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Nefazodone attenuates the behavioral and neurochemical effects of ethanol
Alcohol,Vol. 15, No. l, pp. 77-86, 1998 Copyright© 1998ElsevierScienceInc. Printedin the USA.All rightsreserved 0741-8329/98$19.00+ .00 ELSEVIER
PII S0741-8329(97)00101-8
Nefazodone Attenuates the Behavioral and Neurochemical Effects of Ethanol PETER
OLAUSSON, MIA ERICSON, ANNELIE PETERSSON, ALEXANDER B O S O D E R P A L M A N D J O R G E N A. E N G E L
KOSOWSKI,
Department of Pharmacology, Institute of Physiology and Pharmacology, GOteborg University, Medicinaregatan Z S-413 90 GOteborg, Sweden R e c e i v e d 22 O c t o b e r 1996; A c c e p t e d 17 A p r i l 1997 OLAUSSON, P., M. ERICSON, A. PETERSSON, A. KOSOWSKI, B. SODERPALM AND J. A. ENGEL. Neff
Serotonin
Ethanol
Nefazodone
In vivo microdialysis
P H A R M A C O L O G I C A L experiments have previously demonstrated that ethanol, like other drugs of abuse, increases the activity in the mesocorticolimbic dopamine system, which is part of the brain reward system. This dopamine system originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens and the frontal cortex. Activation of the mesocorticolimbic dopamine system by ethanol has been demonstrated to result in dopamine overflow in the N Acc (4,17,28,68,691, and this effect has been suggested to be involved in mediating reward of drugs of abuse (15,67). Evidence indicates that the brain serotonin (5-hydroxytryptamine; 5-HT) system is also involved in reward-related mechanisms. The neuroanatomical substrate for an interaction between the 5-HT and dopamine systems has been confirmed by several studies, demonstrating that the VTA and the N Acc receive an input from serotonergic axon terminals originating in both the medial and dorsal raphe nuclei (4857). Furthermore, it has been shown that 5-HT terminals in the VTA form synaptic contacts with dopaminergic neurons (24). However, the specific mechanisms underlying this interaction, in terms
Ethanol intake
Rat
of, for example, the 5-HT receptor subtypes involved, appear to be very complex and are yet to be determined. Serotonergic drugs have been observed to modulate both neurochemical (26,27,46,68,69) and behavioral (9,12,46) responses to drugs of abuse, including ethanol (16,35,41,59). Administration of compounds that are believed to facilitate the brain 5-HT activity often decreases ethanol intake and preference in animals (16,35) and humans (2,33,34). Several different 5-HT receptor subtypes, i.e., the serotoninlA (5-HTtA), serotoninm (5-HTm), serotonin2 (5-HT2), and serotonin3 (5-HT3) receptors, have been suggested to be involved in these modulatory actions. Nefazodone is a 1,2A-triazole derivate with antidepressant activity (14). The most potent interaction sites of this compound are antagonism of 5-HT2A receptors and inhibition of the 5-HT reuptake carrier. Acutely, nefazodone also weakly inhibits the norepinephrine reuptake carrier, but it has only low affinity to or is inactive at other central receptor sites (14,23,64). Based on evidence indicating 5-HT as a modulator of the rewarding effects of drugs of abuse, the aims of the present study were to investigate the influence of nefazodone on etha-
Requests for reprints should be addressed to Peter Olausson. Department of Pharmacology, Institute of Physiology and Pharmacology, G6teborg University, Medicinaregatan 7, S-413 90 G(Steborg, Sweden. ]'el: +46-31-773-34-00:Fax: +46-31-82-17-95. 77
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nol-induced accumbal dopamine overflow and on voluntary ethanol intake in the male Wistar rat. METHOD
Animals Male Wistar rats weighing 250-320 g, supplied by BeeKay (Sollentuna, Sweden), were used in all experiments. The rats were housed five per cage until operation or ethanol preference screening, after which the animals were kept in separate cages. The animals were kept under constant cage temperature (25°C), humidity (60-65%), and controlled light-dark conditions (microdialysis studies: light on at 0500 h and off at 1900 h; ethanol drinking studies: light off at 1000 h and on at 2200 h). The rats had free access to food and liquid (microdialysis studies: tap water; ethanol drinking studies: tap water and ethanol solution) at all times. Animals were allowed to adapt for at least 1 week to the animal department facilities before the start of any experiment. In all experiments drug naive animals were used and each animal was used in only one experiment. The study was approved by the Ethics Committee for Animal Experiments, G6teborg, Sweden.
Drugs Nefazodone HC1 (BMY 13754), a gift from Bristol-Myers Squibb, Scandinavia, ethanol (95%; Kemetyl, Stockholm, Sweden), and NaCI (Merck, Darmstadt, Germany) were used in the study. Nefazodone was dissolved in distilled water to 5 mg/ml and injected subcutaneously (SC) in a volume of 10 ml/ kg. Ethanol was diluted to 15% w/v with physiological saline (0.9% NaC1) and injected intraperitoneally (IP) in a volume of 16 ml/kg.
Microdialysis Technique To extract dopamine and dopamine metabolites, a microdialysis probe was implanted into the rat brain. The microdialysis was performed with an I-shaped probe [for details see (65)]. The rats were anaesthetised with ketamin 50 mg/ml (Parke-Davies, Barcelona, Spain) and xylazin 20 mg/ml (Bayer, Leverkusen, Germany) in a mixture of 2:1, that was injected in a volume of 2 ml/kg (IP). The microdialysis probe was then implanted in the N Acc of the brain by a stereotactic operation, using a Kopf stereotaxic instrument, and fixed to the skull using Phosphatine dental cement (Svedia Dental Industri, Sweden). The stereotactic coordinates of the probe relative to bregma were A/P +1.85, L/M -1.4, V/D - 7 . 8 (50). To substitute any loss of body liquids that may occur due to the operation, the rats were injected with 3 ml of 0.9% NaC1 (SC) and were allowed to recover for at least 40 h before the start of the microdialysis experiment. At the beginning of the experiment the inlet of the probe was connected to a perfusion pump C M A 100 (Carnegie Medicin, Stockholm, Sweden) via a swivel, and the outlet was connected to a collecting tube. The swivel allowed the animals to move freely during the experiment. The probe was then perfused with Ringer solution containing in mM: NaC1 140, CaCI 2 1.2, KCI 3.0, and MgC12 1.0 [modified from (40)]. The perfusion rate during the experiment was 2 txl/min and dialysate fractions (40 p.l) were collected every 20 rain.
Biochemical Assay To determine the concentration of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid
(HVA) in the dialysate, two different high pressure liquid chromatography systems with electrochemical detection (HPLC-EC) were used. To identify the peaks in the chromatogram an external standard containing 264 fmol/p~l of dopamine, 297 fmol/p.l of D O P A C , and 274 fmol/txl of H V A was used. The data sampled were analysed with a Dynamax HPLC method manager v. 1.3 (Instrument AB Lambda, Sollentuna, Sweden).
Microdialysis Experimental Design Animals were prepared for the dialysis experiment and connected to the perfusion pump. A minimum of 90 min were allowed for stabilisation before any sample was collected. The dialysate was then collected in 20-min fractions (40 I~1), which were analysed until stable basal values (_+ 10%) were obtained for each animal, at least four samples (80 min), before any drug was injected. When basal values were stable, the rats received nefazodone (25 or 50 mg/kg, SC) or vehicle. Two samples were collected (40 min) before ethanol (15% w/v; 2.5 g/kg, SC) or vehicle was injected. A t least five more samples (100 min) were collected before the microdialysis experiment was ended. The basal levels of dopamine or metabolites before any drug treatment were set to 100%. The biochemical effects of drug or vehicle injections were related to this baseline. All microdialysis values are presented as the mean peak values + SEM, expressed as percent of baseline.
Screening for Ethanol Preference Rats had free access to one bottle of water and one bottle containing a weak ethanol solution at all times. The ethanol concentration was gradually increased ( 2 4 - 6 % v/v) over a 2-week period. During the screening period the animals were housed individually in clear plastic cages. Thereafter, they had continuous access to two bottles (plastic 300-ml bottles with ballvalve sprouts; A L A B , Sweden) containing either tap water or 6% (v/v) ethanol solution. This ethanol concentration was used because previous observations (18) indicate that the ethanol consumption is maximal at approximately this concentration in the strain of rats used in the present study. Water and ethanol intake were then measured during a 4-week period, twice a week, when the bottles were also cleaned and filled with fresh liquids. The positions of the two bottles were also shifted at this occasion (i.e., twice a week). Body weight was recorded once a week throughout the screening period. The amount (grams) of ethanol solution 6% (v/v) consumed in percent of total fluid intake (grams) was used as an index of ethanol preference. Rats were classified as low-preferring (LP; <~20% ethanol) or high-preferring (HP; ~<60% ethanol) based on their preference for the ethanol solution over water.
Ethanol Drinking Study Experimental Design HP and LP rats were injected with nefazodone (50 mg/kg, SC) or vehicle in a counterbalanced cross-over fashion. The dose of nefazodone was chosen based on previous experience from the microdialysis experiment (see the Result section). Injections were given at 0930 h each day for two periods of three consecutive days, four days separating the periods. Ethanol consumption was measured between 1000 and 1700 h all treatment days, and the 3-day mean consumption and preference values for each animal was determined and used in all calculations. Both bottles were present during the whole mea-
NEFAZODONE
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FIG. 1. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced dopamine overflow in the rat N Acc. All values are expressed as means + SEM. Statistics: ANOVA for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal dopamine levels were significantly different compared to control [veh + veh (n = 8) vs. veh + EtOH (n = 9) p = 0.0073; veh + veh (n = 8) vs. nef(25) + EtOH (n = 8) p - 0.0164; veh + EtOH (n = 9) vs. nef(50) + EtOH (n = 9) p = 0.0132]. The inserted histogram shows accumbal dopamine levels in the 60-min fraction (i.e., 60 min after veh/nef and 20 min after veh/EtOH). The symbol $ indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on dopamine levels in the rat N Acc. All values are expressed as means + SEM. Statistics: ANOVA for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal dopamine levels were significantly different compared to control [veh + veh (n = 8) vs. nef(50) + veh (n = 8) p = 0.0199]. The symbol 1" indicates time of injection.
80
O L A U S S O N ET AL.
suring period and the positions of the bottles were shifted at the end of each treatment day. Body weight was recorded before and after each treatment period.
Statistics The microdialysis data were statistically analysed using a two-way analysis of variance ( A N O V A ) , followed by the Fisher's protected least significant difference (PLSD) test implemented for comparisons with unequal ns (1). The data acquired in the ethanol drinking study were evaluated using the Wilcoxon signed rank test. A probability value of p < 0.05 was considered statistically significant.
12.7 g/kg ( p = 0.0251), and ethanol preference by 22%, from 69 to 47% ( p = 0.0251), in HP (n = 8) but not LP (n = 15) rats, when measured between 1000 and 1700 h (Fig. 4a, b). LP rats significantly lowered their water intake by 35%, from 35.0 to 23.0 g/kg (p = 0.0007), after injection of nefazodone (50 mg/kg, SC), whereas the water intake was not altered by nefazodone in the HP rats ( p = 0.6744; Fig. 4c). The total fluid intake was decreased by 30% in HP ( p = 0.0357), and by 33% in LP ( p = 0.0008) rats. The body weight was not affected by the 3-day nefazodone treatment (p = 0.6496, n = 23; data not shown). DISCUSSION
RESULTS
Effects on Extracellular Dopamine Levels" Administration of ethanol (2.5 g/kg, IP) produced a statistically significant increase of the dopamine levels in the first two dialysate samples (p = 0.0073; n = 9), i.e., within 40 min of the ethanol injection. The peak extracellular dopamine level (136 _+ 9%) was observed in the first sample. The concentration of dopamine then gradually decreased and reached baseline levels 60 min after the ethanol injection (Fig. la). Administration of nefazodone (50 mg/kg, SC) significantly increased extracellular dopamine levels ( p = 0.0199: n = 8) in the N Acc. The peak dopamine level (128 _+ 11%) was observed in the fraction collected 40 min after the nefazodone injection (Fig. lb). The low dose of nefazodone (25 mg/kg, SC) significantly increased the accumbal dopamine levels by 15% in the 60-min fraction ( p = 0.0459; n = 6). Nefazodone (50 mg/kg, but not 25 mg/kg, SC) reduced ethanol-stimulated dopamine overflow ( p = 0.0132: n = 9), and the dopamine content observed after coadministration of nefazodone and ethanol did not differ from baseline levels. The nefazodone-pretreated group reached basal dopamine levels 40 min after ethanol administration and the lowest dopamine concentration (71 _+ 8%) was found after 80 rain (Fig. la).
Effects, on Extracellular Levels of Doparnine Metabolites Ethanol (2.5 g/kg, IP) significantly increased D O P A C (p 0.0093: n = 11) and H V A ( p = 0.001; n - 8) overflow (Figs. 2a and 3a). The highest D O P A C content (126 _+ 7%) was observed in the third sample, 60 min after ethanol injection, whereas the highest H V A levels (152 _+ 8%) were observed 100 min after ethanol (e.g., in the 140-rain fraction). A dose of 50 mg/kg (SC) nefazodone did not significantly alter D O P A C or H V A levels, whereas the low dose of nefazodone (25 mg/kg, SC) significantly increased both accumbal D O P A C ( p = 0.0074; n = 6) and H V A ( p = 0.0121: n - 6) during the first 60 and 80 min after injection, respectively (Figs. 2b and 3b). The peak D O P A C and H V A levels were observed in the third sample, collected 60 min after nefazodone injection (127 _+ 15% and 131 -+ 19%, respectively). Administration of nefazodone (50 mg/kg, SC) significantly decreased the ethanol-induced levels of H V A (p = 0.0159; n = 9), but not DOPAC, in the N Acc, whereas nefazodone (25 mg/kg, SC) had no effect on the ethanol-induced changes of the dopamine metabolites (Figs. 2a and 3a).
Effect on Voluntary Ethanol lntake Administration of nefazodone (50 mg/kg, SC) significantly decreased the ethanol solution intake by 51%, from 25.8 to
Ethanol and Accumbal Dopamine Overflow In the present microdialysis experiments ethanol (2.5 g/kg, IP) significantly increased accumbal dopamine overflow in freely moving rats, a result in line with previous reports (4,8, 22,68,69). This effect may involve both increased neuronal firing and enhanced dopamine release, and could be exerted by a direct action on the dopamine system or indirectly via interference with other central neurotransmitter systems. Evidence has accumulated indicating that direct interference with ligand-gated ion channels, such as the 5-HT 3 receptor (8,68,69), the N-methyl-D-aspartate receptor (11), the y-aminobutyric acid (GABA)A/benzodiazepine receptor (58), and the nicotinic acetylcholine receptor (4,5), may be involved in the dopamine activating effects of ethanol. However, the specific mechanisms underlying the ethanol-induced activation of the mesolimbic dopamine system remain to be elucidated. Previous experiments have demonstrated that ethanolinduced accumbal dopamine overflow is associated with increased D O P A C and H V A levels (4,22), supporting the present observations of elevated dopamine metabolite levels in the N Acc after ethanol injection. This increase has been suggested to reflect an elevated level of dopamine metabolism due to the increased dopamine overflow. However, these findings are not entirely consistent with those of Yoshimoto et al. (69), who observed a significant elevation of HVA, but not of DOPAC, levels after acute ethanol administration.
N
NEFAZODONE AND ETHANOL
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FIG. 2. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced increase of D O P A C in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal D O P A C levels were significantly different compared to control [veh + veh (n = 8) vs. veh + E t O H (n = 11) p = 0.0093; veh + veh (n = 8) vs. nef(25) + E t O H (n = 8) p = 0.0018]. The symbol 1" indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on D O P A C levels in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated m e a s u r e s followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal D O P A C levels were significantly different compared to control [veh + veh (n = 8) vs. nef(25) + veh (n = 6) p = 0.0074]. The symbol 1" indicates time of injection.
82
OLAUSSON
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ET AL.
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FIG. 3. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced increase of H V A in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated m e a s u r e s followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal H V A levels were significantly different compared to control [veh + veh (n = 8) vs. veh + E t O H (n = 8) p = 0.0010; veh + veh (n = 8) vs. nef(25) + E t O H (n = 8) p = 0.0046; veh + E t O H (n = 8) vs. nef(50) + E t O H (n = 9) p = 0.0159]. The symbol $ indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on H V A levels in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal H V A levels were significantly different compared to control [veh + veh (n = 8) vs. nef(25) + veh (n - 6) p - 0.0121]. The symbol 7 indicates time of injection.
N E F A Z O D O N E AND E T H A N O L
83
a 30-
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dopaminergic activity (3,29,44). Thus, 5-HT applied both in the VTA or the N Acc increases accumbal dopamine levels (21,49). The dopamine release after 5-HT application in the N Acc most likely involves terminal 5-HT 3 receptors (8,46,55), whereas the effect of 5-HT in the VTA, which is mimicked by 5-HT m agonists (21), could be mediated via activation of 5-HTlb receptors decreasing the descending G A B A tone (30). Although local application of 5-HT and 5-HT agonists produce the effects mentioned above, systemic administration of selective 5-HT reuptake inhibitors (SSRI) generally fail to alter accumbal dopamine overflow (20,61). Thus, the reuptake inhibitory effect of nefazodone is not by itself likely to explain the increased dopamine overflow in the N Acc. It may be suggested that the combined effects of nefazodone may produce a dopamine activating effect larger than the stimulation induced by 5-HT2A receptor antagonism or 5-HT reuptake inhibition alone. As mesolimbic dopamine stimulation is considered to be involved in substance abuse, the dopamine activating property of nefazodone could indicate abuse liability. However, this possibility appears less likely, because nefazodone did not reinforce self-administration in primates (19). The mesolimbic dopamine activating effect of nefazodone could instead be an additional mechanism of importance for the antidepressant property of nefazodone (54,66). Although nefazodone (50 mg/kg, SC) increased accumbal dopamine overflow, the D O P A C or HVA levels were not altered, a pattern in line with that obtained after 5-HT~ antagonists (13). Such a pattern has, however, also been described after dopamine reuptake inhibitors (25). Whether nefazodone interferes with the dopamine reuptake carrier is Unknown, but because the lower dose (25 mg/kg, SC) slightly raised both accumbal dopamine (see above) and metabolite levels, this possibility appears less likely.
Coadministration of Nefazodone and Ethanol and Accumbal Dopamine Overflow HP
LP
C 407
Pretreatment with nefazodone (50 mg/kg, SC, - 4 0 min) strongly attenuated ethanol-stimulated dopamine overflow in the N Acc. The extracellular levels of D O P A C and HVA also tended to be lower in rats pretreated with nefazodone, although this difference was significant for HVA only. It is noteworthy that only the high dose of nefazodone (50 mg/kg, SC), which activated the mesolimbic dopamine system per se, altered the neurochemical response to ethanol. Thus, ethanol
352 --~30,
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FIG. 4. (a) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on ethanol solution (6%) intake between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are
the mean daily ethanol consumption per kg body weight during 3 consecutive days of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxon signed rank test. *p < 0.05 compared to vehicle treatment. (b) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on ethanol solution (6%) preference between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are the mean daily ethanol preference during 3 consecutive clays of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxon signed rank test. *p < 0.05 compared to vehicle treatment. (c) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on water intake between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are the mean daily water consumption per kg of body weight during 3 consecutive days of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxonsigned rank test. ***p < 0.001 compared to vehicle treatment.
84
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was injected during a slightly elevated baseline, which could have masked an enhancement of dopamine overflow after ethanol. However, in these experiments, the dopamine levels induced by nefazodone were lower than those after ethanol, suggesting that further dopaminergic stimulation after ethanol could have been obtained. Instead, according to the present results, the interaction between the two drugs results in a decreased mesolimbic dopamine activity, compared to that after either of the two drugs administered alone. The complex pharmacology of nefazodone suggests that the nefazodone-induced attenuation of ethanol-stimulated accumbal dopamine overflow could result from one or more of its interactions. However, 5-HT2A antagonists have previously been demonstrated to reduce behavioral activation associated with excessive dopaminergic neurotransmission (26,31,46,56), as well as drug-induced enhancement of dopamine release (26,46). Moreover, exogenous 5-HT, as well as 5-HT 2 receptor agonists, potentiate the ethanol-stimulated excitation of dopamine cell-firing in the VTA (6,62), whereas, according to in vitro observations by Brodie and Trifunovic (7,62), ethanolinduced excitation of mesolimbic dopamine neurons could be reduced by the 5-HT 2receptor antagonist ketanserin. Taken together, these results suggest that nefazodone preferentially, by means of its 5-HT2A receptor antagonistic properties, decreases ethanol-stimulated dopamine overflow in the N Acc. In conclusion, nefazodone counteracts the dopamine-activating effects of ethanol, whereas it per se elevates mesolimbic dopamine overflow (i.e.. nefazodone appears to interfere with dopaminergic neurotransmission in an activity-dependent manner). The mechanism underlying this apparently paradoxical effect of nefazodone remains to be elucidated. It is interesting to note, however, that the 5-HT2A receptor antagonists MDL 100,907 and ritanserin have been demonstrated to attenuate the effects of other psychomotor stimulants, such as the dopamine reuptake blocker G B R 12909, the N-methyl-D-aspartate receptor antagonist MK-801, the muscarinic receptor antagonist atropine, and amphetamine (10,53). On the basis of these findings, Carlsson (10) proposed that the 5-HT 2 receptor may have a "permissive" role for psychomotor activation.
Nefazodone and Voluntary Ethanol Intake Interestingly, nefazodone (50 mg/kg), unlike the related drug trazodone (37), decreased both ethanol solution intake and ethanol preference in HP but not LP rats from a nonselected Wistar strain. Thus, although a dose-response relationship was not established, the present results indicate that nefazodone in a dose that prevents ethanol-induced dopamine overflow (see above) also lowers ethanol intake in animals consuming quantities (approx. 1.6 g/kg/7 h) sufficient to enhance dopamine overflow (22,28,69). This indicates, but does not prove, that pharmacological actions of nefazodone on the mesolimbic dopamine system are involved in the behavioral effect observed. It should be noted that nefazodone also decreased water intake in LP but not in HP rats. Thus, in
both LP and HP rats nefazodone reduced the intake of the preferred solution only. Taken together with findings indicating that the mesolimbic dopamine system is activated also by natural rewards (36,39), these results may suggest that nefazodone interferes not only with ethanol reward (HP rats), but also with water reward (LP rats). An alternative explanation to the behavioral findings is that nefazodone decreases general ingestive behaviors and thereby reduces ethanol intake (41), an interpretation supported by the fact that the total fluid intake was decreased both in HP and LP rats. However, in this case also a lowering of the intake of the nonpreferred solution would have been expected, at least in HP rats in which the water intake certainly was large enough to allow a decrement. In LP rats the lack of an effect could be due to a floor effect. Furthermore, even though food intake was not measured, the body weights were not affected during the 3-day treatment period (see Results section), supporting the conclusion that nefazodone interferes mainly with reward-related mechanisms and not with ingestive behaviors in general. Although some previous studies on the effect of 5-HT 2 antagonists, possibly acting in the N Acc (47), on voluntary ethanol intake support the present results (37,38,42,47), there are also contradictory reports available (43,59,60). On the other hand, SSRIs consistently decrease voluntary ethanol intake in both rats and humans (2,16,34,35,41), suggesting that indirect nefazodone-induced stimulation of 5-HT receptors involved in ethanol self-administration [e.g., 5-HTjA receptors (12,16, 33,59)] may also be involved in the effect of nefazodone on voluntary ethanol drinking. CONCLUSION The present study demonstrates that both ethanol and nefazodone increases accumbal dopamine levels, whereas nefazodone pretreatment reduces the ethanol-induced dopamine overflow. Although further experiments are needed to determine the specific mechanism(s) underlying this inhibition, circumstantial evidence suggests that 5-HT2A receptor inhibition may be involved in a dopamine activity-dependent manner. Interestingly, nefazodone also decreases voluntary ethanol intake in HP Wistar rats, suggesting a functional effect of nefazodone possibly related 1o the neurochemical effect described above. Because the models used here have been shown to be of predictive value for the effect of ethanol-intake reducing drugs in man, the present findings suggest that nefazodone could be useful in future pharmacological treatment of alcohol abuse. ACKNOWLEDGEMENTS This study was financially supported by the Swedish Medical Research Council (grants No. 4247 and 11583), the Swedish Society of Medicine, Magnus Bergvalls stiftelse, ~.ke Wibergs stiftelse, Systembolagets fond f6r alkoholforskning, and Bristol-Myers Squibb, Scandinavia. The authors also thank Dr. Lennart Svensson, Dr. Ola Blomqvist, Gun Andersson, and Kenn Johannessen for helpful advice and assistance throughout the study.
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PII S0741-8329(97)00101-8
Nefazodone Attenuates the Behavioral and Neurochemical Effects of Ethanol PETER
OLAUSSON, MIA ERICSON, ANNELIE PETERSSON, ALEXANDER B O S O D E R P A L M A N D J O R G E N A. E N G E L
KOSOWSKI,
Department of Pharmacology, Institute of Physiology and Pharmacology, GOteborg University, Medicinaregatan Z S-413 90 GOteborg, Sweden R e c e i v e d 22 O c t o b e r 1996; A c c e p t e d 17 A p r i l 1997 OLAUSSON, P., M. ERICSON, A. PETERSSON, A. KOSOWSKI, B. SODERPALM AND J. A. ENGEL. Neff
Serotonin
Ethanol
Nefazodone
In vivo microdialysis
P H A R M A C O L O G I C A L experiments have previously demonstrated that ethanol, like other drugs of abuse, increases the activity in the mesocorticolimbic dopamine system, which is part of the brain reward system. This dopamine system originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens and the frontal cortex. Activation of the mesocorticolimbic dopamine system by ethanol has been demonstrated to result in dopamine overflow in the N Acc (4,17,28,68,691, and this effect has been suggested to be involved in mediating reward of drugs of abuse (15,67). Evidence indicates that the brain serotonin (5-hydroxytryptamine; 5-HT) system is also involved in reward-related mechanisms. The neuroanatomical substrate for an interaction between the 5-HT and dopamine systems has been confirmed by several studies, demonstrating that the VTA and the N Acc receive an input from serotonergic axon terminals originating in both the medial and dorsal raphe nuclei (4857). Furthermore, it has been shown that 5-HT terminals in the VTA form synaptic contacts with dopaminergic neurons (24). However, the specific mechanisms underlying this interaction, in terms
Ethanol intake
Rat
of, for example, the 5-HT receptor subtypes involved, appear to be very complex and are yet to be determined. Serotonergic drugs have been observed to modulate both neurochemical (26,27,46,68,69) and behavioral (9,12,46) responses to drugs of abuse, including ethanol (16,35,41,59). Administration of compounds that are believed to facilitate the brain 5-HT activity often decreases ethanol intake and preference in animals (16,35) and humans (2,33,34). Several different 5-HT receptor subtypes, i.e., the serotoninlA (5-HTtA), serotoninm (5-HTm), serotonin2 (5-HT2), and serotonin3 (5-HT3) receptors, have been suggested to be involved in these modulatory actions. Nefazodone is a 1,2A-triazole derivate with antidepressant activity (14). The most potent interaction sites of this compound are antagonism of 5-HT2A receptors and inhibition of the 5-HT reuptake carrier. Acutely, nefazodone also weakly inhibits the norepinephrine reuptake carrier, but it has only low affinity to or is inactive at other central receptor sites (14,23,64). Based on evidence indicating 5-HT as a modulator of the rewarding effects of drugs of abuse, the aims of the present study were to investigate the influence of nefazodone on etha-
Requests for reprints should be addressed to Peter Olausson. Department of Pharmacology, Institute of Physiology and Pharmacology, G6teborg University, Medicinaregatan 7, S-413 90 G(Steborg, Sweden. ]'el: +46-31-773-34-00:Fax: +46-31-82-17-95. 77
78
O L A U S S O N ET AL.
nol-induced accumbal dopamine overflow and on voluntary ethanol intake in the male Wistar rat. METHOD
Animals Male Wistar rats weighing 250-320 g, supplied by BeeKay (Sollentuna, Sweden), were used in all experiments. The rats were housed five per cage until operation or ethanol preference screening, after which the animals were kept in separate cages. The animals were kept under constant cage temperature (25°C), humidity (60-65%), and controlled light-dark conditions (microdialysis studies: light on at 0500 h and off at 1900 h; ethanol drinking studies: light off at 1000 h and on at 2200 h). The rats had free access to food and liquid (microdialysis studies: tap water; ethanol drinking studies: tap water and ethanol solution) at all times. Animals were allowed to adapt for at least 1 week to the animal department facilities before the start of any experiment. In all experiments drug naive animals were used and each animal was used in only one experiment. The study was approved by the Ethics Committee for Animal Experiments, G6teborg, Sweden.
Drugs Nefazodone HC1 (BMY 13754), a gift from Bristol-Myers Squibb, Scandinavia, ethanol (95%; Kemetyl, Stockholm, Sweden), and NaCI (Merck, Darmstadt, Germany) were used in the study. Nefazodone was dissolved in distilled water to 5 mg/ml and injected subcutaneously (SC) in a volume of 10 ml/ kg. Ethanol was diluted to 15% w/v with physiological saline (0.9% NaC1) and injected intraperitoneally (IP) in a volume of 16 ml/kg.
Microdialysis Technique To extract dopamine and dopamine metabolites, a microdialysis probe was implanted into the rat brain. The microdialysis was performed with an I-shaped probe [for details see (65)]. The rats were anaesthetised with ketamin 50 mg/ml (Parke-Davies, Barcelona, Spain) and xylazin 20 mg/ml (Bayer, Leverkusen, Germany) in a mixture of 2:1, that was injected in a volume of 2 ml/kg (IP). The microdialysis probe was then implanted in the N Acc of the brain by a stereotactic operation, using a Kopf stereotaxic instrument, and fixed to the skull using Phosphatine dental cement (Svedia Dental Industri, Sweden). The stereotactic coordinates of the probe relative to bregma were A/P +1.85, L/M -1.4, V/D - 7 . 8 (50). To substitute any loss of body liquids that may occur due to the operation, the rats were injected with 3 ml of 0.9% NaC1 (SC) and were allowed to recover for at least 40 h before the start of the microdialysis experiment. At the beginning of the experiment the inlet of the probe was connected to a perfusion pump C M A 100 (Carnegie Medicin, Stockholm, Sweden) via a swivel, and the outlet was connected to a collecting tube. The swivel allowed the animals to move freely during the experiment. The probe was then perfused with Ringer solution containing in mM: NaC1 140, CaCI 2 1.2, KCI 3.0, and MgC12 1.0 [modified from (40)]. The perfusion rate during the experiment was 2 txl/min and dialysate fractions (40 p.l) were collected every 20 rain.
Biochemical Assay To determine the concentration of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid
(HVA) in the dialysate, two different high pressure liquid chromatography systems with electrochemical detection (HPLC-EC) were used. To identify the peaks in the chromatogram an external standard containing 264 fmol/p~l of dopamine, 297 fmol/p.l of D O P A C , and 274 fmol/txl of H V A was used. The data sampled were analysed with a Dynamax HPLC method manager v. 1.3 (Instrument AB Lambda, Sollentuna, Sweden).
Microdialysis Experimental Design Animals were prepared for the dialysis experiment and connected to the perfusion pump. A minimum of 90 min were allowed for stabilisation before any sample was collected. The dialysate was then collected in 20-min fractions (40 I~1), which were analysed until stable basal values (_+ 10%) were obtained for each animal, at least four samples (80 min), before any drug was injected. When basal values were stable, the rats received nefazodone (25 or 50 mg/kg, SC) or vehicle. Two samples were collected (40 min) before ethanol (15% w/v; 2.5 g/kg, SC) or vehicle was injected. A t least five more samples (100 min) were collected before the microdialysis experiment was ended. The basal levels of dopamine or metabolites before any drug treatment were set to 100%. The biochemical effects of drug or vehicle injections were related to this baseline. All microdialysis values are presented as the mean peak values + SEM, expressed as percent of baseline.
Screening for Ethanol Preference Rats had free access to one bottle of water and one bottle containing a weak ethanol solution at all times. The ethanol concentration was gradually increased ( 2 4 - 6 % v/v) over a 2-week period. During the screening period the animals were housed individually in clear plastic cages. Thereafter, they had continuous access to two bottles (plastic 300-ml bottles with ballvalve sprouts; A L A B , Sweden) containing either tap water or 6% (v/v) ethanol solution. This ethanol concentration was used because previous observations (18) indicate that the ethanol consumption is maximal at approximately this concentration in the strain of rats used in the present study. Water and ethanol intake were then measured during a 4-week period, twice a week, when the bottles were also cleaned and filled with fresh liquids. The positions of the two bottles were also shifted at this occasion (i.e., twice a week). Body weight was recorded once a week throughout the screening period. The amount (grams) of ethanol solution 6% (v/v) consumed in percent of total fluid intake (grams) was used as an index of ethanol preference. Rats were classified as low-preferring (LP; <~20% ethanol) or high-preferring (HP; ~<60% ethanol) based on their preference for the ethanol solution over water.
Ethanol Drinking Study Experimental Design HP and LP rats were injected with nefazodone (50 mg/kg, SC) or vehicle in a counterbalanced cross-over fashion. The dose of nefazodone was chosen based on previous experience from the microdialysis experiment (see the Result section). Injections were given at 0930 h each day for two periods of three consecutive days, four days separating the periods. Ethanol consumption was measured between 1000 and 1700 h all treatment days, and the 3-day mean consumption and preference values for each animal was determined and used in all calculations. Both bottles were present during the whole mea-
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40
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Veh+Veh
O
Nef(25)+Veh
A
Nef(50)+Veh
2O 0
I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
l
veh/nef
?
veh/EtOH
time (min)
FIG. 1. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced dopamine overflow in the rat N Acc. All values are expressed as means + SEM. Statistics: ANOVA for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal dopamine levels were significantly different compared to control [veh + veh (n = 8) vs. veh + EtOH (n = 9) p = 0.0073; veh + veh (n = 8) vs. nef(25) + EtOH (n = 8) p - 0.0164; veh + EtOH (n = 9) vs. nef(50) + EtOH (n = 9) p = 0.0132]. The inserted histogram shows accumbal dopamine levels in the 60-min fraction (i.e., 60 min after veh/nef and 20 min after veh/EtOH). The symbol $ indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on dopamine levels in the rat N Acc. All values are expressed as means + SEM. Statistics: ANOVA for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal dopamine levels were significantly different compared to control [veh + veh (n = 8) vs. nef(50) + veh (n = 8) p = 0.0199]. The symbol 1" indicates time of injection.
80
O L A U S S O N ET AL.
suring period and the positions of the bottles were shifted at the end of each treatment day. Body weight was recorded before and after each treatment period.
Statistics The microdialysis data were statistically analysed using a two-way analysis of variance ( A N O V A ) , followed by the Fisher's protected least significant difference (PLSD) test implemented for comparisons with unequal ns (1). The data acquired in the ethanol drinking study were evaluated using the Wilcoxon signed rank test. A probability value of p < 0.05 was considered statistically significant.
12.7 g/kg ( p = 0.0251), and ethanol preference by 22%, from 69 to 47% ( p = 0.0251), in HP (n = 8) but not LP (n = 15) rats, when measured between 1000 and 1700 h (Fig. 4a, b). LP rats significantly lowered their water intake by 35%, from 35.0 to 23.0 g/kg (p = 0.0007), after injection of nefazodone (50 mg/kg, SC), whereas the water intake was not altered by nefazodone in the HP rats ( p = 0.6744; Fig. 4c). The total fluid intake was decreased by 30% in HP ( p = 0.0357), and by 33% in LP ( p = 0.0008) rats. The body weight was not affected by the 3-day nefazodone treatment (p = 0.6496, n = 23; data not shown). DISCUSSION
RESULTS
Effects on Extracellular Dopamine Levels" Administration of ethanol (2.5 g/kg, IP) produced a statistically significant increase of the dopamine levels in the first two dialysate samples (p = 0.0073; n = 9), i.e., within 40 min of the ethanol injection. The peak extracellular dopamine level (136 _+ 9%) was observed in the first sample. The concentration of dopamine then gradually decreased and reached baseline levels 60 min after the ethanol injection (Fig. la). Administration of nefazodone (50 mg/kg, SC) significantly increased extracellular dopamine levels ( p = 0.0199: n = 8) in the N Acc. The peak dopamine level (128 _+ 11%) was observed in the fraction collected 40 min after the nefazodone injection (Fig. lb). The low dose of nefazodone (25 mg/kg, SC) significantly increased the accumbal dopamine levels by 15% in the 60-min fraction ( p = 0.0459; n = 6). Nefazodone (50 mg/kg, but not 25 mg/kg, SC) reduced ethanol-stimulated dopamine overflow ( p = 0.0132: n = 9), and the dopamine content observed after coadministration of nefazodone and ethanol did not differ from baseline levels. The nefazodone-pretreated group reached basal dopamine levels 40 min after ethanol administration and the lowest dopamine concentration (71 _+ 8%) was found after 80 rain (Fig. la).
Effects, on Extracellular Levels of Doparnine Metabolites Ethanol (2.5 g/kg, IP) significantly increased D O P A C (p 0.0093: n = 11) and H V A ( p = 0.001; n - 8) overflow (Figs. 2a and 3a). The highest D O P A C content (126 _+ 7%) was observed in the third sample, 60 min after ethanol injection, whereas the highest H V A levels (152 _+ 8%) were observed 100 min after ethanol (e.g., in the 140-rain fraction). A dose of 50 mg/kg (SC) nefazodone did not significantly alter D O P A C or H V A levels, whereas the low dose of nefazodone (25 mg/kg, SC) significantly increased both accumbal D O P A C ( p = 0.0074; n = 6) and H V A ( p = 0.0121: n - 6) during the first 60 and 80 min after injection, respectively (Figs. 2b and 3b). The peak D O P A C and H V A levels were observed in the third sample, collected 60 min after nefazodone injection (127 _+ 15% and 131 -+ 19%, respectively). Administration of nefazodone (50 mg/kg, SC) significantly decreased the ethanol-induced levels of H V A (p = 0.0159; n = 9), but not DOPAC, in the N Acc, whereas nefazodone (25 mg/kg, SC) had no effect on the ethanol-induced changes of the dopamine metabolites (Figs. 2a and 3a).
Effect on Voluntary Ethanol lntake Administration of nefazodone (50 mg/kg, SC) significantly decreased the ethanol solution intake by 51%, from 25.8 to
Ethanol and Accumbal Dopamine Overflow In the present microdialysis experiments ethanol (2.5 g/kg, IP) significantly increased accumbal dopamine overflow in freely moving rats, a result in line with previous reports (4,8, 22,68,69). This effect may involve both increased neuronal firing and enhanced dopamine release, and could be exerted by a direct action on the dopamine system or indirectly via interference with other central neurotransmitter systems. Evidence has accumulated indicating that direct interference with ligand-gated ion channels, such as the 5-HT 3 receptor (8,68,69), the N-methyl-D-aspartate receptor (11), the y-aminobutyric acid (GABA)A/benzodiazepine receptor (58), and the nicotinic acetylcholine receptor (4,5), may be involved in the dopamine activating effects of ethanol. However, the specific mechanisms underlying the ethanol-induced activation of the mesolimbic dopamine system remain to be elucidated. Previous experiments have demonstrated that ethanolinduced accumbal dopamine overflow is associated with increased D O P A C and H V A levels (4,22), supporting the present observations of elevated dopamine metabolite levels in the N Acc after ethanol injection. This increase has been suggested to reflect an elevated level of dopamine metabolism due to the increased dopamine overflow. However, these findings are not entirely consistent with those of Yoshimoto et al. (69), who observed a significant elevation of HVA, but not of DOPAC, levels after acute ethanol administration.
N
NEFAZODONE AND ETHANOL
160 140
81
DOPAC m
~120
t
..~1oo 80013
~= 6 0 ~. 4 0 20-
[]
Veh + Veh
O
Veh + EtOH
~k
N e f (25) + E t O H N e f (50) + E t O H
I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
t
t
veh/nef
t i m e (min)
veh/EtOH
b
160
14o 120
-
100
-
"~
80 -
~
60-
•
40-
20 -
DOPAC
[]
Veh +
O
N e f (25) + V e h
A
N e f (50) + V e h
Veh
I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
t veh/nef
t veh/EtOH
t i m e (min)
FIG. 2. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced increase of D O P A C in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal D O P A C levels were significantly different compared to control [veh + veh (n = 8) vs. veh + E t O H (n = 11) p = 0.0093; veh + veh (n = 8) vs. nef(25) + E t O H (n = 8) p = 0.0018]. The symbol 1" indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on D O P A C levels in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated m e a s u r e s followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal D O P A C levels were significantly different compared to control [veh + veh (n = 8) vs. nef(25) + veh (n = 6) p = 0.0074]. The symbol 1" indicates time of injection.
82
OLAUSSON
180
ET AL.
HVA
160
= 140 w 120 100
80 "~
[]
Veh + Veh
O
Veh + EtOH
6O 40N e f (25) + E t O H 20 O 0
I 0
N e f (50) + EtOH I
I
I
I
I
I
I
20
40
60
80
100
120
140
l veh/nef
t time (min)
veh/EtOH
b
160 -
HVA 140 -
~
120 100
.~
80-
60~
[]
Veh + Veh
O
N e f (25) + V e h
A
N e f (50) + V e h
4020I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
I veh/nef
I veh/EtOH
time (min)
FIG. 3. (a) Effect of nefazodone (25 and 50 mg/kg, SC) on ethanol (2.5 g/kg, IP)-induced increase of H V A in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated m e a s u r e s followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal H V A levels were significantly different compared to control [veh + veh (n = 8) vs. veh + E t O H (n = 8) p = 0.0010; veh + veh (n = 8) vs. nef(25) + E t O H (n = 8) p = 0.0046; veh + E t O H (n = 8) vs. nef(50) + E t O H (n = 9) p = 0.0159]. The symbol $ indicates time of injection. (b) Effect of nefazodone (25 and 50 mg/kg, SC) on H V A levels in the rat N Acc. All values are expressed as m e a n s + SEM. Statistics: A N O V A for repeated measures followed by Fisher's PLSD test. Filled symbols indicate the period during which the accumbal H V A levels were significantly different compared to control [veh + veh (n = 8) vs. nef(25) + veh (n - 6) p - 0.0121]. The symbol 7 indicates time of injection.
N E F A Z O D O N E AND E T H A N O L
83
a 30-
~
25
~
20
A
~=152 '~ 10 ~
~5 HP
LP
b 80-
70-
~ 60"~ 50= 40-. O : = 30-_
dopaminergic activity (3,29,44). Thus, 5-HT applied both in the VTA or the N Acc increases accumbal dopamine levels (21,49). The dopamine release after 5-HT application in the N Acc most likely involves terminal 5-HT 3 receptors (8,46,55), whereas the effect of 5-HT in the VTA, which is mimicked by 5-HT m agonists (21), could be mediated via activation of 5-HTlb receptors decreasing the descending G A B A tone (30). Although local application of 5-HT and 5-HT agonists produce the effects mentioned above, systemic administration of selective 5-HT reuptake inhibitors (SSRI) generally fail to alter accumbal dopamine overflow (20,61). Thus, the reuptake inhibitory effect of nefazodone is not by itself likely to explain the increased dopamine overflow in the N Acc. It may be suggested that the combined effects of nefazodone may produce a dopamine activating effect larger than the stimulation induced by 5-HT2A receptor antagonism or 5-HT reuptake inhibition alone. As mesolimbic dopamine stimulation is considered to be involved in substance abuse, the dopamine activating property of nefazodone could indicate abuse liability. However, this possibility appears less likely, because nefazodone did not reinforce self-administration in primates (19). The mesolimbic dopamine activating effect of nefazodone could instead be an additional mechanism of importance for the antidepressant property of nefazodone (54,66). Although nefazodone (50 mg/kg, SC) increased accumbal dopamine overflow, the D O P A C or HVA levels were not altered, a pattern in line with that obtained after 5-HT~ antagonists (13). Such a pattern has, however, also been described after dopamine reuptake inhibitors (25). Whether nefazodone interferes with the dopamine reuptake carrier is Unknown, but because the lower dose (25 mg/kg, SC) slightly raised both accumbal dopamine (see above) and metabolite levels, this possibility appears less likely.
Coadministration of Nefazodone and Ethanol and Accumbal Dopamine Overflow HP
LP
C 407
Pretreatment with nefazodone (50 mg/kg, SC, - 4 0 min) strongly attenuated ethanol-stimulated dopamine overflow in the N Acc. The extracellular levels of D O P A C and HVA also tended to be lower in rats pretreated with nefazodone, although this difference was significant for HVA only. It is noteworthy that only the high dose of nefazodone (50 mg/kg, SC), which activated the mesolimbic dopamine system per se, altered the neurochemical response to ethanol. Thus, ethanol
352 --~30,
20-
"///////, m 10-
5HP
LP
FIG. 4. (a) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on ethanol solution (6%) intake between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are
the mean daily ethanol consumption per kg body weight during 3 consecutive days of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxon signed rank test. *p < 0.05 compared to vehicle treatment. (b) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on ethanol solution (6%) preference between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are the mean daily ethanol preference during 3 consecutive clays of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxon signed rank test. *p < 0.05 compared to vehicle treatment. (c) Effect of nefazodone (50 mg/kg, SC), injected at 0930 h, on water intake between 1000 and 1700 h in HP (n = 8) and LP (n = 15) rats. Open bars are the values during vehicle treatment and hatched bars during nefazodone treatment. Data are the mean daily water consumption per kg of body weight during 3 consecutive days of drug or vehicle treatment. All values are expressed as means + SEM. Statistics: Wilcoxonsigned rank test. ***p < 0.001 compared to vehicle treatment.
84
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was injected during a slightly elevated baseline, which could have masked an enhancement of dopamine overflow after ethanol. However, in these experiments, the dopamine levels induced by nefazodone were lower than those after ethanol, suggesting that further dopaminergic stimulation after ethanol could have been obtained. Instead, according to the present results, the interaction between the two drugs results in a decreased mesolimbic dopamine activity, compared to that after either of the two drugs administered alone. The complex pharmacology of nefazodone suggests that the nefazodone-induced attenuation of ethanol-stimulated accumbal dopamine overflow could result from one or more of its interactions. However, 5-HT2A antagonists have previously been demonstrated to reduce behavioral activation associated with excessive dopaminergic neurotransmission (26,31,46,56), as well as drug-induced enhancement of dopamine release (26,46). Moreover, exogenous 5-HT, as well as 5-HT 2 receptor agonists, potentiate the ethanol-stimulated excitation of dopamine cell-firing in the VTA (6,62), whereas, according to in vitro observations by Brodie and Trifunovic (7,62), ethanolinduced excitation of mesolimbic dopamine neurons could be reduced by the 5-HT 2receptor antagonist ketanserin. Taken together, these results suggest that nefazodone preferentially, by means of its 5-HT2A receptor antagonistic properties, decreases ethanol-stimulated dopamine overflow in the N Acc. In conclusion, nefazodone counteracts the dopamine-activating effects of ethanol, whereas it per se elevates mesolimbic dopamine overflow (i.e.. nefazodone appears to interfere with dopaminergic neurotransmission in an activity-dependent manner). The mechanism underlying this apparently paradoxical effect of nefazodone remains to be elucidated. It is interesting to note, however, that the 5-HT2A receptor antagonists MDL 100,907 and ritanserin have been demonstrated to attenuate the effects of other psychomotor stimulants, such as the dopamine reuptake blocker G B R 12909, the N-methyl-D-aspartate receptor antagonist MK-801, the muscarinic receptor antagonist atropine, and amphetamine (10,53). On the basis of these findings, Carlsson (10) proposed that the 5-HT 2 receptor may have a "permissive" role for psychomotor activation.
Nefazodone and Voluntary Ethanol Intake Interestingly, nefazodone (50 mg/kg), unlike the related drug trazodone (37), decreased both ethanol solution intake and ethanol preference in HP but not LP rats from a nonselected Wistar strain. Thus, although a dose-response relationship was not established, the present results indicate that nefazodone in a dose that prevents ethanol-induced dopamine overflow (see above) also lowers ethanol intake in animals consuming quantities (approx. 1.6 g/kg/7 h) sufficient to enhance dopamine overflow (22,28,69). This indicates, but does not prove, that pharmacological actions of nefazodone on the mesolimbic dopamine system are involved in the behavioral effect observed. It should be noted that nefazodone also decreased water intake in LP but not in HP rats. Thus, in
both LP and HP rats nefazodone reduced the intake of the preferred solution only. Taken together with findings indicating that the mesolimbic dopamine system is activated also by natural rewards (36,39), these results may suggest that nefazodone interferes not only with ethanol reward (HP rats), but also with water reward (LP rats). An alternative explanation to the behavioral findings is that nefazodone decreases general ingestive behaviors and thereby reduces ethanol intake (41), an interpretation supported by the fact that the total fluid intake was decreased both in HP and LP rats. However, in this case also a lowering of the intake of the nonpreferred solution would have been expected, at least in HP rats in which the water intake certainly was large enough to allow a decrement. In LP rats the lack of an effect could be due to a floor effect. Furthermore, even though food intake was not measured, the body weights were not affected during the 3-day treatment period (see Results section), supporting the conclusion that nefazodone interferes mainly with reward-related mechanisms and not with ingestive behaviors in general. Although some previous studies on the effect of 5-HT 2 antagonists, possibly acting in the N Acc (47), on voluntary ethanol intake support the present results (37,38,42,47), there are also contradictory reports available (43,59,60). On the other hand, SSRIs consistently decrease voluntary ethanol intake in both rats and humans (2,16,34,35,41), suggesting that indirect nefazodone-induced stimulation of 5-HT receptors involved in ethanol self-administration [e.g., 5-HTjA receptors (12,16, 33,59)] may also be involved in the effect of nefazodone on voluntary ethanol drinking. CONCLUSION The present study demonstrates that both ethanol and nefazodone increases accumbal dopamine levels, whereas nefazodone pretreatment reduces the ethanol-induced dopamine overflow. Although further experiments are needed to determine the specific mechanism(s) underlying this inhibition, circumstantial evidence suggests that 5-HT2A receptor inhibition may be involved in a dopamine activity-dependent manner. Interestingly, nefazodone also decreases voluntary ethanol intake in HP Wistar rats, suggesting a functional effect of nefazodone possibly related 1o the neurochemical effect described above. Because the models used here have been shown to be of predictive value for the effect of ethanol-intake reducing drugs in man, the present findings suggest that nefazodone could be useful in future pharmacological treatment of alcohol abuse. ACKNOWLEDGEMENTS This study was financially supported by the Swedish Medical Research Council (grants No. 4247 and 11583), the Swedish Society of Medicine, Magnus Bergvalls stiftelse, ~.ke Wibergs stiftelse, Systembolagets fond f6r alkoholforskning, and Bristol-Myers Squibb, Scandinavia. The authors also thank Dr. Lennart Svensson, Dr. Ola Blomqvist, Gun Andersson, and Kenn Johannessen for helpful advice and assistance throughout the study.
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