Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Opioidergic and serotonergic neurotransmissions in the central nervous system have been shown to play significant roles in alcohol abuse or alcoholism (Gianoulakis, 1993; LeMarquand et al., 1994; Nevo and Hamon, 1995a). A number of studies have indicated a link between µ-opioid receptors and serotonin_1A [5-hydroxytryptamine_1A (5-HT_1A)] receptors to alcohol preference, tolerance, and dependence (Wong et al., 1990; Hyytia, 1993; McBride et al., 1998). In addition, both µ-opioid receptors and 5-HT_1A receptors belong to a superfamily of seven-transmembrane-domain (heptahelical) receptors that couple to the same subgroup of G protein, an inhibitory GTP-binding regulatory protein (G_i/G_o). This subfamily of G proteins (G_i/G_o) is sensitive to pertussis toxin and able to mediate a variety of effector systems, such as inhibition of adenylyl cyclase, reduction in calcium channel conductance, and activation of potassium channels when activated by either endogenous or extracellular signals (i.e., hormones, neurotransmitter, or agonists; Aghajanian and Wang, 1986; Hescheler et al., 1987; Harrington et al., 1992). Theoretically, ethanol is assumed to interfere with the process of signal transduction by acting at any part of the cascade chain from the membrane receptor to G protein and thus to the effector. In fact, ethanol was found to influence the signal transduction cascade at the level of receptor through, for example, both µ-opioid receptor and 5-HT_1A receptors, even though their associated cellular functions were not monitored at the same time (Nevo et al., 1995b; Winkler et al., 1998), or at the level of G proteins such as G_s and G_i/G_o (Wand et al., 1993; Ozawa et al., 1994) and at the level of effector such as adenylyl cyclase (Tabakoff and Hoffman, 1979; Wand et al., 1993). However, whether the functional coupling of these two receptors to their G proteins is affected by ethanol has not been investigated. The advent of an assay based on measurement of agonist-stimulated [³⁵S]guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPS) binding allows the coupling efficiency between specific receptors and their G proteins to be monitored by radioligand binding in membranes (Traynor and Nahorski, 1995) or by quantitative autoradiography on brain slices (Sim et al., 1995).

The Fawn-Hooded (FH) rat, a strain of inbred rat with high alcohol preference, consumes large amounts of ethanol in a two-bottle free-choice situation (Rezvani et al., 1990; Chen et al., 1998). In addition, behavioral studies demonstrated that compounds such as opioid receptor antagonists and 5-HT_1A receptor agonists were able to reduce the ethanol consumption in FH rats (Rezvani et al., 1991; Cowen et al., 1999). Therefore, it is hypothesized that the function of both µ-opioid receptors and 5-HT_1A receptors in mesocorticolimbic structures may be affected by ethanol consumption in FH rats. To test this hypothesis, brain sections at the level of the nucleus accumbens and the hippocampus from FH rats under a paradigm of ethanol self-administration with or without 48-h withdrawal were analyzed by means of quantitative in vitro autoradiography. Specifically, a comparative study of µ-opioid receptor- and 5-HT_1A receptor-mediated [³⁵S]GTPS binding was mapped throughout selected brain regions from the different experimental groups.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

All experiments were performed in accordance with the Prevention of Cruelty to Animals Acts 1986 under the guidelines of the Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia.

Materials. [³⁵S]GTPS (1250 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA). GDP, GTP, and [D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) were obtained from Sigma Chemical Co. (St. Louis, MO). L694,247 was purchased from Tocris Cookson (Bristol, UK). ¹⁴C standard microscales were purchased from ARC (St. Louis, MO). Kodak Biomax AR films were obtained from Kodak IBI (New Haven, CT). All other reagents were of either analytical or laboratory grade and obtained from various suppliers.

Ethanol Consumption. Male FH rats (n = 15, 350-400 g; stock parents obtained from Dr. Amir Rezvani, University of the North Carolina School of Medicine, Chapel Hill, NC) were assigned into three groups: alcohol-naïve FH [FH (naïve)], FH after chronic alcohol self-administration [FH (chronic)], and FH with 48-h withdrawal after chronic ethanol [FH (withdrawal)]. In the FH (naïve) group (n = 5), FH rats were never exposed to ethanol, whereas in both the FH (chronic; n = 5) and FH (withdrawal; n = 5) groups, rats were given access to 5% ethanol for 50 days with or without 48-h withdrawal under a two-bottle free-choice paradigm. One drink container was filled with tap water and the other was filled with 5% ethanol. To monitor the fluid consumption, rats were housed individually in a 12-h light/dark cycle cage with free access to standard chow. The respective containers were weighed each day for 50 days to determine daily consumption rates of both ethanol and water. Drink container positions were randomly changed to avoid the development of place-preference. At the end of monitoring, rats were decapitated, and the brains were removed and frozen in isopentane cooled at 35°C and then stored at 80°C until cutting.

Autoradiography of Agonist-Stimulated [³⁵S]GTPS Binding to G Proteins. Brain sections (20 µm) at the level of the nucleus accumbens (bregma, 1.7-1.3 mm) and the hippocampus (bregma, 2.8 to 2.5 mm) (Paxinos and Watson, 1986) were cut on a cryostat at 20°C and thaw-mounted onto gelatin-chrome alum-coated slides. Slides were dried under a vacuum and stored desiccated at 80°C until use.

Data Analysis. Autoradiographic images on developed films were subsequently quantified (using microcomputer imaging device M4 image analysis; Imaging Research, St. Catherine's, Ontario, Canada) by comparison of optical density, under constant illumination, of the autoradiograms compared with the ¹⁴C standard microscales (Miller, 1991). The density of basal and agonist-stimulated [³⁵S]GTPS binding was expressed in dpm/mm² of targeted nucleus and was measured from four consecutive sections in each region in each rat. The results are also expressed as the net percent increase of agonist-stimulated [³⁵S]GTPS binding over the basal level. For each agonist, brain sections from five rats per group were used. Unless otherwise indicated, data are reported as the mean ± S.E. value.

Statistical Analysis. The statistics software program SigmaStat (Jandel Scientific, Costa Madre, CA) was used throughout. The comparison of difference between basal versus stimulated [³⁵S]GTPS binding, as well as differences between treatment groups, was determined by two-way ANOVA followed by Bonferroni's t test. One-way ANOVA was also used to compare the percentage of net increase of [³⁵S]GTPS binding between the different treatment groups. A significance level of P < .05 was used throughout.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Ethanol Consumption. FH rats in the FH (chronic) and FH (withdrawal) groups consumed 110.8 ± 9.8 and 104.2 ± 16.2 (ml/kg b.wt.) of 5% ethanol/day, respectively, corresponding to 4.4 ± 0.4 and 4.1 ± 0.6 (g/kg b.wt.) of ethanol/day. FH rats in both groups had a high preference (>75%) for 5% ethanol.

Autoradiography of DAMGO-Stimulated [³⁵S]GTPS Binding to G Proteins. Figure 1, A and D, shows that the basal [³⁵S]GTPS binding had a low density in most brain areas examined, although the hippocampus and amygdala were relatively higher than other regions. In the presence of 10 µM DAMGO, intense binding of [³⁵S]GTPS was found in the cerebral cortex, striatum, nucleus accumbens, lateral septum, amygdala, and the hippocampus (Fig. 1, B and E). The addition of unlabeled GTPS (10 µM) abolished detectable [³⁵S]GTPS binding, which was defined as nonspecific binding. A similar pattern of discrete distribution of the basal, as well as DAMGO-stimulated [³⁵S]GTPS binding, was obtained from three experimental groups: FH (naïve), FH (chronic), and FH (withdrawal) (Fig. 2, A-C). By quantitative comparison with the basal binding, a significant increase in [³⁵S]GTPS binding stimulated by DAMGO was observed in all tested brain regions in alcohol-naïve, ethanol-withdrawal FH rats and most brain regions in chronic ethanol self-administrated FH rats, except the frontal and parietal cortex and hippocampus (Fig. 2, A-C, and Table 1). Due to relatively high basal binding, the hippocampus and amygdala showed a low percentage of net increase in [³⁵S]GTPS binding, although they had a high density of [³⁵S]GTPS binding. There was no significant difference in basal [³⁵S]GTPS binding among the three experimental groups for each individual brain region, but varied density of basal [³⁵S]GTPS binding existed between different brain regions in each group (Fig. 2, A-C). Chronic ethanol self-administration produced an inhibition of DAMGO-stimulated [³⁵S]GTPS binding in most brain regions except the amygdala and frontal and parietal cortex (Table 1). Significant reduction in DAMGO-stimulated [³⁵S]GTPS binding (P < .05, n = 5) was found in the nucleus accumbens (19%), lateral septum (15%), and striatum (23%) compared with the FH (naïve) control, whereas other regions displayed only a trend toward decrease. The decreased DAMGO-stimulated [³⁵S]GTPS binding recovered to control levels in most regions after 48-h withdrawal from chronic ethanol treatment (Table 1).

View larger version (157K): [in this window] [in a new window]   Fig. 1.   Autoradiograms from coronal brain sections of the alcohol-naïve FH rats at the levels of the nucleus accumbens (A-C) and hippocampus (D-F), exposed to 40 pM [35S]GTPS and 2 mM GDP in either the absence (basal: A and D) or the presence of 10 µM DAMGO (B and E) or 10 µM L694,247 (C and F). Scale bar, 1.2 mm.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Quantitative autoradiographic binding (dpm/mm2) of [35S]GTPS to FH rat brain sections from three experimental groups exposed to 40 pM [35S]GTPS and 2 mM GDP in the absence (open column) or the presence (closed column) of 10 µM DAMGO. A, alcohol-naïve FH rats (n = 5). B, FH rats with chronic ethanol consumption (n = 5). C, FH rats with chronic ethanol consumption followed by 48-h withdrawal (n = 5). *P < .05, comparison between the increase in agonist-stimulated [35S]GTPS binding by 10 µM DAMGO and the basal by two-way ANOVA followed by Bonferroni's t test. AMG, amygdala; FP, frontal/parietal cortex; OT, occipital/temporal cortex; Cx, cortex; Hipp, hippocampus; NAcc, accumbens nucleus; LS, lateral septum.

View this table: [in this window] [in a new window]   TABLE 1 Net percentage of increase in 10 µM DAMGO- or L694,247-stimulated [35S]GTPS binding over basal in rat brain sections from FH (naïve), FH (chronic), or FH (withdrawal) groups Data are mean ± S.E. from four consecutive brain sections obtained from each rat, with five rats in each group.

Autoradiography of L694,247-Stimulated [³⁵S] GTPS Binding to G Proteins. The greatest effect of L694,247 (10 µM)-stimulated [³⁵S]GTPS binding was in the lateral septum (Fig. 1, C and F), which reached 60% binding over basal (P < .05, n = 5). Significant stimulation of [³⁵S]GTPS binding was also observed in the occipital and temporal cortex, cingulate cortex, claustrum, and amygdala. However, in the frontal and parietal cortex, hippocampus, nucleus accumbens, and striatum, L694,247 failed to produce any significant increase of [³⁵S]GTPS binding. Additionally, the pattern of the distribution of L694,247-stimulated [³⁵S]GTPS binding was identical among three experimental groups (Fig. 3, A-C). Chronic ethanol caused a trend toward decrease in L694,247-stimulated [³⁵S]GTPS binding in the cingulate cortex and lateral septum, but the reduction did not reach statistical significance (Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Quantitative autoradiographic binding (dpm/mm2) of [35S]GTPS to FH rat brain sections from three experimental groups exposed to 40 pM [35S]GTPS and 2 mM GDP in the absence (open column) or the presence (closed column) of 10 µM L694,247. A, alcohol-naïve FH rats (n = 5). B, FH rats with chronic ethanol consumption (n = 5). C, FH rats with chronic ethanol consumption followed by 48-h withdrawal (n = 5). *P < .05, comparison between the increase in agonist-stimulated [35S]GTPS binding by 10 µM L694,247 and the basal by two-way ANOVA followed by Bonferroni's t test. AMG, amygdala; FP, frontal/parietal cortex; OT, occipital/temporal cortex; Cx, cortex; Hipp, hippocampus; NAcc, accumbens nucleus; LS, lateral septum.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This study represents the first examination of agonist-stimulated [³⁵S]GTPS binding in the alcohol-preferring FH rats. Furthermore, the data indicate that µ-opioid receptor-stimulated binding is sensitive to chronic ethanol consumption, whereas 5-HT_1A receptor-stimulated binding is unaffected in this paradigm.

A significant increase in [³⁵S]GTPS binding was induced by the µ-opioid receptor agonist DAMGO (10 µM), which could be detected in the striatum, nucleus accumbens, and the claustrum and cingulate cortex, in agreement with previous findings (Sim et al., 1995) and parallels the dense distribution of µ-opioid receptors in these brain regions (Mansour et al., 1987). Moreover, the DAMGO-stimulated increase in [³⁵S]GTPS binding appears to be specifically mediated by the µ-opioid receptor (Sim et al., 1995). Similarly, the 5-HT_1A/1B/1D receptor agonist L694,247 was able to produce a significant increase of [³⁵S]GTPS binding over basal in the present study as well. However, in contrast to DAMGO, the increased [³⁵S]GTPS binding induced by L694,247 was found in only several mesocorticolimbic regions where 5-HT_1A receptors are densely distributed, especially in the lateral septum. It is worthwhile to mention that it was L694,247, a 5-HT_1A/1B/1D receptor agonist, but not a conventional 5-HT_1A agonist such 8-OH-DPAT or flesinoxan, that was selected to stimulate 5-HT_1A receptors in the present study. Previous studies have demonstrated that L694,247-stimulated [³⁵S]GTPS binding appears to be mediated by 5-HT_1A receptors rather than 5-HT_1B/1D receptors (Sim et al., 1997; Dupuis et al., 1998; Mize and Alper, 1999).

In the receptor-mediated [³⁵S]GTPS binding stimulated by either DAMGO or L694,247, the net increase in [³⁵S]GTPS binding was generally proportional to the regional density of corresponding receptors in the rat brain. Exceptionally, the increase in [³⁵S]GTPS binding stimulated by either DAMGO or L694,247 in the hippocampus, where both µ-opioid receptors and 5-HT_1A receptors are densely located, was less obvious compared with the high net increase seen in other brain regions. In comparison with other brain nuclei, the hippocampus exhibited a greater level of basal [³⁵S]GTPS binding. The topographic patterns of the distribution of [³⁵S]GTPS binding stimulated by DAMGO and L694,247 are quite different from each other, but the [³⁵S]GTPS binding produced by either of the individual agonists exhibited a similar pattern among alcohol-naïve, chronic ethanol, and withdrawal rat groups.

Under the long-term self-administration of ethanol, most brain regions showed a reduced percentage of net increase in [³⁵S]GTPS binding induced by DAMGO. Several basal ganglia/limbic structures (i.e., the caudate-putamen, nucleus accumbens, and lateral septum) were significantly affected by chronic ethanol exposure, whereas other regions, such as the frontal cortex and amygdala, displayed only a trend toward decrease. The decrease in DAMGO-stimulated [³⁵S]GTPS binding was diminished after 48-h ethanol withdrawal and back to control levels in most brain regions. Interestingly, a trend toward a slight "rebound" increase was produced by ethanol withdrawal in the amygdala and claustrum compared with alcohol-naïve FH rats. It is important to note that the reduced DAMGO-stimulated [³⁵S]GTPS binding after chronic ethanol does not reflect down-regulation of µ-opioid receptors. On the contrary, a previous study from our laboratory demonstrated that chronic ethanol consumption in FH rats leads to an up-regulation of µ-opioid receptors in such limbic regions (Cowen et al., 1999). Therefore, these data apparently indicate a reduced capacity of the receptor to couple G protein. Furthermore, these factors together suggest a substantial effect of ethanol consumption on µ-opioid receptor function, because [³⁵S]GTPS binding is reduced in the face of increased receptor number. As such, the present data may reflect an underestimation of the true impact of ethanol on µ-opioid receptor function and, therefore, physiological consequences. In contrast to DAMGO, L694,247-stimulated [³⁵S]GTPS binding was not significantly affected by chronic ethanol or withdrawal in all tested brain regions, whereas it showed a trend toward reduction in the cingulate cortex and lateral septum. Although 5-HT_1A receptors were reduced in the septum, subregions of the frontal cortex, and hippocampus in Wistar rats after 2 days of exposure to ethanol (Ulrichsen et al., 1997), whether this occurs in FH rats awaits confirmation. There are, however, several explanations for the results of L694,247-stimulated [³⁵S]GTPS binding. First, 5-HT_1A receptor functional coupling to G protein may not be impaired by chronic ethanol consumption in FH rats. Second, there may be a substantial redundancy of 5-HT_1A receptors such that a subtle change in receptor function may not be detected by the present technique.

It is likely that the direct effect of ethanol may not play a major role in the alteration of [³⁵S]GTPS binding stimulated by either agonist because both basal [³⁵S]GTPS binding and agonist-stimulated [³⁵S]GTPS binding are sensitive to direct application of ethanol (unpublished observations; Selley et al., 1996). Considering the fact that the ethanol-induced effect on [³⁵S]GTPS binding occurs in a receptor- and region-specific manner, it suggests that secondary mechanisms downstream of ethanol may be involved. Because agonist-induced increase in [³⁵S]GTPS binding is a measure of the interaction between the activated receptor and G protein, particularly the -subunit of G_i/G_o proteins (G_i/G_o), any changes occurring at the level of receptors or G proteins would influence coupling efficiency. Because agonist-stimulated [³⁵S]GTPS binding could also be regulated by G proteins themselves, it is understandable that any change in either the number or the conformational structures of G proteins might alter the ability of G proteins to bind [³⁵S]GTPS. Furthermore, the ratio of receptors to G proteins could influence agonist-stimulated [³⁵S]GTPS binding; for example, the potency of full agonist-stimulated [³⁵S]GTPS binding varied in Chinese hamster ovary cell lines under differential ratio of recombinant 5-HT_1A receptor to G proteins, which was assumed to derive from the change in the receptor/G protein stoichiometry (Newman-Tancredi et al., 1997). Evidence also supports that the number of G_i/G_o subunits are affected by chronic ethanol. In the study of Wand et al. (1993), an increased level of G_i was found in ethanol-sensitive mouse brain with chronic ethanol treatment, which was considered to be attributed to the decreased adenylyl cyclase activity induced by ethanol. In addition, data from postmortem brain demonstrated that ethanol-enhanced guanine nucleotide binding in human cortical membranes and the increase in G_s and G_i/G_o binding were decreased in all cortical regions of alcoholic patients (Ozawa et al., 1994). These conflicting results may come from the use of different techniques and subjects in different studies. It is noteworthy that the ethanol-induced alteration of G_i could vary according to different cultured cell lines when exposed to a high concentration of ethanol (100 mM) in in vitro studies (Charness et al., 1988). Whether such a phenomenon can be invoked to explain the differential alteration of [³⁵S]GTPS binding by chronic ethanol in brain sections in the present study is worth consideration. The unchanged basal [³⁵S]GTPS binding in the present study may suggest that the level of G proteins remained unchanged after ethanol consumption. However, the possibility of the regulation of the number of G_i/G_o proteins could not be completely ruled out from the present study because [³⁵S]GTPS is assumed to bind indiscriminately to the entire pool of G proteins, including G_i/G_o and G_s (Raymond, 1995), even though G_s was not readily detected by this measurement (Selley et al., 1997). The reduction in DAMGO-stimulated [³⁵S]GTPS binding might also reflect a reduced affinity of G_i/G_o to [³⁵S]GTPS, such that the significant reduction in µ-opioid receptor-mediated [³⁵S]GTPS binding or a desensitization of G_i/G_o proteins induced by chronic ethanol in the striatum, nucleus accumbens, and lateral septum may be ascribed to the intrinsic change in G_i/G_o (i.e., the ability of G_i/G_o to bind [³⁵S]GTPS; Tirone et al., 1988).

In summary, the functional couplings of both µ-opioid receptors (DAMGO-stimulated) and 5-HT_1A receptors (L694,247-stimulated) to their respective G proteins have been mapped through selected brain regions. Chronic ethanol caused a significant reduction in DAMGO-stimulated [³⁵S]GTPS binding in the striatum, nucleus accumbens, and lateral septum but did not cause any significant reduction in L694,247-stimulated [³⁵S]GTPS binding in all tested regions. DAMGO-stimulated [³⁵S]GTPS binding recovered to FH (naïve) levels after ethanol withdrawal. The mechanism underlying the region-specific and receptor-specific suppression of agonist-stimulated [³⁵S]GTPS binding by chronic ethanol remains unclear. The different subpools of G_i/G_o proteins coupling to a specific receptor or to the same receptor but located in different brain regions may determine the variable sensitivity to ethanol treatment. Other mechanisms may also be involved in ethanol-induced inhibitory effects on DAMGO-stimulated [³⁵S]GTPS binding, such as the number and affinity of receptors or G proteins.

In conclusion, this investigation is the first study to demonstrate the neuroanatomical functional coupling of G proteins to either µ-opioid receptors or 5-HT_1A receptors at the level of two main mesocorticolimbic regions in alcohol-preferring FH rat brain sections by measuring agonist-stimulated [³⁵S]GTPS binding via quantitative autoradiography. The reduced functional coupling of µ-opioid receptors to G proteins by chronic ethanol drinking in some brain regions, particularly in the mesocorticolimbic regions, may have some pathophysiological role in the effects of ethanol consumption in FH rats.