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Consequences of monosodium glutamate or goldthioglucose arcuate nucleus lesions on ethanol-induced locomotion
Drug and Alcohol Dependence 68 (2002) 189 /194 www.elsevier.com/locate/drugalcdep
Consequences of monosodium glutamate or goldthioglucose arcuate nucleus lesions on ethanol-induced locomotion Carles Sanchis-Segura, Carlos M.G. Aragon * Area de Psicobiologia, Universitat Jaume I., Campus de Borriol, Apartat. 8029 AP, Castello´ 12071, Spain Received 12 October 2001; accepted 22 June 2002
Abstract It has been suggested that the endogenous opioid system, especially b-endorphins, may play an important role in the behavioral effects of ethanol. The main site of b-endorphin synthesis in the brain is the hypothalamic arcuate nucleus (ARC). In the present study, we used the neurotoxins monosodium glutamate (MSG) or goldthioglucose (GTG) to produce a selective ARC lesion and to assess its effects on the locomotion observed after ethanol administration. The results show that MSG or GTG pre-treatment produces a blockade of the increased locomotion produced by the injection of low and moderate doses of ethanol (0.5 and 1.5 g/kg, respectively). These effects were observed in the absence of any change in blood ethanol levels. On the other hand, MSG (but not GTG) pre-treatment enhanced the locomotor depression produced by higher doses of this alcohol (2.5 g/kg). Finally, caffeine (10 mg/kg)-induced locomotion was unaffected by the aforementioned neurotoxic agents. Thus, taken together, the present results suggest that MSG and GTG administration produce a blockade of the stimulating effects of ethanol on locomotion in mice and thus provides further support for a role of the ARC in the behavioral effects observed after ethanol administration. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Locomotion; Monosodium glutamate; Goldthioglucose; b-Endorphin; Arcuate
1. Introduction Ethanol exerts a wide range of behavioral effects through its interaction with several neurotransmitters and neuromodulators in the Central Nervous System (CNS). It seems that the endogenous opiate system and especially the proopiomelanocortin (POMc)-derived peptide b-endorphin may be of particular significance (Gianoulakis, 1996; Herz, 1997). Several lines of evidence suggest this interaction. For instance, ethanol modifies the binding properties of opioid receptors, as well as endogenous opioid synthesis and secretion (Tabakoff and Hoffman, 1983; Gianoulakis, 1989; de Waele et al., 1992; de Waele and Gianoulakis, 1993; Rasmussen et al., 1998). Furthermore, a large number of studies have shown that opioid antagonists reduce ethanol behavioral effects such as voluntary ethanol consumption or ethanol-induced locomotor activity
* Corresponding author. Tel.: /34-964-729337; fax: /34-964729350 E-mail address: [email protected] (C.M.G. Aragon).
(Kiianmaa et al., 1983; Prunell et al., 1987; Ulm et al., 1995; Davidson and Amit, 1997). Conversely, other authors have reported that low doses of opioid agonists, especially m-receptor ligands, can enhance some of these ethanol effects (Hubbell et al., 1987; Kuribara et al., 1991; Stromberg et al., 1997). In addition, studies in rodents as well as in humans reveal that subjects with higher endogenous opioid system sensitivity to ethanol are at a higher risk of excessive ethanol consumption than are subjects with lower sensitivity (Froelich et al., 1995; Gianoulakis, 1996). In fact, the opioid receptor antagonist naltrexone has become one of the most used therapeutic approaches for coping with excessive ethanol drinking and alcoholism (Herz, 1997; Stromberg et al., 1997). The brain sites of POMc synthesis include the hypothalamic Arcuate nucleus (ARC) and the medullary nucleus tractus solitary (NTS) (Khatchaturian et al., 1985). The ARC is located at the base of the hypothalamus, in close apposition to the median eminence and the pituitary gland. It surrounds the third ventricle and is directly connected to the pituitary gland,
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some thalamic and hypothalamic nuclei, the midbrain periaqueductual gray area, the autonomic nuclei of brain stem and the limbic system, including the amygdala, the nucleus accumbens and the ventral tegmental area (Chronwal, 1985; Khatchaturian et al., 1985). Conversely, the projections of the NTS system appear to be local within the medulla oblongata, with possible projections to the parabranchial area nuclei and to the spinal cord (Khatchaturian et al., 1985). It therefore seems that the ARC may be a more trustworthy system than the NTS for assessing the interactions between alcohol and the POMc-related peptides in ethanolrelated behaviors (Gianoulakis, 1996; Herz, 1997). In accordance with this rationale, one of our previous studies showed that selective destruction of ARC / POMc containing neurons using estradiol valerate (EV) (Desjardins et al., 1993) results in a blockade of the stimulatory effects of low or moderate ethanol doses on locomotion in female mice, but does not affect the depressant effects of higher doses of ethanol (SanchisSegura et al., 2000). Thus the aim of these experiments is to assess whether a more extensive destruction of the ARC (as produced by monosodium glutamate, MSG (Holzwarth-McBride et al., 1976; Peruzzo et al., 2000), or goldthioglucose, GTG (Young, 1989)) reproduces this effect. The present study is therefore an attempt to verify if the effects on ethanol-induced locomotion observed following EV administration (Sanchis-Segura et al., 2000) are due to its effect on the ARC. Moreover, the present study can provide additional evidence about the possible role of other neurotransmitters contained in the ARC on ethanol-induced locomotion.
2. Methods 2.1. Subjects For the MSG experiments, mice pups were obtained from pregnant female Swiss /Webster mice, purchased from Janvier Espan˜a S.L. (Madrid, Spain). For the GTG study, 4-week-old male Swiss /Webster mice were purchased from Janvier Espan˜a S.L. (Madrid, Spain) and housed in groups (four animals per cage), with standard laboratory rodent chow and tap water available ad libitum. 2.2. Drugs MSG, GTG (aureothioglucose) and caffeine were purchased from Sigma Aldrich S.A., Spain. Ethanol solution (20% v/v) was prepared from ethanol 96% (Panreac Qu’mica S.A., Spain). All these drugs were dissolved in saline (0.9%) solution.
2.3. Procedure For the MSG experiment, newborn pups received subcutaneous injections of MSG (4 mg/g body weight), or equivalent volumes of a saline solution (0.9%), on days 1, 3, 5, 7 and 9 after birth (Holzwarth-McBride et al., 1976; Crabbe and Dorsa, 1986). At 3 weeks of age, male mice were separated and housed in groups with four animals per cage. At 8 weeks after birth the behavioral tests were performed. For the GTG experiment, after 7 days of habituation to laboratory conditions, male mice were divided into two groups and saline or GTG was administered intraperitoneally (IP). GTG was dissolved at a concentration of 300 mg/ml and was injected IP to mice at a dose of 500 mg/kg (Young, 1989). The behavioral tests were performed 8 weeks later. The differences in the source of the subjects and drug administration correspond to the previously described requirements of each substance to produce an effective lesion of the hypothalamic ARC (Holzwarth-McBride et al., 1976; Crabbe and Dorsa, 1986; Young, 1989). For instance, it has been described that the ability of MSG to damage the arcuate neurons is related to the developmental status of the blood brain barrier in this perinatal period (Peruzzo et al., 2000), whereas GTG seems able to produce an effective ARC lesion when independent of an animal’s age (Young, 1989). 2.4. Apparatus The open field apparatus and locomotion measures have been described previously (Sanchis-Segura et al., 2000). Briefly, it consisted of a glass cylinder (25 cm diameter) the floor of which was divided in four equal quadrants by two intersecting lines drawn across it. A count was scored when a subject crossed a line with all four paws. Male mice pre-treated with saline, MSG or GTG were challenged with ethanol (0.0, 0.5, 1.5 or 2.5 g/ kg; IP; n /8 per group). Immediately after this treatment, mice were placed individually in the open field chambers for 20 min. Individual measures of locomotor activity were recorded for the last 10-min period. This delay was chosen in order to decrease the effects of animal handling and the environmental novelty of the open field (Dudek and Tritto, 1994) and because at this time brain ethanol levels are maximal after an IP administration (Yim and Gonzales, 2000). In a second study, we assessed the effect of MSG or GTG pretreatment on caffeine-induced locomotion (10 mg/kg; IP). Test conditions were as described above. 2.5. Blood ethanol levels Additional mice pre-treated with saline, MSG or GTG were used to assess blood ethanol levels after the
C. Sanchis-Segura, C.M.G. Aragon / Drug and Alcohol Dependence 68 (2002) 189 /194
administration of an ethanol injection (0.5, 1.5 or 2.5 g/ kg). Trunk blood was collected at 20 min following this ethanol challenge. Plasma ethanol levels were enzymatically assessed with an Alcohol Diagnostic Kit from Sigma-Aldrich Qu’mica (Spain) (Sanchis-Segura et al., 2000). 2.6. Histology The effects of repeated saline or MSG injections to mice pups during the perinatal days were assessed. Thus a separate group of mice pups were treated as described in the procedure section and 8 weeks later animals were anesthetized and perfused with an heparynized (1000 u/ L) saline solution. Coronal slices (40 mm) were obtained using a microtome and mounted on to slides. The slides were stained using a cresyl violet standard procedure (Davies and Stewart, 1997) and the coverslipped slides were viewed under 25 / or 100 / magnification.
3. Results Mice treated with MSG showed, 8 weeks later, several morphological alterations. It has been demonstrated that these changes reflect the extent of ARC lesion after MSG administration (Dubovicky et al., 1997). Thus, the nose /anus distance was significantly reduced [T (13) / 4.60, P B/0.01] in mice pre-treated with MSG compared to saline pre-treated mice (mean9/SEM were 8.539/0.37 and 10.609/0.22 cm, respectively; n /5/6 per group). The tail length was also shorter in MSG pre-treated mice [T (13) /8.40, P B/0.01] than in control mice (mean9/SEM were 6.509/0.13 and 8.419/0.19 cm, respectively; n /5 /6 per group). Moreover, the histological assessment of coronal brain sections of mice treated with MSG revealed a significant cell loss in the ARC nucleus compared to saline treated mice (Fig. 1). On the other hand, after 2 months, mice treated with GTG showed a significant increase in body weight compared to saline pre-treated mice [T(38) / /3.93, P B/0.01]. Thus, mean9/SEM body weights (n /20 per group) were 44.149/0.78 and 52.149/1.87 g for saline and GTG pre-treated mice, respectively. In accordance with previous reports (Bergen et al., 1998), this increase in body weight may be understood as an index of the destruction of the ARC produced by GTG. Fig. 2A depicts the effects of perinatal MSG pretreatment on ethanol-induced locomotion. As the figure shows, ethanol-induced a biphasic effect on locomotor activity in animals pre-treated with saline. However, MSG-treated mice did not show any ethanol induction of locomotion, in fact to the contrary they displayed an ethanol dose-dependent reduction of locomotion. A two-way ANOVA revealed a significant effect of the pre-treatment (saline or MSG) [F (1, 56) /74.87, P B/
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0.01] and the ethanol dose (0.0, 0.5, 1.5 or 2.5 g/kg) [F (3, 56) /16.28, P B/0.01] factors, as well as a significant interaction between them [F (3, 56) /8.71, P B/ 0.01]. Fisher’s LSD test showed that MSG blocked the increase of locomotion produced by low or moderate doses of ethanol (0.5 and 1.5 g/kg) whereas it enhanced the depressant effects produced by a higher dose of ethanol (2.5 g/kg). On the other hand, MSG pretreatment did not affect spontaneous locomotor activity (ethanol dose 0.0 g/kg). Similarly, as shown in Fig. 2B, GTG-treated mice did not show any ethanol induction of locomotion. Thus, a two-way ANOVA of pretreatment (saline or GTG) /ethanol dose (0.0,05, 1.5 or 2.5 g/kg) revealed a significant effect of the pretreatment [F (1, 56) /14.58, P B/0.01] and the ethanol dose [F (3, 56) /7.38, P B/0.01] factors, as well as a significant interaction between them [F (3, 56) /4.91, P B/0.01]. Fisher’s LSD test showed that GTG pretreatment reduced the increase in locomotion observed after low or moderate doses of ethanol, whereas it did not modify the locomotion observed after higher doses of ethanol (2.5 g/kg) or after a saline injection. Fig. 3A presents the locomotor activity values observed in saline or MSG pre-treated mice after an acute caffeine injection (10 mg/kg). A two-way ANOVA revealed that caffeine produced a significant enhancement of mice locomotion [F (1, 28) /14.18, P B/0.01]. This effect was unmodified by MSG. As shown in Fig. 3B, similar results were obtained when comparing the effects of a saline or GTG pre-treatment on the locomotion of mice challenged with the same dose of caffeine. Again, a two-way ANOVA revealed that caffeine produced a significant enhancement of locomotion [F (1, 28) /10.32, P B/0.01] and that this effect was unmodified by the pre-treatment conditions. Table 1 presents the effects of MSG or GTG pretreatment on blood ethanol levels. In animals pretreated with MSG, a two-way ANOVA revealed that the ethanol dose factor [F (1, 2) /118.07; P B/0.01] and its interaction with the group factor [F (1, 2) /12.13; P B/0.01] yielded significant effects. Mean comparisons using Fisher’s LSD test revealed that animals pretreated with MSG and administered with 2.5 g/kg of ethanol had higher blood ethanol levels than their controls. On the other hand, ethanol blood levels of animals pre-treated with GTG or saline did not show any difference, and only the ethanol dose factor yielded a significant effect [F (1, 2) /46.89; P B/0.01].
4. Discussion The results of the present study show that MSG or GTG treatment produces an antagonistic effect on the stimulant effects of low and moderate ethanol doses in mice locomotion. At present, the explanation of these
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Fig. 1. Cresyl violet coronal hypothalamic sections of animals perinatally treated with saline (A and D) or MSG (B, C, E, F) as described in Section 2. As can be observed in pictures B and C, MSG produced an evident lesion of the ARC (magnification 25 /). The loss of cells produced by perinatal MSG administration can be better observed at higher (100 /) magnification (pictures D, E and F). Circles and arrows have been included to better indicate the ARC location.
Fig. 2. Effects of MSG (A) or GTG (B) pre-treatment on ethanol (0.5 /2.5 g/kg)-induced locomotor activity. Bars depict mean9/SEM locomotor activity (counts/10 min) for all treatment groups (n /8 per group). White and dashed bars represent saline and GTG or MSG pretreated animals, respectively. (*P B/0.01 significantly different from saline pre-treated group.)
Fig. 3. Effects of MSG (A) or GTG (B) pre-treatment on caffeine (10 mg/kg)-induced locomotor activity. Bars depict mean9/SEM locomotor activity (counts/10 min) for all treatment groups (n /8 per group). (*P B/0.01 significantly different from saline pre-treated group.)
C. Sanchis-Segura, C.M.G. Aragon / Drug and Alcohol Dependence 68 (2002) 189 /194 Table 1 Mean9SEM of blood ethanol levels (mg/dl) in (A) MSG or (B) GTG pre-treated mice as described in the methods section and their respective control groups Pre-treatment
Ethanol dose (g/kg) 0.5
1.5
2.5
A Saline MSG
66.88911.51 60.5393.11
277.84939.25 234.5796.73
316.04911.58 472.98930.69*
B Saline GTG
77.44917.04 64.19916.42
181.0298.09 245.17916.64
332.40914.75 341.79958.38
Blood samples were taken 20 min after ethanol administration (0.5 / 2.5 g/kg; IP). (* P B 0.01)
results remains open but it could be related to the lesion that MSG and GTG administration produces in the ARC. In this respect, it has been shown that the MSG treatment conditions used in the present study results in the destruction of 80 /90% of ARC neurons (Desjardins et al., 1992). Similar results have been found after GTG administration, which may produce an even more extensive lesion which includes the ventromedial hypothalamus and other adjacent areas (Young, 1989). In the present study, we confirmed previous observations about cell loss in the ARC following MSG treatment (Desjardins et al., 1992; Peruzzo et al., 2000) in addition to some of the morphological changes that occur after MSG or GTG administration and which have been clearly correlated with the extent of the ARC lesion (Dubovicky et al., 1997; Bergen et al., 1998). The behavioral data obtained in the present study will therefore be discussed within this conceptual framework. In our data, GTG or MSG had no effect on the spontaneous locomotion of mice. With regard to our results it is necessary to note that the effect of MSG on the spontaneous locomotor activity of rodents is unclear and that conflicting findings have been published. For example, increases (Dubovicky et al., 1997), decreases (Barnhart and Pizzi, 1982; Crabbe and Dorsa, 1986) and the absence of an effect have all been reported (Barnhart and Pizzi, 1982; Dubovicky et al., 1999; Ali et al., 2000). On the other hand, although the effects of GTG on the spontaneous locomotion of mice have no precedent they do seem to provide further support to the notion that the ARC lesion does not alter the spontaneous locomotion of mice. Conversely, both MSG and GTG produced a deep change in the behavioral effects of low and moderate doses of ethanol. Thus, mice treated with either of these two neurotoxins showed a blockade of the stimulatory effects on mice locomotion observed after ethanol (0.5 or 1.5 g/kg) administration. Since, at these doses, blood ethanol levels were unmodified, it can be suggested that
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the changes on ethanol-induced locomotion following MSG or GTG administration may be related to an effect of these substances in the brain, probably at the ARC. Moreover, in the present study, MSG enhanced the depressant effects of a higher (2.5 g/kg) ethanol dose on mice locomotion, whilst GTG did not. This result could be related to the increase in blood ethanol levels observed after a high dose of ethanol in MSG-, but not GTG-treated mice. This suggestion is in accordance with a previous report (Crabbe and Dorsa, 1986), which showed that MSG enhances blood ethanol levels in addition to boosting the reduction of locomotion produced by high doses of ethanol in mice. Finally, it was observed that after a caffeine challenge the locomotion of MSG- or GTG pre-treated mice was no different from that of saline pre-treated mice. The assessment of these effects was also unprecedented and these results suggest that the ARC does not belong to a general system activated by all drugs, which are able to increase mice locomotion. In general, there is a close parallel between the present results and those observed in a previous study using EV. EV produces a selective destruction of the POMcsynthesizing neurons of the ARC (Desjardins et al., 1990, 1993) and blocks ethanol-induced locomotion (Sanchis-Segura et al., 2000). Moreover, this substance does not alter the effects of caffeine on mice locomotion (Sanchis-Segura et al., 2000). Thus, although there are some differences in the pattern of destruction of the ARC caused by these neurotoxins, they all block ethanol- but not caffeine-induced locomotion and destroy the ARC POMc-synthesizing neurons. Because the POMc derived peptide b-endorphin has been proposed as a possible mediator of some of the behavioral effects of ethanol (Gianoulakis, 1996; Herz, 1997), it seems possible that the effects of MSG, GTG and EV on ethanol-induced locomotion could be related to their ability to lesion the endorphin-synthesizing neurons. Additional although indirect support for this hypothesis seems to be provided by experiments which show that opioid receptor blockade results in a decrease in ethanol- but not caffeine-induced locomotion (Kiianmaa et al., 1983; Sawynok et al., 1995). In summary, the present results show that MSG or GTG administration, under the treatment conditions used in this study, produces a blockade of ethanol effects on mice locomotion. Because of the only known common effect of MSG and GTG is their ability to produce an ARC lesion, it seems reasonable to suggest that their identical consequences on ethanol-induced locomotion are related to this capacity. Therefore, these data argue for a role of the ARC nucleus in the behavioral effects of ethanol. Although the exact role of the ARC remains to be elucidated, it could be related to its ability to synthesize b-endorphins and other
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POMc-derived peptides. Future experiments will be dedicated to exploring this topic.
Acknowledgements This research was funded by a grant from Fondo de Investigacio´n Sanitaria. Ministerio de Sanidad y Consumo. (FIS 001101), Spain. C.S.-S. was supported by a fellowship from the Conselleria d’ Educacio´ i Cie`ncia de la Generalitat Valenciana, Spain. The authors gratefully acknowledge the assistance of Dr J.K. Young for his technical comments about GTG dosage and effects.
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Consequences of monosodium glutamate or goldthioglucose arcuate nucleus lesions on ethanol-induced locomotion Carles Sanchis-Segura, Carlos M.G. Aragon * Area de Psicobiologia, Universitat Jaume I., Campus de Borriol, Apartat. 8029 AP, Castello´ 12071, Spain Received 12 October 2001; accepted 22 June 2002
Abstract It has been suggested that the endogenous opioid system, especially b-endorphins, may play an important role in the behavioral effects of ethanol. The main site of b-endorphin synthesis in the brain is the hypothalamic arcuate nucleus (ARC). In the present study, we used the neurotoxins monosodium glutamate (MSG) or goldthioglucose (GTG) to produce a selective ARC lesion and to assess its effects on the locomotion observed after ethanol administration. The results show that MSG or GTG pre-treatment produces a blockade of the increased locomotion produced by the injection of low and moderate doses of ethanol (0.5 and 1.5 g/kg, respectively). These effects were observed in the absence of any change in blood ethanol levels. On the other hand, MSG (but not GTG) pre-treatment enhanced the locomotor depression produced by higher doses of this alcohol (2.5 g/kg). Finally, caffeine (10 mg/kg)-induced locomotion was unaffected by the aforementioned neurotoxic agents. Thus, taken together, the present results suggest that MSG and GTG administration produce a blockade of the stimulating effects of ethanol on locomotion in mice and thus provides further support for a role of the ARC in the behavioral effects observed after ethanol administration. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Locomotion; Monosodium glutamate; Goldthioglucose; b-Endorphin; Arcuate
1. Introduction Ethanol exerts a wide range of behavioral effects through its interaction with several neurotransmitters and neuromodulators in the Central Nervous System (CNS). It seems that the endogenous opiate system and especially the proopiomelanocortin (POMc)-derived peptide b-endorphin may be of particular significance (Gianoulakis, 1996; Herz, 1997). Several lines of evidence suggest this interaction. For instance, ethanol modifies the binding properties of opioid receptors, as well as endogenous opioid synthesis and secretion (Tabakoff and Hoffman, 1983; Gianoulakis, 1989; de Waele et al., 1992; de Waele and Gianoulakis, 1993; Rasmussen et al., 1998). Furthermore, a large number of studies have shown that opioid antagonists reduce ethanol behavioral effects such as voluntary ethanol consumption or ethanol-induced locomotor activity
* Corresponding author. Tel.: /34-964-729337; fax: /34-964729350 E-mail address: [email protected] (C.M.G. Aragon).
(Kiianmaa et al., 1983; Prunell et al., 1987; Ulm et al., 1995; Davidson and Amit, 1997). Conversely, other authors have reported that low doses of opioid agonists, especially m-receptor ligands, can enhance some of these ethanol effects (Hubbell et al., 1987; Kuribara et al., 1991; Stromberg et al., 1997). In addition, studies in rodents as well as in humans reveal that subjects with higher endogenous opioid system sensitivity to ethanol are at a higher risk of excessive ethanol consumption than are subjects with lower sensitivity (Froelich et al., 1995; Gianoulakis, 1996). In fact, the opioid receptor antagonist naltrexone has become one of the most used therapeutic approaches for coping with excessive ethanol drinking and alcoholism (Herz, 1997; Stromberg et al., 1997). The brain sites of POMc synthesis include the hypothalamic Arcuate nucleus (ARC) and the medullary nucleus tractus solitary (NTS) (Khatchaturian et al., 1985). The ARC is located at the base of the hypothalamus, in close apposition to the median eminence and the pituitary gland. It surrounds the third ventricle and is directly connected to the pituitary gland,
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some thalamic and hypothalamic nuclei, the midbrain periaqueductual gray area, the autonomic nuclei of brain stem and the limbic system, including the amygdala, the nucleus accumbens and the ventral tegmental area (Chronwal, 1985; Khatchaturian et al., 1985). Conversely, the projections of the NTS system appear to be local within the medulla oblongata, with possible projections to the parabranchial area nuclei and to the spinal cord (Khatchaturian et al., 1985). It therefore seems that the ARC may be a more trustworthy system than the NTS for assessing the interactions between alcohol and the POMc-related peptides in ethanolrelated behaviors (Gianoulakis, 1996; Herz, 1997). In accordance with this rationale, one of our previous studies showed that selective destruction of ARC / POMc containing neurons using estradiol valerate (EV) (Desjardins et al., 1993) results in a blockade of the stimulatory effects of low or moderate ethanol doses on locomotion in female mice, but does not affect the depressant effects of higher doses of ethanol (SanchisSegura et al., 2000). Thus the aim of these experiments is to assess whether a more extensive destruction of the ARC (as produced by monosodium glutamate, MSG (Holzwarth-McBride et al., 1976; Peruzzo et al., 2000), or goldthioglucose, GTG (Young, 1989)) reproduces this effect. The present study is therefore an attempt to verify if the effects on ethanol-induced locomotion observed following EV administration (Sanchis-Segura et al., 2000) are due to its effect on the ARC. Moreover, the present study can provide additional evidence about the possible role of other neurotransmitters contained in the ARC on ethanol-induced locomotion.
2. Methods 2.1. Subjects For the MSG experiments, mice pups were obtained from pregnant female Swiss /Webster mice, purchased from Janvier Espan˜a S.L. (Madrid, Spain). For the GTG study, 4-week-old male Swiss /Webster mice were purchased from Janvier Espan˜a S.L. (Madrid, Spain) and housed in groups (four animals per cage), with standard laboratory rodent chow and tap water available ad libitum. 2.2. Drugs MSG, GTG (aureothioglucose) and caffeine were purchased from Sigma Aldrich S.A., Spain. Ethanol solution (20% v/v) was prepared from ethanol 96% (Panreac Qu’mica S.A., Spain). All these drugs were dissolved in saline (0.9%) solution.
2.3. Procedure For the MSG experiment, newborn pups received subcutaneous injections of MSG (4 mg/g body weight), or equivalent volumes of a saline solution (0.9%), on days 1, 3, 5, 7 and 9 after birth (Holzwarth-McBride et al., 1976; Crabbe and Dorsa, 1986). At 3 weeks of age, male mice were separated and housed in groups with four animals per cage. At 8 weeks after birth the behavioral tests were performed. For the GTG experiment, after 7 days of habituation to laboratory conditions, male mice were divided into two groups and saline or GTG was administered intraperitoneally (IP). GTG was dissolved at a concentration of 300 mg/ml and was injected IP to mice at a dose of 500 mg/kg (Young, 1989). The behavioral tests were performed 8 weeks later. The differences in the source of the subjects and drug administration correspond to the previously described requirements of each substance to produce an effective lesion of the hypothalamic ARC (Holzwarth-McBride et al., 1976; Crabbe and Dorsa, 1986; Young, 1989). For instance, it has been described that the ability of MSG to damage the arcuate neurons is related to the developmental status of the blood brain barrier in this perinatal period (Peruzzo et al., 2000), whereas GTG seems able to produce an effective ARC lesion when independent of an animal’s age (Young, 1989). 2.4. Apparatus The open field apparatus and locomotion measures have been described previously (Sanchis-Segura et al., 2000). Briefly, it consisted of a glass cylinder (25 cm diameter) the floor of which was divided in four equal quadrants by two intersecting lines drawn across it. A count was scored when a subject crossed a line with all four paws. Male mice pre-treated with saline, MSG or GTG were challenged with ethanol (0.0, 0.5, 1.5 or 2.5 g/ kg; IP; n /8 per group). Immediately after this treatment, mice were placed individually in the open field chambers for 20 min. Individual measures of locomotor activity were recorded for the last 10-min period. This delay was chosen in order to decrease the effects of animal handling and the environmental novelty of the open field (Dudek and Tritto, 1994) and because at this time brain ethanol levels are maximal after an IP administration (Yim and Gonzales, 2000). In a second study, we assessed the effect of MSG or GTG pretreatment on caffeine-induced locomotion (10 mg/kg; IP). Test conditions were as described above. 2.5. Blood ethanol levels Additional mice pre-treated with saline, MSG or GTG were used to assess blood ethanol levels after the
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administration of an ethanol injection (0.5, 1.5 or 2.5 g/ kg). Trunk blood was collected at 20 min following this ethanol challenge. Plasma ethanol levels were enzymatically assessed with an Alcohol Diagnostic Kit from Sigma-Aldrich Qu’mica (Spain) (Sanchis-Segura et al., 2000). 2.6. Histology The effects of repeated saline or MSG injections to mice pups during the perinatal days were assessed. Thus a separate group of mice pups were treated as described in the procedure section and 8 weeks later animals were anesthetized and perfused with an heparynized (1000 u/ L) saline solution. Coronal slices (40 mm) were obtained using a microtome and mounted on to slides. The slides were stained using a cresyl violet standard procedure (Davies and Stewart, 1997) and the coverslipped slides were viewed under 25 / or 100 / magnification.
3. Results Mice treated with MSG showed, 8 weeks later, several morphological alterations. It has been demonstrated that these changes reflect the extent of ARC lesion after MSG administration (Dubovicky et al., 1997). Thus, the nose /anus distance was significantly reduced [T (13) / 4.60, P B/0.01] in mice pre-treated with MSG compared to saline pre-treated mice (mean9/SEM were 8.539/0.37 and 10.609/0.22 cm, respectively; n /5/6 per group). The tail length was also shorter in MSG pre-treated mice [T (13) /8.40, P B/0.01] than in control mice (mean9/SEM were 6.509/0.13 and 8.419/0.19 cm, respectively; n /5 /6 per group). Moreover, the histological assessment of coronal brain sections of mice treated with MSG revealed a significant cell loss in the ARC nucleus compared to saline treated mice (Fig. 1). On the other hand, after 2 months, mice treated with GTG showed a significant increase in body weight compared to saline pre-treated mice [T(38) / /3.93, P B/0.01]. Thus, mean9/SEM body weights (n /20 per group) were 44.149/0.78 and 52.149/1.87 g for saline and GTG pre-treated mice, respectively. In accordance with previous reports (Bergen et al., 1998), this increase in body weight may be understood as an index of the destruction of the ARC produced by GTG. Fig. 2A depicts the effects of perinatal MSG pretreatment on ethanol-induced locomotion. As the figure shows, ethanol-induced a biphasic effect on locomotor activity in animals pre-treated with saline. However, MSG-treated mice did not show any ethanol induction of locomotion, in fact to the contrary they displayed an ethanol dose-dependent reduction of locomotion. A two-way ANOVA revealed a significant effect of the pre-treatment (saline or MSG) [F (1, 56) /74.87, P B/
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0.01] and the ethanol dose (0.0, 0.5, 1.5 or 2.5 g/kg) [F (3, 56) /16.28, P B/0.01] factors, as well as a significant interaction between them [F (3, 56) /8.71, P B/ 0.01]. Fisher’s LSD test showed that MSG blocked the increase of locomotion produced by low or moderate doses of ethanol (0.5 and 1.5 g/kg) whereas it enhanced the depressant effects produced by a higher dose of ethanol (2.5 g/kg). On the other hand, MSG pretreatment did not affect spontaneous locomotor activity (ethanol dose 0.0 g/kg). Similarly, as shown in Fig. 2B, GTG-treated mice did not show any ethanol induction of locomotion. Thus, a two-way ANOVA of pretreatment (saline or GTG) /ethanol dose (0.0,05, 1.5 or 2.5 g/kg) revealed a significant effect of the pretreatment [F (1, 56) /14.58, P B/0.01] and the ethanol dose [F (3, 56) /7.38, P B/0.01] factors, as well as a significant interaction between them [F (3, 56) /4.91, P B/0.01]. Fisher’s LSD test showed that GTG pretreatment reduced the increase in locomotion observed after low or moderate doses of ethanol, whereas it did not modify the locomotion observed after higher doses of ethanol (2.5 g/kg) or after a saline injection. Fig. 3A presents the locomotor activity values observed in saline or MSG pre-treated mice after an acute caffeine injection (10 mg/kg). A two-way ANOVA revealed that caffeine produced a significant enhancement of mice locomotion [F (1, 28) /14.18, P B/0.01]. This effect was unmodified by MSG. As shown in Fig. 3B, similar results were obtained when comparing the effects of a saline or GTG pre-treatment on the locomotion of mice challenged with the same dose of caffeine. Again, a two-way ANOVA revealed that caffeine produced a significant enhancement of locomotion [F (1, 28) /10.32, P B/0.01] and that this effect was unmodified by the pre-treatment conditions. Table 1 presents the effects of MSG or GTG pretreatment on blood ethanol levels. In animals pretreated with MSG, a two-way ANOVA revealed that the ethanol dose factor [F (1, 2) /118.07; P B/0.01] and its interaction with the group factor [F (1, 2) /12.13; P B/0.01] yielded significant effects. Mean comparisons using Fisher’s LSD test revealed that animals pretreated with MSG and administered with 2.5 g/kg of ethanol had higher blood ethanol levels than their controls. On the other hand, ethanol blood levels of animals pre-treated with GTG or saline did not show any difference, and only the ethanol dose factor yielded a significant effect [F (1, 2) /46.89; P B/0.01].
4. Discussion The results of the present study show that MSG or GTG treatment produces an antagonistic effect on the stimulant effects of low and moderate ethanol doses in mice locomotion. At present, the explanation of these
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Fig. 1. Cresyl violet coronal hypothalamic sections of animals perinatally treated with saline (A and D) or MSG (B, C, E, F) as described in Section 2. As can be observed in pictures B and C, MSG produced an evident lesion of the ARC (magnification 25 /). The loss of cells produced by perinatal MSG administration can be better observed at higher (100 /) magnification (pictures D, E and F). Circles and arrows have been included to better indicate the ARC location.
Fig. 2. Effects of MSG (A) or GTG (B) pre-treatment on ethanol (0.5 /2.5 g/kg)-induced locomotor activity. Bars depict mean9/SEM locomotor activity (counts/10 min) for all treatment groups (n /8 per group). White and dashed bars represent saline and GTG or MSG pretreated animals, respectively. (*P B/0.01 significantly different from saline pre-treated group.)
Fig. 3. Effects of MSG (A) or GTG (B) pre-treatment on caffeine (10 mg/kg)-induced locomotor activity. Bars depict mean9/SEM locomotor activity (counts/10 min) for all treatment groups (n /8 per group). (*P B/0.01 significantly different from saline pre-treated group.)
C. Sanchis-Segura, C.M.G. Aragon / Drug and Alcohol Dependence 68 (2002) 189 /194 Table 1 Mean9SEM of blood ethanol levels (mg/dl) in (A) MSG or (B) GTG pre-treated mice as described in the methods section and their respective control groups Pre-treatment
Ethanol dose (g/kg) 0.5
1.5
2.5
A Saline MSG
66.88911.51 60.5393.11
277.84939.25 234.5796.73
316.04911.58 472.98930.69*
B Saline GTG
77.44917.04 64.19916.42
181.0298.09 245.17916.64
332.40914.75 341.79958.38
Blood samples were taken 20 min after ethanol administration (0.5 / 2.5 g/kg; IP). (* P B 0.01)
results remains open but it could be related to the lesion that MSG and GTG administration produces in the ARC. In this respect, it has been shown that the MSG treatment conditions used in the present study results in the destruction of 80 /90% of ARC neurons (Desjardins et al., 1992). Similar results have been found after GTG administration, which may produce an even more extensive lesion which includes the ventromedial hypothalamus and other adjacent areas (Young, 1989). In the present study, we confirmed previous observations about cell loss in the ARC following MSG treatment (Desjardins et al., 1992; Peruzzo et al., 2000) in addition to some of the morphological changes that occur after MSG or GTG administration and which have been clearly correlated with the extent of the ARC lesion (Dubovicky et al., 1997; Bergen et al., 1998). The behavioral data obtained in the present study will therefore be discussed within this conceptual framework. In our data, GTG or MSG had no effect on the spontaneous locomotion of mice. With regard to our results it is necessary to note that the effect of MSG on the spontaneous locomotor activity of rodents is unclear and that conflicting findings have been published. For example, increases (Dubovicky et al., 1997), decreases (Barnhart and Pizzi, 1982; Crabbe and Dorsa, 1986) and the absence of an effect have all been reported (Barnhart and Pizzi, 1982; Dubovicky et al., 1999; Ali et al., 2000). On the other hand, although the effects of GTG on the spontaneous locomotion of mice have no precedent they do seem to provide further support to the notion that the ARC lesion does not alter the spontaneous locomotion of mice. Conversely, both MSG and GTG produced a deep change in the behavioral effects of low and moderate doses of ethanol. Thus, mice treated with either of these two neurotoxins showed a blockade of the stimulatory effects on mice locomotion observed after ethanol (0.5 or 1.5 g/kg) administration. Since, at these doses, blood ethanol levels were unmodified, it can be suggested that
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the changes on ethanol-induced locomotion following MSG or GTG administration may be related to an effect of these substances in the brain, probably at the ARC. Moreover, in the present study, MSG enhanced the depressant effects of a higher (2.5 g/kg) ethanol dose on mice locomotion, whilst GTG did not. This result could be related to the increase in blood ethanol levels observed after a high dose of ethanol in MSG-, but not GTG-treated mice. This suggestion is in accordance with a previous report (Crabbe and Dorsa, 1986), which showed that MSG enhances blood ethanol levels in addition to boosting the reduction of locomotion produced by high doses of ethanol in mice. Finally, it was observed that after a caffeine challenge the locomotion of MSG- or GTG pre-treated mice was no different from that of saline pre-treated mice. The assessment of these effects was also unprecedented and these results suggest that the ARC does not belong to a general system activated by all drugs, which are able to increase mice locomotion. In general, there is a close parallel between the present results and those observed in a previous study using EV. EV produces a selective destruction of the POMcsynthesizing neurons of the ARC (Desjardins et al., 1990, 1993) and blocks ethanol-induced locomotion (Sanchis-Segura et al., 2000). Moreover, this substance does not alter the effects of caffeine on mice locomotion (Sanchis-Segura et al., 2000). Thus, although there are some differences in the pattern of destruction of the ARC caused by these neurotoxins, they all block ethanol- but not caffeine-induced locomotion and destroy the ARC POMc-synthesizing neurons. Because the POMc derived peptide b-endorphin has been proposed as a possible mediator of some of the behavioral effects of ethanol (Gianoulakis, 1996; Herz, 1997), it seems possible that the effects of MSG, GTG and EV on ethanol-induced locomotion could be related to their ability to lesion the endorphin-synthesizing neurons. Additional although indirect support for this hypothesis seems to be provided by experiments which show that opioid receptor blockade results in a decrease in ethanol- but not caffeine-induced locomotion (Kiianmaa et al., 1983; Sawynok et al., 1995). In summary, the present results show that MSG or GTG administration, under the treatment conditions used in this study, produces a blockade of ethanol effects on mice locomotion. Because of the only known common effect of MSG and GTG is their ability to produce an ARC lesion, it seems reasonable to suggest that their identical consequences on ethanol-induced locomotion are related to this capacity. Therefore, these data argue for a role of the ARC nucleus in the behavioral effects of ethanol. Although the exact role of the ARC remains to be elucidated, it could be related to its ability to synthesize b-endorphins and other
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POMc-derived peptides. Future experiments will be dedicated to exploring this topic.
Acknowledgements This research was funded by a grant from Fondo de Investigacio´n Sanitaria. Ministerio de Sanidad y Consumo. (FIS 001101), Spain. C.S.-S. was supported by a fellowship from the Conselleria d’ Educacio´ i Cie`ncia de la Generalitat Valenciana, Spain. The authors gratefully acknowledge the assistance of Dr J.K. Young for his technical comments about GTG dosage and effects.
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