Comparative gene expression in brain regions of human alcoholics.

The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.

Acute exposure to alcohol during early postnatal life causes a deficit in the total number of cerebellar Purkinje cells in the rat.

Alcohol taken regularly over a lengthy period of time has been claimed to cause the loss of neurons in both the adult and developing brain. However, it remains uncertain whether acute, as opposed to chronic, exposure to alcohol at specified periods can also cause disruption in the neuronal population of the developing brain. This question was investigated by exposing Wistar rat pups to 7.5 g/kg body weight of ethanol administered as a 10% solution via an intragastric cannula over an 8 hour period either on the 5th (PND5) or the 10th (PND10) postnatal day of age. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mothers was used as "suckle controls." All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes, all pups were returned to the lactating dams and weaned at 21 days of age. Between 52 and 54 days of age, the rats were anaesthetised with an intraperitoneal injection with Nembutal and killed by intracardiac perfusion with 3% glutaraldehyde in 0.1 M phosphate buffer. The relatively unbiased stereological procedure known as the "fractionator" method was used to estimate the total number of Purkinje cells in the cerebellum of each animal. The Purkinje cell nucleolus was used as the counting unit; it was assumed that each Purkinje cell contained only one nucleolus. PND10 ethanol-treated rats and gastrostomy and suckle controls had between about 210,000-232,000 Purkinje cells in the cerebellum. However, the PND5 ethanol-treated rats had only about 137,000 Purkinje cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate early gene expression during morphine withdrawal.

The expression of immediate early genes (IEG)s c-fos, c-jun and zif/268 was studied during naloxone-precipitated morphine withdrawal in various organs of the rat. Dependence was induced over a period of 6 days by a graded regimen of 6-hourly injections. Northern analysis revealed peak expression of all IEGs occurred in the forebrain plus cerebellum at 20 min and at 60 min in the brain stem following morphine withdrawal. Increased levels of c-fos and c-jun mRNA were observed in the spinal cord at 40 min of morphine withdrawal. An increase in c-fos and c-jun but not zif/268 mRNAs was seen in the jejunum between 20 and 60 min. Elevated levels of the IEG protein products in the cerebral cortex, hippocampus, thalamus, cerebellum, brain stem and spinal cord were observed at 60 min following morphine withdrawal. These data emphasize the temporal and spatial variation in IEG expression in different tissues during opiate withdrawal.

The induction of immediate early genes in postischemic and transplanted livers in rats. Its relation to organ survival.

The protein products of the immediate early genes (IEG)s have been proposed to play an important role in long-term tissue plasticity such as cell repair or programmed cell death. The expression of liver IEGs was studied following liver ischemia (LI) or OLT in rats. In LI, 60 min of warm ischemia was induced in shunted rats (shunt LI group; 100% survival) and nonshunted rats (nonshunted LI group; poor survival). In OLT, donor livers were transplanted into the recipients within 1 hr (fresh liver OLT group; 100% survival) or after 24 hr of storage using University of Wisconsin solution (preserved liver OLT group; poor survival). Using both models, IEG mRNAs (c-fos and c-jun) were analyzed by Northern blot hybridization at various times before and after reperfusion. The expression of liver IEGs was not induced by warm ischemia and cold preservation alone. Reperfusion of livers following warm ischemia or cold preservation resulted in a distinctly different pattern of gene expression in viable and nonviable livers. In shunted LI and fresh liver OLT groups (viable), c-fos and c-jun mRNAs increased markedly to a peak value within 1-2 hr of reperfusion, returning to basal level by 3 hr. In nonviable livers, the level of these mRNAs was detected continuously at 3 hr of reperfusion in the nonshunted LI model and also at 6 hr after reperfusion in the preserved liver OLT group. Our data suggest that a protracted pattern of expression of c-fos and c-jun in the liver at the early stage of reperfusion might be correlated with the severity of liver transplant-related insults and subsequent graft failure.

Ethanol and synaptosomal calcium homeostasis.

The effect of ethanol on synaptosomal calcium homeostasis was studied in the rat using the fluorescent dye, fura-2, and 45Ca uptake. The mitochondrial poison, cyanide, caused a substantial rise in intracellular free Ca2+ concentration, [Ca2+]i, over that of control synaptosomes. This rise was enhanced by ethanol. Ethanol also augmented the rise in [Ca2+]i induced by ouabain, indicating that modulation of Na(+)-Ca2+ exchange is probably not the underlying mechanism. The Ca2+ channel blockers, verapamil and La3+, also failed to inhibit the rise in [Ca2+]i caused by ethanol. Preincubation of synaptosomes with caffeine, however, caused a significant decrease in the rise of [Ca2+]i due to ethanol, suggesting that ethanol exerts effects on Ca2+ homeostasis at the level of the endoplasmic reticulum.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation.

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-1 DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP.

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse.

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.

Egr transcription factors in the nervous system.

The Egr proteins, Egr-1, Egr-2, Egr-3 and Egr-4, are closely related members of a subclass of immediate early gene-encoded, inducible transcription factors. They share a highly homologous DNA-binding domain which recognises an identical DNA response element. In addition, they have several less-well conserved structural features in common. As immediate early proteins, the Egr transcription factors are rapidly induced by diverse extracellular stimuli within the nervous system in a discretely controlled manner. The basal expression of the Egr proteins in the developing and adult rat brain and the induction of Egr proteins by neurotransmitter analogue stimulation, physiological mimetic and brain injury paradigms is reviewed. We review evidence indicating that Egr proteins are subject to tight differential control through diverse mechanisms at several levels of regulation. These include transcriptional, translational and post-translational (including glycosylation, phosphorylation and redox) mechanisms and protein-protein interaction. Ultimately the differentially co-ordinated Egr response may lead to discrete effects on target gene expression. Some of the known target genes of Egr proteins and functions of the Egr proteins in different cell types are also highlighted. Future directions for research into the control and function of the different Egr proteins are also explored.

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain.

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [(3)H]flunitrazepam, [(3)H]GABA or [(3)H]muscimol to extensively washed synaptic membranes. Neither the affinity (K(d)) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [(3)H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [(3)H]flunitrazepam or [(3)H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA-benzodiazepine receptor ionophore-complex in the brain.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain.

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Sp1. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity.

Ethanol and protein kinase C in rat brain.

The effect of chronic ethanol consumption on the catalytic activity of protein kinase C isolated from rat brain was studied in two different ways. Enzyme activity was first measured by phosphorylation of Histone IIIS in vitro. There was no change in the activity of the cytosolic enzyme. Membrane-associated enzyme activity was reduced in the ethanol-treated animal. This difference was not evident if the enzyme was stimulated by arachidonate. The reduction in enzyme activity was confirmed by analysis of the phosphorylation of endogenous substrates in intact synaptosomes. When the binding of the ligand [3H]phorbol dibutyrate was measured by quantitative autoradiography, increased binding to membrane-associated protein kinase C was observed in the CA1 region of the hippocampus but not in other brain regions. These results indicate that ethanol treatment results in a general reduction in membrane-associated protein kinase C activity as measured in vitro but the effect may not be consistent in all brain regions. The differential effect in the CA1 region of the hippocampus may be a reflection of a disruption in the normal regulation of protein kinase C activity in this area and may indicate that this region is a sensitive target for the action of ethanol.

The effect of chronic ethanol consumption on muscarinic receptors in rat brain.

Ethanol (15% v/v) was administered in the drinking water to male Wistar rats over period of 3 months. Binding properties of muscarinic receptors were studied in synaptosomes from selected brain areas using [(3)H]quinuclidinyl benzilate and its displacement by the selective antagonist, pirenzepine and the agonist, carbachol. Dissociation constants (K(d)) of all three ligands in the cerebral cortex, hippocampus and striatum of ethanol-treated groups did not differ from those in controls. Density of [(3)H]quinuclidinyl benzilate binding sites in the cortex of ethanol-treated animals was approx. 50% higher than in controls (2.06 +/- 0.2 and 1.32 +/- 0.2 pmol/mg of protein respectively, mean +/- SD, n = 6, P < 0.001). This was largely attributable to an increase in M(1) binding sites as shown by pirenzepine displacement studies. In the hippocampus and striatum binding capacity of muscarinic receptors was not affected by ethanol treatment. Synthesis of acetylcholine in cerebral cortex prisms from ethanol-treated animals was not inhibited under resting conditions, but stimulation of synthesis by high K(+) concentration was significantly altenuated by comparison with controls. These results suggest that chronic ethanol consumption induces changes in cholinergic neurotransmission in selected brain areas.

Autoradiographic studies on the effect of ethanol on muscarinic receptors in rat brain.

The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [(3)H]QNB to the cortex of 4- and 6-week-old animals, but not 11-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [(3)H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M(1) sites in the younger animals possibly linked to age-related plasticity of the nervous system.

Effects of chronic ethanol treatment and aging on brain phosphoinositide turnover and adenylate cyclase activity.

Inositol phosphate accumulation and adenylate cyclase activity were investigated in the cortex of young and aged ethanol-treated rats. Three months of ethanol treatment of young rats decreased maximal stimulation of inositol phosphate accumulation by carbachol by 26%, from 494 +/- 76% of basal turnover in control animals to 396 +/- 54% in ethanol-treated animals (mean +/- SD). In aged rats ethanol-related changes were no longer observed but age-related changes were evident. EC(50) was significantly higher than in young animals and maximal stimulation was significantly lower. Basal adenylate cyclase activity in cortical membranes of all groups of animals was not different. Forskolin-stimulated adenylate cyclase activity was not affected by ethanol treatment, but was higher in aged animals. The activity of forskolin-stimulated adenylate cyclase in the presence of carbachol was higher in both young and aged ethanol-treated animals, when compared to young controls. These results suggest that both ethanol and aging impair the efficiency of receptor/effector coupling.

Chronic ethanol administration alters binding of [(35)S]t-butylbicyclophosphorothionate to the GABA-benzodiazepine receptor complex in rat brain.

Chronic treatment of male Wistar rats with ethanol (15% v/v) in drinking fluid for a period of 3 months affected the binding of the chloride channel antagonist, [(35)S]TBPS, to well-washed synaptic membranes and slide-mounted brain slices, the affinity for [(35)S]TBPS in brains of ethanol-treated animals was significantly decreased in comparison to controls while receptor density was increased (P < 0.001). However, other well described treatments, viz. an ethanol-containing liquid diet and chronic inhalation of ethanol failed to demonstrate changes in the binding of [(35)S]TBPS in brain preparations. Our findings suggest that long-term administration of ethanol can induce alterations in the characteristics of the ionophore component of the GABA-benzodiazepine receptor complex. This may have relevance to ethanol-induced neuronal damage.

Effects of chronic ethanol inhalation on the enhancement of benzodiazepine binding to mouse brain membranes by GABA.

Chronic ethanol inhalation produced no change in the number or affinity of [(3)H]flunitrazepam binding sites on well-washed synaptic membranes prepared from male Quackenbush mice, but produced a significant decrease in the capacity of GABA to enhance [(3)H]flunitrazepam binding. This decrease was characterised by a higher EC(50) (1.4 ? M compared to 0.6 ? M) and a lower maximal level of enhancement (162% compared to 172%) for tissue from the chronically treated animals compared to tissue from control animals. Acute ethanol treatment or ethanol incubated in vitro with the brain membranes did not produce changes in any of the [(3)H]flunitrazepam binding parameters. These results support other findings that chronic ethanol may affect the coupling of various sites on GABA-A receptor-ionophore complexes in brain.

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 effectors, 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.

Increased NMDA-induced excitability during ethanol withdrawal: a behavioural and histological study.

Intrahippocampal injections of N-methyl-D-aspartic acid (NMDA) leads to neurodegeneration in a dose-dependent manner. Chronic administration of ethanol to animals leads to CNS tolerance and dependence. Hyperexcitability following ethanol withdrawal is thought to be related to increased sensitivity of the NMDA receptors. The purpose of this study was to investigate this predisposition to hyperexcitability by intrahippocampal injection of low dose of NMDA. Using control and ethanol-withdrawn male Wistar rats, behavioural indices were determined immediately after injection and morphological damage was assessed after a period of recovery. There was significantly increased hyperactivity in the ethanol-treated rats immediately after injection. Morphological damage resulting from 5 nmol of NMDA was significantly greater in the CA3 region of the hippocampus in these animals. These data support the hypothesis that ethanol dependence and subsequent withdrawal is associated with increased sensitivity to NMDA which may underlie ethanol withdrawal-associated brain damage.

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 pair-fed 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.

Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect of N-methyl-D-aspartic acid in vivo.

Chronic administration of ethanol in animals leads to CNS tolerance and physical dependence. Subsequent withdrawal of ethanol causes hyperexcitability which is thought to be related to increased sensitivity of N-methyl-D-aspartic acid (NMDA) receptors. The purpose of this study was to investigate sensitivity to NMDA in ethanol-treated animals by detecting damage after intrahippocampal injection of NMDA. Choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) specific activity was used as markers of cholinergic and gamma-aminobutyric acid neurons, respectively. Ethanol-dependent animals were more liable to die following intrahippocampal injection of either 120 or 240 nmol of NMDA. There was a significantly greater decrease in hippocampal GAD but not ChAT specific activity in the surviving animals. These data support the hypothesis that ethanol dependence is associated with increased sensitivity to NMDA which may be responsible for excitotoxic brain damage and death.