Chronic ethanol consumption reduces delta-and mu-opioid receptor-stimulated G-protein coupling in rat brain.

BACKGROUND: Ethanol consumption is thought to enhance the release of endogenous opioids acting at opioid receptors (ORs) in the central nervous system. Prior studies have shown that chronic ethanol consumption in alcohol-preferring rats uncouples mu-ORs from Gi proteins. The purpose of this study was to investigate the potential for uncoupling of the delta- and the mu-OR after chronic ethanol consumption in a nonpreferring rat strain. METHODS: We used radiohistochemical methods to study mu- and delta-OR-stimulated G-protein coupling in brain tissue of rats ingesting liquid diets containing 6.7% ethanol (v/v) for 16 days, as compared with 0% ethanol pair-fed control rats. Sections of brain from pair-fed and ethanol-treated rats were incubated with guanylyl 5'-[gamma-[35S]-thio]-triphosphate ([35S]-GTPgammaS) in the absence and presence of d-Pen2,d-Pen5 enkephalin (DPDPE), a delta-OR agonist, or Tyr-d-Ala-Gly-N(me)Phe-Gly-ol-enkephalin (DAMGO), a mu-OR agonist. RESULTS: DPDPE significantly stimulated [35S]-GTPgammaS binding in the hippocampal dentate gyrus (DG), CA1, cerebellum, and inferior colliculus of untreated pair-fed controls. By contrast, DPDPE-stimulated [35S]-GTPgammaS binding was reduced significantly in those brain regions in the ethanol-consuming group. DAMGO stimulated [35S]-GTPgammaS binding in cortex, caudate, nucleus accumbens, DG, CA1, and superior and inferior colliculi, whereas the DG, CA1, and colliculi showed a significant reduction of binding after chronic ethanol. Basal [35S]-GTPgammaS binding was not different between the two diet groups. CONCLUSIONS These data are the first to demonstrate functional uncoupling of delta-ORs from G proteins after chronic ethanol consumption. Uncoupling may result from modulation of receptors, possibly by internalization or phosphorylation. Alterations in functional coupling of both delta- and mu-ORs and subsequent effects may contribute to continued ethanol consumption.

Delta opioid receptor localization in the rat cerebellum.

Opioid receptors have been localized to a number of brain regions in rats as well as in other species. In situ hybridization has demonstrated the presence of mRNA for the delta receptor subtype in adult rat cerebellar cortex and in several deep nuclei, but there are no reports on localization of the delta receptor protein in cerebellar regions. In the present study, both quantitative immunohistochemistry and Western blots reveal the presence of delta receptors in the adult rat cerebellum, using a specific affinity-purified antibody. Purkinje cells and processes, as well as cells in the granule cell layer, were positively stained with the antibody. Quantitation of confocal microscopy images illustrated a lower relative level of delta receptor immunoreactivity in cerebellar cortical neurons as compared to neurons in hippocampal regions, striatum and cerebral cortex. Stimulation of delta receptors with a selective agonist, DPDPE, in frozen sections of rat brain, induced a significant increase in binding of [35S]-GTPgammaS in the cerebellar cortex as compared to basal binding levels, thereby demonstrating coupling of the receptor subtype to G-protein. Functional implications for the delta receptor in the cerebellum are discussed, particularly in light of evidence for the presence of a cerebellar opioid receptor for the endogenous opioid methionine enkephalin during early postnatal life.

Ionotropic glutamate receptor subunit expression in the rat hippocampus: lack of an effect of a long-term ethanol exposure paradigm.

BACKGROUND: Studies have shown that acute ethanol exposure inhibits ionotropic glutamate receptor function and that long-term ethanol exposure results in maladaptive increases in the expression of some of these receptors in neurons. It has been postulated that these changes, when unopposed by ethanol, contribute, in part, to the hyperexcitability associated with ethanol withdrawal. In this study, we compared the effect of long-term ethanol exposure on the hippocampal expression levels of subunits belonging to the three families of ionotropic glutamate receptors. METHODS: Adult male Sprague-Dawley rats were fed an ethanol-containing diet for 16 days. This diet contained 0% ethanol on days 1 and 2, 3% on days 3 and 4, 5% on days 5 to 7, and 6.7% on days 8 to 16. Control rats received an equivalent amount of an isocaloric diet without ethanol. Rats were killed on day 16 at the peak of ethanol consumption. Hippocampal homogenates were prepared by sonication and analyzed by Western immunoblotting techniques. On a separate group of rats, we measured withdrawal scores and audiogenic seizures on day 17. RESULTS: Ethanol-exposed rats had significantly higher withdrawal scores, and a significantly higher percentage of them developed audiogenic seizures; this indicates that the 16-day ethanol diet induces ethanol dependence. Unexpectedly, we found that expression of NR1 (including the expression of NR1 subunits containing the N1, C1, and C2 inserts), NR2A, NR2B, NR2C, GluR1, GluR2/3, GluR5, GluR6/7, and KA2 subunits was not altered in hippocampal homogenates from ethanol-exposed rats. CONCLUSIONS: These results indicate that maladaptive changes in the hippocampal expression levels of ionotropic glutamate receptor subunits do not always occur in ethanol-dependent rats. Consequently, other mechanisms must mediate the hyperexcitability state associated with ethanol withdrawal in these animals.

Conditioned place preference for cocaine is attenuated in mice over-expressing the 5-HT(3) receptor.

The serotonin 5-HT(3) receptor is thought to play a role in the reward pathway and drug abuse by modulating dopamine release within the mesolimbic pathway. Dopamine release stimulated by cocaine and methamphetamine is blocked by administration of 5-HT(3) receptor antagonists. Animal studies demonstrate that 5-HT(3) receptor antagonists decrease cocaine and methamphetamine preference. We have developed a 5-HT(3) receptor over-expressing mouse to study the role of this receptor in substance abuse. No changes in either the dopamine receptors (D1, D2, D3, and D4) or in the dopamine transporter (DAT) were found over a wide range of brain regions. 5-HT(3) receptor over-expressing mice failed to develop conditioned place preference to 10 mg/kg or 6 mg/kg cocaine but showed a modest preference for 4 mg/kg cocaine. 5HT(3) receptor over-expressing mice were more sensitive to the locomotor activating effects of low dose cocaine and methamphetamine. Further, brain slices from the transgenic mice release more dopamine in response to low concentrations of cocaine. These data suggest that 5HT(3) receptor over-expression in the forebrain decreases cocaine preference and increases acute sensitivity with a corresponding increase in the amount of dopamine released in response to cocaine.

Fear conditioning-induced alterations of phospholipase C-beta1a protein level and enzyme activity in rat hippocampal formation and medial frontal cortex.

We investigated the effects of one-trial fear conditioning on phospholipase C-beta1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-beta1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-beta1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-beta1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-beta1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-beta1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-beta1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-beta1a protein level in hippocampal formation and phospholipase C-beta1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-beta1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-beta1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-beta1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-beta1a signal transduction.

Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices.

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.

Fetal alcohol exposure alters neurosteroid modulation of hippocampal N-methyl-D-aspartate receptors.

The actions of ethanol on brain ligand-gated ion channels have important roles in the pathophysiology of alcohol-related neurodevelopmental disorders and fetal alcohol syndrome. Studies have shown that N-methyl-d-aspartate (NMDA) receptors are among the ligand-gated ion channels affected by prenatal ethanol exposure. We exposed pregnant dams to an ethanol-containing liquid diet that results in blood ethanol levels near the legal intoxication limit in most states (0.08%). Primary cultures of hippocampal neurons were prepared from the neonatal offspring of these dams, and NMDA receptor function was assessed by patch clamp electrophysiological techniques after 6-7 days in culture in ethanol-free media. Unexpectedly, we did not detect any changes in hippocampal NMDA receptor function at either the whole-cell or single-channel levels. However, we determined that fetal alcohol exposure alters the actions of the neurosteroids pregnenolone sulfate and pregnenolone hemisuccinate, which potentiate NMDA receptor function. Western immunoblot analyses demonstrated that this alteration is not due to a change in the expression levels of NMDA receptor subunits. Importantly, in utero ethanol exposure did not affect the actions of neurosteroids that inhibit NMDA receptor function. Moreover, the actions of pregnenolone sulfate on type A gamma-aminobutyric acid and non-NMDA receptor function were unaltered by ethanol exposure in utero, which suggests that the alteration is specific to NMDA receptors. These findings are significant because they provide, at least in part, a plausible mechanistic explanation for the alterations in the behavioral responses to neurosteroids found in neonatal rats prenatally exposed to ethanol and to other forms of maternal stress (Zimmerberg, B., and McDonald, B. C. (1996) Pharmacol. Biochem. Behav. 55, 541-547).

A review of the effects of prenatal or early postnatal ethanol exposure on brain ligand-gated ion channels.

BACKGROUND: Ligand-gated ion channels mediate fast excitatory and inhibitory synaptic transmission in the developing central nervous system. These channels have been shown to have roles in neuronal proliferation, differentiation, and programmed cell death. Numerous studies over the past 10 years indicate that prenatal and/or early postnatal ethanol exposure affects neurotransmitter-gated ion channels. METHODS: We conducted a review of the relevant literature, identified by a computer-assisted literature search. This review presents an overview of studies performed with experimental preparations from the brains of rodents exposed to ethanol in utero and/or during the neonatal period and summarizes some of the salient issues that have developed in the course of these investigations. Differences in ethanol exposure paradigms and blood alcohol concentrations obtained in these studies are highlighted, and directions for future research are suggested. RESULTS: Most studies have focused on the effects of prenatal or early postnatal ethanol exposure on NMDA receptors. These studies show that ethanol exposure affects ligand binding, subunit expression, and function of this receptor. Fewer studies have examined ethanol's effects on ligand-gated ion channels other than NMDA receptors. For instance, a study reported changes in ligand binding to hippocampal kainate receptors. Another study found alterations in modulation of GABA(A) receptors by benzodiazepines and neurosteroids. CONCLUSIONS: These studies suggest that the effects of ethanol on brain ion channels may have a role in the pathophysiology of Alcohol-Related Neurodevelopmental Disorders and Fetal Alcohol Syndrome.

Prenatal ethanol exposure diminishes activity-dependent potentiation of amino acid neurotransmitter release in adult rat offspring.

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies suggest that these deficits are, in part, linked to neurochemical abnormalities that reduce the ability to sustain long-term potentiation (LTP) in hippocampal formation of adult offspring. One presynaptic component of LTP that manifests during the first half-hour after tetanic stimulation is an enhancement of amino acid neurotransmitter release. Given that the onset of enhanced neurotransmitter release correlates temporally with the decay of hippocampal LTP in prenatal ethanol-exposed offspring, we tested the hypothesis that prenatal ethanol exposure reduces tetanus stimulus-induced potentiation of electrically evoked amino acid release in hippocampal slices. Rat dams consumed 1 of 3 diets throughout gestation: (1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories) that produces a maternal peak blood ethanol concentration of 83 mg/dl; (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) Purina rat chow ad libitum. Hippocampal slices were prepared from adult offspring from each experimental diet group. Neither the amount of hippocampal slice tissue protein nor the incorporation of [3H]-D-aspartate (D-ASP) was affected by prenatal ethanol exposure. Furthermore, spontaneous efflux and electrically evoked D-ASP release were similar among the three diet groups. However, tetanus stimulus-induced potentiation of evoked D-ASP release in prenatal ethanol-exposed offspring was reduced to about one-third of the potentiation of D-ASP release observed in the control diet groups. These results suggest that prenatal ethanol exposure produces long-lasting deficits in the neurochemical mechanisms responsible for activity-dependent potentiation of amino acid transmitter release without affecting the synaptic machinery responsible for amino acid uptake, storage, and release.

Prenatal ethanol exposure decreases GAP-43 phosphorylation and protein kinase C activity in the hippocampus of adult rat offspring.

Consumption of moderate quantities of ethanol during pregnancy produces deficits in long-term potentiation in the hippocampal formation of adult offspring. Protein kinase C (PKC)-mediated phosphorylation of the presynaptic protein GAP-43 is critical for the induction of long-term potentiation. We tested the hypothesis that this system is affected in fetal alcohol-exposed (FAE) rats by measuring GAP-43 phosphorylation and PKC activity in the hippocampus of adult offspring of rat dams that had consumed one of three diets throughout gestation: (a) a 5% ethanol liquid diet, which produced a maternal blood ethanol concentration of 83 mg/dl (FAE); (b) an isocalorically equivalent 0% ethanol diet (pair-fed); or (c) lab chow ad libitum. Western blot analysis using specific antibodies to PKC-phosphorylated GAP-43 revealed that FAE rats had an approximately 50% reduction in the proportion of phosphorylated GAP-43. Similarly, we found that PKC-mediated incorporation of 32P into GAP-43 was reduced by 85% in hippocampal slices from FAE rats compared with both control groups. FAE animals also showed a 50% reduction in total hippocampal PKC activity, whereas the levels of six major PKC isozymes did not change in any of the diet groups. These results suggest that GAP-43 phosphorylation deficits in rats prenatally exposed to moderate levels of ethanol are not due to alterations in the expression of either the enzyme or substrate protein, but rather to a defect in kinase activation.

Prenatal ethanol exposure alters the modulation of the gamma-aminobutyric acidA1 receptor-gated chloride ion channel in adult rat offspring.

We examined the effect of prenatal ethanol exposure on gamma-aminobutyric acid (GABA)-stimulated 36Cl- flux. Sprague-Dawley rat dams were fed either a liquid diet containing 5% ethanol, pair-fed an isocalorically equivalent 0% ethanol diet or rat chow ad libitum throughout gestation. Membrane vesicles were prepared from medial frontal cortex, cerebellum and hippocampal formation of adult offspring in each diet group. GABA-stimulated 36Cl- flux was not significantly affected by prenatal ethanol exposure in any of the three brain regions examined. Positive allosteric modulation of GABA-stimulated 36Cl- flux by flunitrazepam or alphaxalone, as well as negative modulation by FG-7142 or pregnenolone, were all diminished in medial frontal cortex of 5% ethanol diet offspring compared with both ad libitum and pair-fed control groups. In cerebellum, prenatal ethanol exposure attenuated the modulatory effects of both benzodiazepines, but did not affect neurosteroid modulation. In hippocampus, prenatal ethanol exposure enhanced the effects of flunitrazepam and alphaxalone, whereas negative modulatory effects were either decreased (FG-7142) or unchanged (pregnenolone). These results indicate that moderate ethanol consumption during gestation can produce long-lasting alterations in neuromodulatory influences on GABAA receptor-mediated inhibitory neurotransmission in adult offspring. In hippocampal formation, the heightened sensitivity to positive modulatory influences may contribute to synaptic plasticity deficits in fetal ethanol-exposed rat offspring. We speculate that these prenatal ethanol-induced changes may be either a consequence of differential GABAA receptor subunit expression or receptor uncoupling in different brain regions. Furthermore, offspring exposed to ethanol in utero may display differential sensitivities to benzodiazepines and possibly other centrally active therapeutic agents.

Effects of prenatal ethanol exposure on phospholipase C-beta 1 and phospholipase A2 in hippocampus and medial frontal cortex of adult rat offspring.

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A2 (PLA2) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-beta 1 and PLA2 activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-beta 1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca(2+)-dependent PLA2 activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA2 form. Cytosolic PLA2 activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca(2+)-dependent PLA 2 and PLC-beta 1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.

Ethanol-mediated transplacental induction of CYP2E1 in fetal rat liver.

We examined the potential for the widely consumed xenobiotic ethanol to transplacentally induce fetal rat CYP2E1. Throughout gestation, rat dams were fed a liquid diet containing 5% ethanol or two separate control diets. At 2 days before term, the dams were killed, and maternal and embryonic tissues were collected. Immunoblot analysis of microsomes from fetal liver, placenta and maternal brain revealed a band that comigrated with adult liver CYP2E1. The identity of the immunoreactive protein in placenta, brain and fetal liver was substantiated as CYP2E1 through restriction enzyme digestion of a reverse transcription-polymerase chain reaction product. Quantification of immunoblots containing microsomes from maternal and fetal liver of ethanol-treated dams displayed a 1.4- and 2.4-fold increase in CYP2E1, respectively, compared with microsomes from pair-fed controls. Chlorzoxazone and low substrate concentrations of N-nitrosodimethylamine were used as metabolic probes for CYP2E1. The rate of chlorzoxazone metabolism by maternal hepatic microsomes from dams fed the 5% ethanol diet was 2.6-fold greater than that of controls. Conversely, a negligible increase was observed in the rate of metabolism by hepatic microsomes from ethanol-exposed fetuses compared with pair-fed animals. When N-nitrosodimethylamine demethylation was examined, these same fetal samples exhibited greater rates of activity (1.5-fold) compared with microsomes from control animals. However, this increase was not as great as expected considering the 2.4-fold increase in CYP2E1 protein. Collectively, fetuses exposed to a 5% ethanol diet throughout gestation exhibited transplacental induction of an hepatic CYP2E1 that may possess different catalytic properties from the analogous adult enzyme.

Prenatal exposure to moderate levels of ethanol can have long-lasting effects on hippocampal synaptic plasticity in adult offspring.

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies using animal models of fetal ethanol exposure suggest that these deficits are, at least in part, linked to neurochemical abnormalities in the hippocampal formation. We explored whether prenatal exposure to moderate quantities of ethanol produced functional deficits at the entorhinal cortical perforant path-dentate granule cell connection by examining some electrophysiological properties, including the induction of long-term potentiation (LTP). Rat dams consumed one of three diets throughout gestation: 1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories), which produces a maternal peak blood ethanol concentration of 83 mg/dl; 2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or 3) Purina rat chow ad libitum. Adult offspring (120-150 days of age) from each experimental diet group were anesthetized with urethane and field excitatory postsynaptic potentials (EPSPs) and population spikes were measured in the dentate gyrus in response to ipsilateral perforant path stimulation. We examined input-output functions using a wide range of single pulse stimulation intensities and induction of LTP using high-frequency stimulation. In the 50-500 microA range of single pulse intensities, there were no significant differences among the diet groups in dentate gyrus evoked potentials. In response to high-frequency stimulation, prenatal ethanol-exposed rats showed a smaller increase in field EPSPs and population spikes compared with rats from either of the two control groups. Thus, prenatal exposure to moderate ethanol levels can produce a long-lasting deficit in synaptic enhancement in a neural pathway believed to be critical in certain forms of learning and memory. This deficit in hippocampal synaptic plasticity may, in part, account for cognitive impairments seen in children whose mothers consumed ethanol during pregnancy.

Chronic imipramine treatment induces downregulation of alpha-2 receptors in rat's locus coeruleus and A2 region of the tractus solitarius.

Imipramine is an effective antidepressant agent that blocks the reuptake of monoamines. In order to understand some of its basic mechanisms of action, we investigated the effects of chronic imipramine administration (10 mg/kg, i.p.; 21 days) on the alpha-2 receptor population of several brain sites. Alpha-2 receptor density was estimated by in vitro autoradiography using [3H]Idazoxan. The densitometric analysis revealed a decreased receptor density in the A2 region of the tractus solitarius (20%) and locus coeruleus (16%). No changes were observed in the amygdala, pyriform cortex, periacueductal gray and the bed nucleus of the stria terminalis. These results suggest that chronic imipramine treatment selectively modulates the alpha-2 receptor population localized in the brain stem norepinephrine-rich nuclei and not in the population present on limbic structures innervated by noradrenergic terminal projections. The possible physiological consequences of this selective modulation of alpha-2 receptors are discussed.

Dose- and age-dependent effects of prenatal ethanol exposure on hippocampal metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis.

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal formation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP1) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP1 accumulation in 10- to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP1 accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP1 accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in spleen norepinephrine and lymphocyte [3H]dihydroalprenolol binding site number in genetically epilepsy-prone rats.

Noradrenergic neurotransmission plays an important role in normal immune reactivity. Genetically epilepsy-prone (GEPR-9) rats exhibit deficits in central noradrenergic systems and diminished plaque-forming cell responses following immunization in vivo. In the present study we examined the hypothesis that immunosuppression in GEPR-9 rats is associated with alterations in the splenic noradrenergic system. The content of norepinephrine (NE) in spleens of GEPR-9 age-matched nonepileptic Sprague-Dawley control rats was determined by high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC). In addition, we measured the number of beta-adrenergic receptors on splenic lymphocyte membranes of GEPR-9 and control rats using the beta-adrenergic receptor antagonist dihydroalprenolol ([3H]DHA). HPLC-EC analysis revealed that splenic norepinephrine content was significantly greater in GEPR-9 rats than in controls. Results from receptor binding studies indicated a 33% reduction in specific binding of [3H]DHA to splenic lymphocyte membranes of GEPR-9 rats. Saturation of binding studies revealed a significant decrease in the maximum number of [3H]DHA binding sites on splenic lymphocyte membranes from GEPR-9 rats. These results indicate that the noradrenergic system in GEPR-9 rat spleen is altered. Whether either or both of these changes contribute to reduced immune reactivity in GEPR-9 rats remains to be determined.

Decrease in locus coeruleus [3H]idazoxan binding site density in genetically epilepsy-prone (GEPR) rats.

Deficits in norepinephrine synthesis, transmitter level, turnover and reuptake have been reported in the brain of genetically epilepsy-prone (GEPR) rats. We investigated the hypothesis that these alterations may trigger a compensatory downregulation of locus coeruleus alpha 2-adrenergic receptors and an upregulation of postsynaptic alpha 2-adrenergic receptor density in forebrain regions of GEPR rat brain. alpha 2-adrenergic receptor density was measured in the locus coeruleus and 7 forebrain regions of control and GEPR rats by in vitro [3H]idazoxan autoradiography. Specific [3H]idazoxan binding site density was decreased significantly in the locus coeruleus of both GEPR-3 and GEPR-9 rats compared to controls. No significant differences in specific [3H]idazoxan binding were observed in the 7 forebrain regions of GEPR-9 rats compared to control. Reduced locus coeruleus alpha 2-adrenergic receptor density in GEPR rats may produce a net increase in locus coeruleus noradrenergic cell firing, an effect which could, in part, offset the impact of reduced noradrenergic influence in GEPR rat forebrain. Additionally, decreased norepinephrine levels in GEPR rat brain may be a long-term consequence of reduced alpha 2-adrenergic receptor-mediated inhibition of locus coeruleus firing activity.

Effects of prenatal ethanol exposure on hippocampal ionotropic-quisqualate and kainate receptors.

Previous studies from our laboratories have shown that the consumption of moderate quantities of ethanol by rat dams during pregnancy reduces N-methyl-D-aspartate (NMDA) agonist receptor binding and NMDA-mediated electrophysiological responses in the hippocampal formation of adult offspring. We hypothesized that prenatal ethanol exposure would produce similar effects on receptor number and agonist-mediated responses of two so-called "non-NMDA" subtypes of glutamate receptors, the ionotropic-quisqualate (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive and the kainate-sensitive receptors. Sprague-Dawley rats were fed either a liquid diet containing 3.35% ethanol, an isocalorically matched liquid diet, or lab chow ad libitum throughout gestation. No significant differences between offspring from these three groups in the agonist concentration-response curves for either AMPA-induced or kainate-induced depolarization of hippocampal CA1 pyramidal neurons were observed. Furthermore, no significant differences in the density of [3H]-AMPA or [3H]-vinylidene kainic acid binding sites in any of the apical dendritic field regions of dorsal or ventral hippocampal formation were observed between the groups. These results indicate that the ionotropic quisqualate and kainate receptors, located in the apical dendritic field regions of the principal hippocampal neurons, are not affected by the same degree of prenatal ethanol exposure, which is known to reduce NMDA receptor binding and function in these same regions.

Evidence of altered T-lymphocyte number and proliferative responses in genetically epilepsy-prone rats.

Genetically epilepsy-prone (GEPR-9) rats exhibit decreased antibody plaque-forming cell responses following immunization. We examined the hypothesis that this immunosuppression was due to deficits in the number or proliferative responses of T-lymphocytes. Splenocyte responses to concanavalin A and pokeweed mitogen were significantly greater in GEPR-9 rats than controls. Flow cytometric analysis indicated that GEPR-9 rats possess an increase in T-cells associated with the T-helper phenotype. The increased proportion of T-helper cells in GEPR-9 rats may underlie their enhanced proliferative responses to T-cell mitogens. These results clearly indicate that the failure of the GEPR-9 rat to respond to a T-dependent antigen in vivo is not due to a lack of T-helper activity.