EFFECTS OF ETHANOL ON EXTRACELLULAR AMINO ACID LEVELS IN HIGH-AND LOW-ALCOHOL SENSITIVE RATS: A MICRODIALYSIS STUDY

Abdelkader Dahchour*, Alex Hoffman1, Richard Deitrich1 and Philippe de Witte

Université catholique de Louvain, Laboratoire de Biologie du Comportement, 1 place Croix du Sud, 1348 Louvain-la-Neuve, Belgium and
1 University of Colorado Health Sciences Center, Department of Pharmacology and Alcohol Research Center, 4200 E. 9th Avenue, Denver, CO 80262, USA

Received 20 September 1999; in revised form 19 April 2000; accepted 25 May 2000


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Selectively bred high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats possess a number of behavioural and electrophysiological differences in their responses to alcohol. Using a microdialysis technique, we have evaluated whether the levels of the amino acids aspartate, glutamate, arginine, taurine, and alanine in HAS and LAS rats differ in their response to ethanol administration (2 g/kg, i.p.). The basal concentrations of each amino acid in these two groups were statistically similar. Following ethanol injection, alanine, arginine, and glutamate were significantly decreased in HAS rats, whereas, alanine, glutamate, and taurine were significantly increased in LAS rats by the end of the experiment. Interestingly, an increase in the sulphonated amino acid taurine was only evident 20 min after ethanol administration in the HAS rats, when compared to saline controls. No changes were observed in the other amino acids studied, aspartate and glycine, after ethanol administration. These data suggest that, in addition to differential behavioural responses to alcohol, HAS and LAS rats also differ in their neurochemical responses to ethanol.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animal models are useful tools to study the mechanism of action of ethanol, and may provide information to comprehend such mechanisms (Deitrich, 1993Go). Although the genetic selection of experimental animals for the study of alcohol responses has been based on different alcohol-related behavioural phenotypes in both mice (McClearn and Kakihana, 1981Go; McClearn et al., 1982Go; Crabbe et al., 1985Go, 1987Go, 1990Go) and rats (Riley et al., 1976Go, 1977Go; Bass and Lester, 1981Go; Eriksson and Rusi, 1981Go; Li et al., 1981Go; Tampier et al., 1981Go; Sinclair et al., 1989Go), the possibility of a genetic selection based on a neurochemical phenotype would contribute to the understanding of the mechanism of action of ethanol.

One of the most successful selective breeding techniques is that based on the initial sensitivity to the hypnotic (sleep time) effects of acute doses of ethanol. Such rats have been developed at the University of Colorado Health Sciences Center, USA (Hansen and Spuhler, 1984Go; Deitrich et al., 1988Go; Draski et al., 1992Go), and this has resulted in two lines of rats with extremes of hypnotic sensitivity, high-alcohol sensitivity rats (HAS) which have a long sleep time and low alcohol sensitivity rats (LAS) which have a short sleep time following ethanol ingestion.

These two lines (HAS and LAS) show many differences in their sensitivity to other hypnotics, such as pentobarbital (Draski et al., 1992Go), halothane (Deitrich et al., 1994Go; Liu and Deitrich, 1998Go), and phenobarbital (Draski et al., 1997Go). In addition, differences in GABA activated Cl channels induced by flunitrazepam, ethanol, and pentobarbital, and ethanol potentiation of muscimol-stimulated chloride uptake have also been demonstrated (Allan et al., 1988Go, 1991Go). However, other parameters which have been assessed in these HAS and LAS rats, such as sensitivity to propofol (Liu and Deitrich, 1998Go), behavioural responses to acute doses of ethanol (Krimmer, 1990Go, 1991Go; Schechter, 1992Go; Schechter and Krimmer, 1992Go), ethanol-induced dependence (Aufrère et al., 1994Go), and taste reactivity tests (Kiefer and Badia, 1997Go) have not shown statistically significant differences between these two lines of rats.

Alterations in excitatory and inhibitory amino acid neurotransmitters and their receptors have been implicated in the ethanol-induced effects on the central nervous system (CNS) and are proposed to underlie a wide range of behavioural alterations, such as depression, hyperexcitability, seizure, and withdrawal symptoms. It is therefore possible that HAS and LAS rats are genetically different with respect either to the basal concentrations of excitatory and inhibitory amino acids or to changes induced by administration of ethanol. To test this hypothesis, HAS and LAS rats were investigated to ascertain firstly whether the basal concentrations of the amino acids in the nucleus accumbens (NAC) differed and secondly whether the levels of these amino acids in response to an acute i.p. injection of ethanol or saline were altered.

The NAC was chosen, because it receives direct glutamatergic connections from the frontal cortex and hippocampus, and plays an important role in mediating the reinforcing effects of a large number of drugs, including alcohol (Koob and Bloom, 1988Go; North, 1992Go).


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animal surgery and microdialysis experiment
Male HAS and LAS rats, bred at the University of Colorado Health Science Center, were used at generation 30 for all experiments. The rats had been selectively bred for 24 generations and maintained by a rotation breeding programme thereafter. Rats of both replicate lines, 3 to 4 months of age, weighing 270–360 g, were housed individually in plastic cages and kept in a temperature- and light-controlled environment (12 h light/12 h dark cycle). Surgery was performed to insert the microdialysis probe directly into the NAC (A/P 1.2 mm; M/L 1.2 mm; D/V –7.7 mm) (Paxinos and Watson, 1982Go), under anaesthesia with urethane 25% (1.25 g/kg i.p.) The microdialysis experiment started by connecting the probe to a microinfusion pump (Infusion syringe pump 22, Harvard apparatus), continuously delivering a Ringer's solution (145 mM NaCl, 4 mM KCl; 1.3 mM CaCl2; pH 7.2) at 1 µl/min. Microdialysis samples were collected, every 20 min during a 5-h period, and analysed by high-performance liquid chromatography (HPLC) with electrochemical detection following o-phthaldialdehyde/ß-mercaptoethanol (OPA/BME) pre-column derivatization, as previously described (Dahchour et al., 1996Go). Rats received either i.p. ethanol 2 g/kg (10 HAS, 8 LAS) or saline (8 HAS, 9 LAS) 2 h after the probe insertion. The basal concentration was estimated for each amino acid by taking the mean concentration of microdialysate collected at 80, 100, and 120 min after insertion of the microdialysis probe. This value was corrected for recovery (see below).

Blood-alcohol assay
Seven HAS and 7 LAS male rats were anaesthetized with urethane and injected i.p. with 2 g/kg of ethanol as a 15% (w/v) solution in saline. A 10-µl blood sample was taken in duplicate, every 30 min for 6 h. The blood-ethanol levels were assayed by the alcohol dehydrogenase method (Boehringer Mannheim, Germany).

Statistical analysis
Data are presented as means ± SEM and analysed by analysis of variance (MANOVA) (time, treatment) with repeated measures followed by the least-significant difference test of multiple comparison (Fisher's LSD protected test) (GB-STAT, Dynamic Microsystems, Silver Spring, MD, USA).

Corrections for recoveries
The term recovery is defined as the ratio between the concentration of a particular substance in the outflow solution and the concentration of the same substance in the solution outside the probe. Absolute recovery is the amount of substance detected in the outflow/unit of time.

Recoveries in the present microdialysis experiments were achieved by continuously perfusing the probe with a Ringer's solution. The probe was inserted into a standard solution containing 5.10–6 M amino acid mixture (Cint). Samples were collected every 20 min. The amino acid concentrations in the outflow (Cout) were determined by HPLC analysis and the recovery in vitro (R in vitro) was calculated as:


Using this formula, the recovery of each amino acid from the amino acid standard solution was 16.89% ± 1.55 for aspartate, 13.72% ± 1.42 for glutamate, 36.00% ± 4.15 for arginine, 27.10% ± 1.67 for taurine, 20.09% ± 2.44 for alanine, 9.34% ± 0.080 for GABA, and 15.4% ± 1.47 for glycine. The correction for recovery for each amino acid (C) was carried out using the following formula:


The validity of the current calculation relies upon the assumption that the conditions in vitro are equal to those in vivo. This is clearly not true, since relative recoveries do not reach a steady state but decline consistently. This explains the high levels of amino acids assayed initially after the insertion of the probe, which decline to a relatively steady state after 2 h. The relative recovery of various substances will vary due to differences in their molecular weight as well as their diffusion through the membrane, which may increase the polydispersity. Hence, mass transport into the fibre is less in vivo when compared with in vitro. Whereas relative recovery is independent of the outer substance concentration, absolute recovery is proportional to it. Many other factors also need to be considered, such as substances which may react with the dialysis membrane, the temperature of the in vivo and in vitro studies, e.g. carried out at 25°C or 37°C, the perfusion flow rate, the membrane area, and the average mass transfer coefficient. In addition, it is important that the medium used for the recovery experiment is of similar viscosity and ionic concentration to that of the brain interstitial fluid. The composition of the perfusion fluid (in having no amino acids) may cause a continuous passage of these compounds from the brain interstitial space to the perfusion fluid, such that, with time, there will be a diminishing level of neurotransmitters.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Basal amino acid concentrations in HAS and LAS rats
The mean basal concentrations of aspartate, glutamate, glycine, arginine, taurine, and alanine are shown in Table 1Go. The basal amino acid concentrations were not statistically different between the HAS and LAS rats. These basal concentrations are presented in two ways, corrected for recoveries in Table 1AGo and not corrected for recoveries in Table 1BGo.


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Table 1. Basal concentration of amino acids and {gamma}-aminobutyric acid (GABA) in dialysates
 
Changes in amino acid concentrations of the NAC microdialysates after injection of ethanol or saline in HAS and LAS rats
Figure 1Go represents the percentage changes in alanine (Fig. 1aGo), arginine (Fig. 1cGo), glutamate (Fig. 1dGo), and taurine (Fig. 1fGo) from the NAC microdialysate of HAS rats and also represents the changes in alanine (Fig. 1bGo), glutamate (Fig. 1eGo), and taurine (Fig. 1gGo) from LAS rats after an i.p. injection of ethanol (2 g/kg) or saline, 2 h after the probe insertion.



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Fig. 1. Time course of changes in alanine, arginine, glutamate, and taurine microdialysate levels up to 5 h after saline or ethanol administration. Ethanol (2 g/kg body weight as a 15% v/v solution in saline) or an equal volume of saline was injected i.p. into HAS (n = 10) and LAS (n = 8) rats. Results are presented as % of baseline levels ± SEM. Significance of the differences from the corresponding saline group *P < 0.05; **P < 0.01. Sal, saline and EtOH, ethanol injection.

 
Ethanol injection (2 g/kg, i.p.) induced a significant decrease of alanine (Fig. 1aGo) in HAS rats compared to the saline controls [F (1, 224) = 11.667; P = 0.035]. A significant difference in time [F (14, 269) = 4.774; P < 0.0001] and in interaction between time and treatment [F (14, 269) = 2.25; P = 0.007)] was observed for alanine. Ethanol administration also reduced the glutamate level in HAS rats (Fig. 1dGo) by comparison to their saline controls [F (1, 224) = 7.85; P = 0.013]. A significant difference between HAS rats and their controls was also observed [F (14, 269) = 13.916; P < 0.001]. The interaction of time and treatment was also significant [F (14, 269) = 4.878; P < 0.0032]. The effect of ethanol on other amino acids in HAS rats was only significant in time [F (14, 254) = 3.945; P < 0.0001] for arginine (Fig. 1cGo) and [F (14, 269) = 2.128; P = 0.011] for taurine (Fig. 1fGo). No significant effects were observed for glycine (data not shown) in both HAS and LAS rats and for arginine (data not shown) in LAS rats. However, ethanol administration to LAS rats produced a significant increase in alanine (Fig. 1bGo), glutamate (Fig. 1eGo), and taurine (Fig. 1gGo) by the end of the experiment and the significance was [F (14, 254) = 2.136; P < 0.05], [F (14, 254) = 2.455; P < 0.01], and [F (14, 254) = 2.188; P < 0.05] respectively for alanine, glutamate, and taurine.

Blood-alcohol level
Figure 2Go shows the rate of elimination of ethanol in the HAS and LAS rats. The maximum blood-alcohol level for each group of rats at 30 min after a 2 g/kg dose of ethanol was not significantly different: 318 ± 10 and 326 ± 15 mg/dl respectively. Although LAS rats showed a higher rate of clearance of ethanol from the blood than HAS rats the difference was not statistically significant.



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Fig. 2. Rate of elimination of ethanol in HAS and LAS rats. HAS and LAS male rats (n = 7 each) were injected i.p. with 2 g/kg of ethanol as a 15% (w/v) solution in saline. A 10-µl blood sample was taken in duplicate every 30 min for up to 6 h. Blood-ethanol levels are presented as means ± SEM (bars) in mg/dl.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
In this study, the extracellular levels of aspartate, glutamate, glycine, arginine, taurine, and alanine in the NAC of HAS and LAS rats, which were selected for their sensitivity to ethanol, have been compared after injection of ethanol, using a microdialysis technique.

The basal concentration (Table 1Go) of each amino acid was statistically similar in both HAS and LAS rats. Furthermore, these basal amino acid concentrations were comparable to Wistar rats which were ethanol naive (Dahchour et al., 1996Go). However, the changes in amino acids assayed during the 300-min microdialysis period in the NAC of these two groups of rats with differing sensitivities to ethanol showed significant differences in their responses to ethanol administration. Apart from glycine, each of the other amino acids showed (Fig. 1Go) discordant responses between the HAS and LAS rats after ethanol injection, in comparison to their saline controls. In the HAS rats, taurine concentration in the microdialysate responded quickly, 20 min after i.p. ethanol injection. Such a response is consistent with our previous data with alcohol-naive Wistar rats after a comparable acute ethanol dose (Dahchour et al., 1994Go, 1996Go). However, in the LAS rats, microdialysate, levels of taurine showed a delayed, but significant, increase at 120–180 min after ethanol administration. Such differences between the HAS and LAS rats were not due to alterations in ethanol metabolism (Draski et al., 1992Go; Thomas et al., 1998Go), since the blood-ethanol contents were similar in both groups at 30 and 60 min. In our previous studies, we suggested that the cause of the increased taurine microdialysate concentration was due to ethanol-induced changes in osmolarity, ultimately evoking taurine efflux from the cell (Dahchour et al., 1994Go, 1996Go). The LAS rats did not exhibit the efflux of taurine, even when ethanol concentrations were high, at 30 min, which might indicate another aetiology for the ethanol-induced taurine efflux. Since the clearance of ethanol by LAS rats was not significantly faster than in HAS rats, one possibility is a higher acetaldehyde concentration in the NAC, which evokes changes in the taurine microdialysate. Another possibility is an enhanced change in osmolarity leading to flux of taurine from the cell (Ward et al., 1997Go).

Glutamate levels were significantly decreased in HAS rats after ethanol administration. However, ethanol administration produced a delayed increase in LAS rats in comparison to their saline controls. These changes in glutamate are in accordance with the hypothesis that excitatory amino acids, particularly glutamate, take part in genetically determining the sensitivity to ethanol, and support the hypothesis that ethanol exerts its effects in part by altering glutamatergic neurotransmission (Wilson et al., 1990Go).

There were decreases in the levels of both arginine and alanine from NAC after ethanol administration in both lines of rats, but a greater decrease in HAS rats. Such changes in arginine could be related to alterations in nitric oxide (NO) production by nitric oxide synthases (NOS). Currently, it is unknown how brain NOS are affected after acute doses of ethanol given to alcohol-naive rats. However, such significant changes in arginine in our two animal lines may indicate alterations in the generation of NO from the substrate arginine by NOS after ethanol administration. Whether this reflects an increase or a decrease in NO production remains to be elucidated. An elevation in NO would be detrimental, while studies have shown beneficial effects after supplementation with NOS inhibitors during alcohol withdrawal (Uzbay et al., 1997Go). This might explain the vulnerability and the resistance to ethanol of HAS and LAS rats respectively and suggests that some of the behavioural effects of ethanol may be mediated by alteration in arginine content. Our findings are consistent with studies which showed that ethanol (25–200 mM) inactivated NOS and that this could be prevented by l-arginine (Fataccioli et al., 1997Go). Stimulation of NMDA receptors enhances the production of NO (Pantazis et al., 1998Go) while their inhibition by ethanol diminishes NOS activity in cortical neurons and decreases NO formation in the brain (Green et al., 1997Go).

The neurochemical role of alanine in the brain remains unknown. However, high concentrations of this amino acid were demonstrated in the microdialysate from these two groups of rats. Although the basal concentrations (Table 1Go) of these amino acids were not statistically different between HAS and LAS rats, ethanol administration markedly reduced alanine in HAS (Fig. 1aGo) rats when compared to the saline group. We do not know what role alanine might play in the sensitivity of HAS rats to ethanol; nevertheless, alanine has been proposed to play the role of precursor of glutamate and could produce an increase in aspartate under hypoxic and ischaemic conditions (Griffin et al., 1998Go). The enantiomer d-alanine could play a role in Alzheimer's disease (D'Aniello et al., 1992Go). The significantly decreased level of alanine brought about by ethanol in HAS, but not LAS, rats, raises the question whether this amino acid plays a role in the sensitivity of these rats to ethanol.

This microdialysis study of rats, which have been selected for their sensitivity to ethanol, clearly demonstrated that HAS and LAS rats respond differentially to ethanol with regard to many amino acids in the NAC. Our results suggest that such changes in these amino acids after ethanol injection may play an important part in their different sensitivities to ethanol and may reflect their different genetic make up. Since the animals were selected only for differences in ethanol sensitivity, any neurochemical difference between the lines is presumed to be related to the mechanism of action of ethanol. The other possibility is that the differences are due to genetic drift and fixation. This possibility is considerably diminished, since the genetically independent replicate HAS and LAS lines did not differ.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
We thank Dr R. Ward for her help in the preparation of this manuscript and Dr Greg Gerhardt for his assistance in the work. This work was supported by the Fonds de la recherche Scientifique et Médicale (1997–2000), L'Institut de Recherches Economiques sur les Boissons (IREB), LIPHA and the National Institute of Alcohol Abuse and Alcoholism (#AA00093, #AA11464).


    FOOTNOTES
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 ABSTRACT
 INTRODUCTION
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* Author to whom correspondence should be addressed. Back


    REFERENCES
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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