Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pairfed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs, that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity. (C) 1999 Elsevier Science B.V. All rights reserved.

Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effecters, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence. (C) 2000 Elsevier Science Ltd. All rights reserved.

Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B Synapses in the adult central amygdala

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes.

Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge

Amyloid-beta peptide (A beta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic A beta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/ peptide ratios of > 0.6:1 by EPR spectroscopy. The toxicity of the A beta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi- or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl- 1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. P-31 magic angle spinning solid-state NMR showed that A beta and A beta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the A beta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved. (C) 1997 Elsevier Science B.V.

Equal Numbers of Neuronal and Nonneuronal Cells Make the Human Brain an Isometrically Scaled-Up Primate Brain

The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells (""neurons"") and 84.6 +/- 9.8 billion NeuN-negative (""nonneuronal"") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. J. Comp. Neurol. 513:532-541, 2009. (c) 2009 Wiley-Liss, Inc.

Inhibitory network interactions shape the auditory processing of natural communication signals in the songbird auditory forebrain

The role of GABA in the central processing of complex auditory signals is not fully understood. We have studied the involvement of GABA(A)-mediated inhibition in the processing of birdsong, a learned vocal communication signal requiring intact hearing for its development and maintenance. We focused on caudomedial nidopallium (NCM), an area analogous to parts of the mammalian auditory cortex with selective responses to birdsong. We present evidence that GABA(A)-mediated inhibition plays a pronounced role in NCM`s auditory processing of birdsong. Using immunocytochemistry, we show that approximately half of NCM`s neurons are GABAergic. Whole cell patch-clamp recordings in a slice preparation demonstrate that, at rest, spontaneously active GABAergic synapses inhibit excitatory inputs onto NCM neurons via GABA(A) receptors. Multi-electrode electrophysiological recordings in awake birds show that local blockade of GABA(A)-mediated inhibition in NCM markedly affects the temporal pattern of song-evoked responses in NCM without modifications in frequency tuning. Surprisingly, this blockade increases the phasic and largely suppresses the tonic response component, reflecting dynamic relationships of inhibitory networks that could include disinhibition. Thus processing of learned natural communication sounds in songbirds, and possibly other vocal learners, may depend on complex interactions of inhibitory networks.

Muscarinic acetylcholine neurotransmission enhances the late-phase of long-term potentiation in the hippocampal-prefrontal cortex pathway of rats in vivo: A possible involvement of monoaminergic systems

The prefrontal cortex is continuously required for working memory processing during wakefulness, but is particularly hypoactivated during sleep and in psychiatric disorders such as schizophrenia. Ammon`s horn CA1 hippocampus subfield (CA1) afferents provide a functional modulatory path that is subjected to synaptic plasticity and a prominent monoaminergic influence. However, little is known about the muscarinic cholinergic effects on prefrontal synapses. Here, we investigated the effects of the muscarinic agonist, pilocarpine (PILO), on the induction and maintenance of CA1-medial prefrontal cortex (mPFC) long-term potentiation (LTP) as well as on brain monoamine levels. Field evoked responses were recorded in urethane-anesthetized rats during baseline (50 min) and after LTP (130 min), and compared with controls. LTP was induced 20 min after PILO administration (15 mg/kg, i.p.) or vehicle (NaCl 0.15 M, i.p.). In a separate group of animals, the hippocampus and mPFC were microdissected 20 min after PILO injection and used to quantify monoamine levels. Our results show that PILO potentiates the late-phase of mPFC UP without affecting either post-tetanic potentiation or early LTP (20 min). This effect was correlated with a significant decrease in relative delta (1-4 Hz) power and an increase in sigma (10-15 Hz) and gamma (2540 Hz) powers in CA1. Monoamine levels were specifically altered in the mPFC. We observed a decrease in dopamine, 5-HT, 5-hydroxyindolacetic acid and noradrenaline levels, with no changes in 3,4-hydroxyphenylacetic acid levels. Our data, therefore, suggest that muscarinic activation exerts a boosting effect on mPFC synaptic plasticity and possibly on mPFC-dependent memories, associated to monoaminergic changes. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Pyramidal neurones in macaque visual cortex: Interareal phenotypic variation of dendritic branching patterns

The basal dendritic arbors of over 500-layer III pyramidal neurones of the macaque cortex were compared by fractal analyses, which provides a measure of the space filling (or branching pattern) of dendritic arbors. Fractal values (D) of individual cells were compared between the cytochrome oxidase (CO)-rich blobs and CO-poor interblobs, of middle and upper layer III, and between sublaminae, in the primary visual area (Vi). These data were compared with those in the CO compartments in the second visual area (V2), and seven other extrastriate cortical areas. (V4, MT, LIP, 7a, TEO, TE and STP). There were significant differences in the fractal dimensions, and therefore the dendritic branching patterns, of cells in striate and extrastriate areas. Of the 55 possible pairwise comparisons of fractal dimension of neurones in different cortical areas (or CO compartments), 39 proved to be significantly different. The markedly different morphologies of pyramidal cells in the different cortical areas may be one of the features that determine the functional signatures of these cells by influencing the number of inputs received by, and propagation of potentials through, their dendritic arbors.

Neonatal ethanol exposure reduces AMPA but not NMDA receptor levels in the rat neocortex

Fetal alcohol syndrome (FAS) is the leading cause of mental retardation in western society. We investigated possible changes in glutamate receptor levels in neonatal animals following ethanol exposure using radioligand binding and western blot analysis. We used a vapor chamber to administer ethanol to neonatal Wistar rats 3 h a day from postnatal day (PND) 4-9. A separation control group was separated from their mothers for the same time and duration as the vapor treatment, while a normal control group was left to develop normally. Daily ethanol administrations resulted in decreased brain weight and body weight, as well as microencephaly (decreased brain:body weight ratio). Neither the affinity nor maximum binding of [H-3]MK-801 (dizoclipine maleate) in the cortex of PND10 rats differed between treatment groups. Western blot analysis also failed to reveal any changes in NMDAR1, NMDAR2A, or NMDAR2B receptor levels. In contrast, the AMPA receptor subunit GluR1 was greatly reduced in vapor-treated pups compared with control pups, as revealed by western blot analysis. A similar reduction was found in westerns with an antibody recognizing the GluR2 and 4 subunits. These results indicate that ethanol reduces AMPA rather than NMDA receptors in the developing neocortex, possibly by blocking NMDA receptors during development. (C) 2002 Elsevier Science B.V. All rights reserved.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Functional and structural studies of two enzymes: membrane bound kinase and Z-DNA/Z-RNA-binding protein

Dissertação para obtenção do Grau de Doutor em Sistemas de Bioengenharia

Pathfinding of corticothalamic axons relies on a rendezvous with thalamic projections.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Effect of rufinamide on gating properties of voltage-gated sodium channel Na(v)1.7

Background: Voltage-gated sodium channels (Nav1.x) are important players in chronic pain. A particular interest has grown in Nav1.7, expressed in nociceptors, since mutations in its gene are associated to two inherited pain syndromes or insensitivity to pain. Rufinamide, a drug used to treat refractory epilepsy such as the Lennox-Gastaut syndrome, has been shown to reduce the number of action potentials in cortical neurons without completely blocking Na channels. Aim: The goal of this study was to investigate the effect of rufinamide on Nav1.7 current. Methods and results: Whole-cell patch clamp experiments were performed using HEK293 cells stably expressing Nav1.7. Rufinamide significantly decreased peak sodium current by 28.3, 21.2 and 12.5% at concentrations of 500, 100 and 50μM respectively (precise EC50 could not be calculated since higher rufinamide concentrations could not be achieved in physiological buffer solution). No significant difference on the V1/2 of voltage-dependence of activation was seen; however a shift in the steady-state inactivation curve was observed (-82.6 mV to -88.8 mV and -81.8 to -87.6 mV for 50 and 100 μM rufinamide respectively, p <0.005). Frequency-dependent inhibition of Nav1.7 was also influenced by the drug. One hundred μM rufinamide reduced the peak sodium current (in % of the peak current taken at the first sweep of a train of 50) from 90.8 to 80.8% (5Hz), 88.7 to 71.8% (10 Hz), 69.1 to 49.2% (25 Hz) and 22.3 to 9.8% (50 Hz) (all p <0.05). Onset of fast inactivation was not influenced by the drug since no difference in the time constant of current decay was observed. Conclusion: In the concentration range of plasma level in human treated for epilepsy, 15 μM, rufinamide only minimally blocks Nav1.7. However, it stabilizes the inactivated state and exerts frequencydependent inhibition of Nav1.7. These pharmacological properties may be of use in reducing ectopic discharges as a causal and symptom related contributor of neuropathic pain syndrome.

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.