and it colocalizes with elements of the microfilament and microtubule matrices including filamentous actin (F-actin), alpha-tubulin, tau, and microtubule-associated protein 2 (MAP2). Withdrawal of Wistar rats after alcohol dependence induced by alcohol vapor produced elevated levels of rNP22 mRNA and protein in the cortex, CA2, and dentate gyrus regions of the hippocampus. In contrast, there was decreased rNP22 expression in the striatum after chronic ethanol exposure. Chronic ethanol exposure did not markedly alter rNP22 colocalization with F-actin, alpha-tubulin, or MAP2, although colocalization at the periphery of the neuronal soma with F-actin was observed only after chronic ethanol exposure and withdrawal. Rat NP22 colocalization with MAP2 was reduced during withdrawal, whereas association with alpha-tubulin and actin was maintained. These findings suggest that the effect of chronic ethanol exposure and withdrawal on rNP22 expression is region selective. Rat NP22 may affect microtubule or microfilament function, thereby regulating the neuroplastic changes associated with the development of alcohol dependence and physical withdrawal.