Effects of chronic ethanol exposure on GABA receptors and GABAB receptor modulation of 3H-GABA release in the hippocampus.

Chronic ethanol treatment (CET), sufficient for decreasing long-term potentiation (LTP) in rats, also enhances 3H-GABA release from hippocampal slices in these same animals. The mechanism for an increase in GABA release may involve changes in presynaptic receptors. Therefore, we characterized presynaptic autoreceptor modulation of 3H-GABA release in hippocampal slices from control and CET rats. The effects of a GABAB receptor agonist (baclofen) and antagonist [2-hydroxy (OH)-saclofen] were tested for their ability to modulate electrically stimulated 3H-GABA release from superfused hippocampal slices. Baclofen decreased stimulated release in a dose-dependent manner and 2-OH-saclofen increased release consistent with the existence of presynaptic GABAB autoreceptors in hippocampus. The GABAA antagonist bicuculline did not significantly modulate basal or stimulated release. When the effects of baclofen and 2-OH-saclofen were measured in animals 48 hr after withdrawal from CET, presynaptic modulation of release by baclofen and 2-OH-saclofen was decreased. In addition, we examined the density of 3H-baclofen and 3H-bicuculline binding in the hippocampal formation using quantitative autoradiographic techniques. We found that the density of 3H-baclofen binding sites was not affected by CET, whereas the density of 3H-bicuculline binding sites was increased by 28% in ethanol-treated rats. These data may explain how CET increases presynaptic regulation of GABA release from hippocampus that may contribute to the decrease in LTP seen in rats after CET.

Neurochemical basis of disruption of hippocampal long term potentiation by chronic alcohol exposure.

The aim of this review is to summarize the possible mechanisms underlying the long-term impairment of learning and memory resulting from chronic ethanol treatment (CET) especially that involving decrements in long-term potentiation (LTP) in hippocampus. CET for a 28-week duration affects the rat hippocampal formation in such a way as to decrease the magnitude of LTP; an effect that can last as long as 7 months after ethanol withdrawal. It appears that NMDA receptor number in hippocampus is unchanged after CET whereas the data suggest a more pronounced role for changes in GABAergic and cholinergic synaptic transmission in determining how CET influences the induction of LTP in hippocampus. In particular, changes in presynaptic modulation of neurotransmitter release in hippocampus may be one mechanism by which CET inhibits LTP. Thus, the mechanisms underlying the effect of CET on LTP are a result of changes in a number of neurotransmitter systems in hippocampus (GABAergic and cholinergic) rather than based solely on changes in glutamate transmission.

Long-term effects of chronic ethanol on muscarinic receptor binding in rat brain.

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([3H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [3H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.

Stability of [3H]MK-801 binding sites following chronic ethanol consumption.

Previous work has demonstrated that short periods (1-2 weeks) of exposure to ethanol produce an upregulation of the N-methyl-D-aspartate (NMDA) receptor complex in hippocampus; an alteration that appears to be associated with the development of physical dependence, because a return to control levels occurs over a 24- to 48-hr abstinence period. Prolonged periods of chronic ethanol treatment (CET; 4-8 months of treatment) have been shown to produce severe and permanent alterations in the morphological and functional characteristics of hippocampal pyramidal neurons. Several lines of research have demonstrated that the NMDA receptor complex is involved in excitotoxic cell loss during certain pathological states. On the basis of this evidence, we hypothesized that prolonged ethanol exposure would be accompanied by an enduring increase in NMDA receptors and that NMDA receptor binding in cells surviving CET would be altered. To test this hypothesis, we measured the binding characteristics of the NMDA receptor complex in a variety of brain structures following CET. Animals were fed a nutritionally complete, ethanol-containing diet for 28 weeks and then allowed a 48-hr abstinence period. A control group was fed the same diet, except sucrose was isocalorically substituted for ethanol. We first examined the effect of CET on the binding properties of a noncompetitive antagonist to the NMDA receptor channel, [3H]diclozipene ([3H]MK-801). Next, as an indirect examination of NMDA receptor function, we measured the ability of glutamate to stimulate channel opening and thus [3H]MK-801 binding. In all brain structures examined, neither the Kd nor the Bmax of [3H]MK-801 binding to the NMDA receptor was altered following CET. In addition, no effect of treatment was seen on the ability of glutamate to stimulate [3H]MK-801 binding.

Chronic ethanol exposure enhances [3H]GABA release and does not affect GABAA receptor mediated 36Cl uptake.

Chronic ethanol treatment (CET) produces a permanent reduction in hippocampal long-term potentiation (LTP). The CET produced reduction of LTP can be eliminated acutely by pharmacological blockade of gamma-aminobutyric acid (GABA)ergicA synaptic transmission with the specific antagonist, bicuculline methiodide. Since LTP induction is normally modulated by activation of GABAergic synaptic transmission, we hypothesized that CET reduced LTP by enhancing either postsynaptic GABAA channel function or GABA release from presynaptic terminals. In the present study, we examined the long term effects of CET on GABAA channel function by measuring the efficacy of GABA to stimulate and bicuculline to antagonize GABA-stimulated 36Cl- uptake in hippocampal and cortical membrane preparations. CET did not affect basal uptake of chloride or the efficacy of either GABA or bicuculline at the GABAA channel. We next measured the long term effects of CET on basal and stimulated GABA release. When basal and electrically-stimulated [3H]GABA release were measured in superfused hippocampal slices, stimulated release was increased by 30% in CET rats. Basal release was unaffected. Thus it appears that CET may be reducing LTP by enhancing plasticity-related GABA release from presynaptic terminals.

Effects of chronic ethanol ingestion on long-term potentiation remain even after a prolonged recovery from ethanol exposure.

Previous work in our laboratory has demonstrated that memory formation, a behavioral process thought to be at least in part attributed to hippocampal functioning, is severely attenuated following 5 months of chronic ethanol treatment (CET) and 2 months of recovery from CET. Additionally, 48 h following CET, a well-recognized physiological correlate of memory formation, long-term potentiation (LTP), is reduced in the hippocampus. We hypothesized that the reduction in LTP may in part contribute to the behavioral deficit in memory formation, which is a permanent consequence of CET. In order for the reduction of LTP to be involved with the permanent deficit in memory acquisition, it too must be present following a prolonged period of ethanol abstinence. The present study examined the permanent effect of CET on LTP. Animals were fed a nutritionally complete, ethanol containing diet for 28 weeks and then allowed a 5-7 month abstinence period. A control group was fed the same diet except sucrose was isocalorically substituted for ethanol. Neurophysiological methods measured the capacity of synaptic connections onto CA1 pyramidal cells to support LTP in response to a variety of conditioning trains. The magnitude of LTP was reduced in CET animals as compared with pair-fed controls. LTP induction is mediated by activation of the N-methyl-d-aspartate (NMDA) receptor complex and is modulated by activation of gamma-aminobutyric acid (GABA)ergic synaptic transmission. The effect of CET on LTP magnitude may be due to effects on the NMDA-induced induction itself or on the GABAergic modulation of induction.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel nicotinic agonist facilitates induction of long-term potentiation in the rat hippocampus.

Long-term potentiation (LTP) can be modulated by a number of neurotransmitter receptors including muscarinic and GABAergic receptor types. We have found that a novel nicotinic agonist, 2,4-dimethoxybenzylidene anabaseine (DMXB), facilitated the induction of LTP in the hippocampus in a dose-dependent and mecamylamine-sensitive manner. DMXB displaced high affinity nicotinic [125I]alpha-bungarotoxin and [3H]acetylcholine binding in rat brain. Xenopus oocyte studies demonstrated that DMXB has agonist activity at alpha 7 but not alpha 4/beta 2 nicotinic receptor subtypes. These results indicated that DMXB is a novel nicotinic agonist with apparent specificity for the alpha 7/alpha-bungarotoxin nicotinic receptor subtype and indicate that nicotinic receptor activation is capable of modulating the induction of long-term potentiation.

Effects of chronic ethanol on cholinergic actions in rat hippocampus: electrophysiological studies and quantification of m1-m5 muscarinic receptor subtypes.

The effects of chronic ethanol treatment (CET) on cholinergic modulation of CA1 evoked field potentials and recurrent inhibition were investigated in rat hippocampal slices. Densities of muscarinic receptor subtypes were quantified in remaining hippocampal tissue by immunoprecipitation. Iontophoretic application of ACh in stratum pyramidale results in facilitation of single evoked population spikes; application in stratum radiatum results in depression of field EPSPs. CET decreased cholinergic facilitation of population spikes, while cholinergic inhibition of field EPSPs remained unaffected. Integrity of feedback (recurrent) inhibitory circuitry was evaluated by paired-pulse stimulation. As previously demonstrated, recurrent inhibition was significantly reduced after CET; cholinergic disinhibition was also significantly reduced. Thus, CET appears to disrupt a subset of cholinergic effector systems within hippocampal neurons. The reductions in cholinergic function produced by CET does not appear to be due to receptor loss, since muscarinic receptor subtype densities were not found to be significantly altered in this tissue. These results support the hypothesis that muscarinic receptor function is impaired in CA1 pyramidal cells through a disruption of intracellular signal transduction mechanisms. While it is unclear whether cholinergic function is reduced in interneurons directly, these results suggest that modulation of neuronal firing in the hippocampus is markedly altered following CET due to impairment of both cholinergic and GABAergic systems.

Effect of chronic ethanol on the septohippocampal system: a role for neurotrophic factors?

The mechanisms by which chronic ethanol exposure produces neuronal damage have not been established. Potentially ethanol may reduce normal neurotrophic influences necessary for neuronal survival, growth, and function. We hypothesized that chronic ethanol exposure might produce a decrease in the synthesis, availability, upregulation, delivery, and/or the biological activity of normally occurring neurotrophic factors, or may alter the capacity of target neurons to respond to these factors. The available evidence leading to this hypothesis and supporting data from our laboratory are discussed.

Chronic ethanol consumption reduces the neurotrophic activity in rat hippocampus.

The effect of chronic ethanol treatment (CET) for 21-26 weeks on the neurotrophic activity contained in the rat hippocampus (HPC) was determined with a bioassay in cultures of dissociated dorsal root ganglion cells (DRG) obtained from E7-8 chick embryos. Extracts of the HPC from CET or pair-fed control rats were used as experimental media, and neuronal survival and neurite-outgrowth of DRG cultures were determined. Both neuronal survival (-25%) and neurite-outgrowth (-50%) were reduced in the presence of HPC extracts from CET rats relative to controls. These data suggest that CET reduces the neurotrophic content of the HPC which may result in damage to septohippocampal neurons.

Chronic ethanol treatment reduces inhibition in CA1 of the rat hippocampus.

The effect of chronic ethanol exposure on inhibition in the rat hippocampal slice was investigated using paired-pulse stimulation techniques with stimulation in stratum radiatum or stratum oriens of CA1. Experimental animals were fed ethanol in a liquid diet for 20 weeks and were withdrawn for at least 8 weeks prior to electrophysiological recording. Prior ethanol treatment had no effect on basic input-output relationships for the extracellular population spike. Ethanol treatment significantly reduced the recurrent inhibition produced by antidromic stimulation in a manner dependent upon stimulus intensity. In addition, with orthodromic paired-pulse stimulation of either stratum radiatum or oriens, a trend toward an augmentation of the facilitation of population spike amplitude was observed, suggesting that feedforward inhibition may also be reduced. These results are similar to those found with treatments that reduce inhibition. Therefore, we conclude that chronic ethanol exposure produces an enduring disruption of inhibitory neuronal function in the rat hippocampus.

Chronic ethanol treatment differentially affects muscarinic receptor responses in rat hippocampus.

Sensitivity of hippocampal field potentials to local (iontophoretic) application of acetylcholine (ACh) was investigated in chronic ethanol treated (CET) and sucrose-fed (control) rats. CET and control rats were fed a liquid diet containing either ethanol or sucrose for 28 weeks. Five to six months after ethanol or sucrose was withdrawn, hippocampal slices were taken and ACh was applied in stratum pyramidale or stratum radiatum of CA1 to observe population spike facilitation or field EPSP inhibition, respectively. Population spikes were facilitated to a considerably lesser extent in CET slices relative to controls, while no treatment differences were observed for dendritic EPSP inhibition. These data suggest that ACh response properties in CA1 exhibit differential sensitivity to CET, and may reflect a distinct susceptibility of muscarinic receptor subtypes to the neurotoxic effects of ethanol.

Binding of [125I]-insulin-like growth factor-1 (IGF-1) in brains of Alzheimer's and alcoholic patients.

Patients with chronic alcoholism and/or Alzheimer's disease suffer from degenerative changes in the cerebral cortex and hippocampus. To investigate possible changes in IGF-1 receptor binding sites in brain tissue of patients with these pathological conditions, the binding of [125I]-IGF-1 was determined in tissues obtained from control, Alzheimer's and/or patients with a history of alcoholism. The four experimental groups examined consisted of patients from similar age groups. Specific binding of [125I]-IGF-1 to cerebral cortical membranes from Alzheimer's patients had significantly more binding sites than age-matched controls, alcoholic patients and alcoholic patients with Alzheimer's disease. Regression analyses indicated that there were no significant differences in [125I]-IGF-1 binding in cerebral cortex with regard to age of patients (1.1% of total variance with a range of 52 to 92 years). Likewise, the time interval between death and autopsy contributed only 1.4% to the total variance in IGF-1 binding. No statistical differences in [125I]-IGF-1 binding were noted in hippocampal tissue from the various patient groups. Thus, human IGF-1 binding sites in cerebral cortex and hippocampus appear to be relatively stable for a number of variables. The increase in cerebral cortical [125I]-IGF-1 binding sites could be due to upregulation of IGF-1 receptors resulting from a decrease in IGF-1 levels in Alzheimer's patients.

Neither chronic exposure to ethanol nor aging affects type I or type II corticosteroid receptors in rat hippocampus.

Both chronic exposure to ethanol and aging are reported to result in a loss of hippocampal pyramidal neurons and an elevation in plasma corticosteroid concentration. Aging has also been reported to result in a reduction in corticosterone receptors and corticosterone-concentrating cells in the hippocampus. Since these aging-associated effects have been hypothesized to be due to the cumulative exposure to corticosteroids over the life span, in the present studies, we investigated the hypothesis that corticosteroids play a similar role in the loss of hippocampal neurons during chronic ethanol ingestion. In contrast to our expectations, when a liquid diet containing either ethanol or sucrose was administered to male Long-Evans rats for 20-24 weeks, a period of treatment found previously to result in hippocampal neuronal loss, there were no ethanol-associated effects on the specific binding of [3H]-aldosterone to Type I or of [3H]dexamethasone to Type II receptors in cytosol derived from either whole hippocampus or dorsal versus ventral hippocampus. Allowing the rats to withdraw from the chronic ethanol exposure for 12 weeks also failed to reveal any ethanol-associated effects on corticosteroid receptor concentrations in the hippocampus. The basal morning concentrations of corticosterone in blood plasma were not affected by any of these treatments. In view of these null findings we next investigated the effects of aging on hippocampal corticosteroid receptors in male Long-Evans and Fischer 344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative.

Biocytin, a naturally occurring low molecular weight analog of biotin, was evaluated as a neuroanatomical tract-tracing marker in the adult rat brain. Since it retains high-affinity binding to avidin, biocytin can be labelled with avidinylated visualization reagents. Iontophoretic or pressure injections resulted in filling of cell bodies and dendrites around the injection site and their efferent axonal processes and boutons. Retrogradely labelled neurons were occasionally observed at a distance but only with large injections. Anterograde tracing with biocytin is successful even in animals that are quite old, in contrast to lectins and HRP conjugates, and offers advantages in delivery, tissue processing, selection of light and/or electron microscopic labels, time to obtain results, and cost over many conventional tracers.

Acetylcholinesterase stain intensity variation in the rat dentate gyrus: a quantitative description based on digital image analysis.

Three-dimensional patterns of variation in the intensity of acetylcholinesterase histochemical staining and the width of stain-defined subregions were quantified for the dentate gyrus of the adult male Long-Evans rat. Matched tissue sections sampled through the central hippocampal formation of five rats were measured with a digital image analysis computer system. The width and stain intensity were determined for defined portions of the dentate gyrus related to gross acetylcholinesterase staining patterns and the known distribution of dentate afferents. Normalized values reflecting stain intensity at defined positions within this standardized sampling array were examined to investigate regional differences in acetylcholinesterase distribution along the primary dendritic axis of dentate granule neurons. The data illustrate quantitative differences in the partitioning of acetylcholinesterase as a function of intrahippocampal position. The variation is more pronounced in the septal-temporal axis than the granule cell layer crest-tip axis. Furthermore, the septal-temporal variations in acetylcholinesterase intensity demonstrate some independence according to proximal-distal location within the molecular layer. The results suggest that acetylcholinesterase distribution within the dentate gyrus may reflect local physiological characteristics of those afferent systems related to this enzyme, including but not necessarily limited to those that are specifically cholinergic.

Alterations and recovery of dendritic spine density in rat hippocampus following long-term ethanol ingestion.

Neuronal loss and dendritic pathology are often observed in humans and animals after long-term ethanol ingestion. It is not known, however, if surviving but damaged neurons can recover normal structure during ethanol abstinence. We quantified dendritic spine density in two neuronal populations in rat hippocampus to investigate whether reversibility from the cellular neurotoxic sequelae of chronic ethanol exposure was possible. Male Long-Evans rats were maintained for 20 weeks on an ethanol-containing liquid diet. Controls were pair-fed a liquid diet with sucrose substituted isocalorically for ethanol. One-half of each group was sacrificed at the end of the 20-week treatment and one-half was given a 20-week ethanol-free recovery period period to sacrifice. Analysis of rapid Golgi material revealed a decreased spine density in CA1 pyramidal cells that increased to control level during abstinence, and an increased spine density in dentate gyrus granule cells that was reduced toward control level during abstinence. Thus, despite the fact that chronic ethanol exposure produced differential initial effects, the return toward normal spine density in each region is consistent with the concept of neuronal recovery and reorganization during abstinence from ethanol.

Ethanol exposure following unilateral entorhinal deafferentation alters synaptic reorganization in the rat dentate gyrus: a quantitative analysis of acetylcholinesterase histochemistry.

The phenomenon of reactive synaptogenesis in the dentate gyrus following partial deafferentation was used to assess quantitatively the effects of ethanol exposure on neuronal plasticity. Adult male Long-Evans rats received unilateral lesions of the entorhinal cortex, followed by 40 days of dietary ethanol exposure. Synaptic reorganization of the commissural/associational (C/A) and cholinergic (primarily septal) afferents was evaluated in the dentate gyrus processed for acetylcholinesterase (AChE) histochemistry. The widths and optical densities of several laminae in the molecular layer were measured using a computer-based digital imaging system. The degree of reorganization was assessed by comparing the lesioned side to the unlesioned side in each animal (difference scores were calculated). In the exposed blade of the dentate gyrus, the lesion-induced expansion of the C/A width was reduced significantly in the ethanol group. However, in the buried blade, there was an actual shrinkage of the C/A zone in the ethanol group. There was also a tendency for less "clearing" or lightening of the AChE staining in the C/A zone of both blades. On the lesioned side, the ethanol group also displayed a significantly greater condensation or darkening of the AChE staining in the proximal outer molecular layer than the pair-fed control group. The supragranular layer of both blades ipsilateral to the lesion was reduced in width in the ethanol group. These results indicate that exposure to ethanol following partial deafferentation disrupts the normal pattern of synaptic reorganization in the dentate gyrus.

Computerized 24-hour esophageal pH monitoring: a new ambulatory technique using radiotelemetry.

A computerized telemetric system using a portable microprocessor receiving unit has been designed for clinical use in ambulatory 24-hour esophageal pH monitoring. The receiving unit can be used with either a pH-sensitive radiotelemetry pill or a standard intraesophageal pH probe. Esophageal pH data is collected under microprocessor control while the patient is in the usual home or work environment. Computerized plotting and analysis of the compiled pH data is performed at the conclusion of the ambulatory testing. The system has been successfully used in patients with symptoms of esophageal reflux and healthy volunteers with no symptoms. The development of a computer-based radiotelemetry system for prolonged monitoring of esophageal pH represents a substantial advance in existing investigational technology. It allows the use of new radiotelemetric techniques of esophageal pH recording with increased patient comfort and acceptance over a 24-hour ambulatory testing period. Microprocessor control of both data collection and analysis allows rapid interpretation of complex 24-hour esophageal pH tracings and greatly facilitates the performance of esophageal pH monitoring as a routine clinical investigation.