Cone Laboratory for Neurosurgical Research, Department of Neurology and Neurosurgery, and Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, H3A 2B4 Canada
Received 2 August 2002; in revised form 4 November 2002; accepted 14 November 2002
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ABSTRACT |
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INTRODUCTION |
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In alcohol consumption-related studies (Myers and Veale, 1968), the administration of substances that increase serotonergic neurotransmission, such as the 5-HT precursor tryptophan, uptake inhibitors or 5-HT releasers, has generally been found to reduce ethanol consumption. Conversely, pharmacological manipulations that reduce cerebral 5-HT neurotransmission, such as lesions of serotonergic neurons, provoke an increase in alcohol intake (Badawy, 1998
; Pihl and LeMarquand, 1998
). Due to the mediation of the central actions of 5-HT through the activation of several receptor subtypes, previous studies attempted to establish which of the receptors may be involved in the mechanisms that modulate alcohol consumption. This was achieved by examining the effects of alcohol consumption on the serotonergic system (McBride et al., 1993
; Meert, 1993
). In relation to these studies, knowledge regarding the chronic effects of alcohol on 5-HT synthesis in the discrete regions of the brain may provide some insight into the basic mechanism by which alcohol acts on the brain serotonergic system, as a change in 5-HT synthesis could result in changes in 5-HT receptors.
An autoradiographic method using labelled -[14C]methyl-l-tryptophan (
-[14C]MTrp), a Trp analogue, as a tracer for the measurement of 5-HT synthesis was developed (Diksic et al., 1990
). It had been established that
-[14C]MTrp as a tracer follows the 5-HT biosynthetic route from Trp to 5-HT (Diksic et al., 2000
; Tohyama et al., 2002
), and that the brain trapping of the tracer correlates with Trp conversion to 5-HT but not with Trp incorporation into proteins. The method permits in vivo measurements of 5-HT synthesis rates in a large number of brain structures with a good anatomical resolution (approximately 0.1 mm) and without any additional pharmacological treatment.
In the present work, using the -MTrp method, 5-HT synthesis rates were measured in discrete regions of rat brain following a continuous treatment with ethanol, delivered continuously by osmotic mini-pumps until decapitation. The aim was to obtain information on the effect of continuous and constant delivery of ethanol on 5-HT synthesis in rat brain. This method of alcohol delivery was selected to ensure a continuous and constant exposure of the brain to alcohol. This procedure also ensures a stable concentration of ethanol in the blood, without the need for frequent handling of the animals. Because there is no other study in which blood alcohol was kept constant for a prolonged period of time and during 5-HT synthesis measurements, a direct comparison of the results presented here and those from previous investigations may be somewhat difficult. Any other administration schedule would produce effects associated both with alcohol exposure and alcohol withdrawal (Badawy and Evans, 1976
; Badawy et al., 1980
; Bano et al., 1996
).
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MATERIALS AND METHODS |
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SpragueDawley male rats (Charles River, St-Constant, Quebec, Canada) weighing between 130 and 150 g at the time of mini-pump implantation were used in the study. The rats were housed in the animal facility (room temperature of 22°C with a 12-h light12-h dark cycle) for at least 3 days before being used in the experiments. The rats had free access to solid food and drinking water. Twenty-four rats (130150 g) were implanted subcutaneously with osmotic mini-pumps (type 2ML2; Alza, Palo Alto, CA, USA) filled with 50% ethanol (in distilled water). It should be noted that the ethanol mixture was delivered to the rats continuously until their decapitation. This means that the ethanol was delivered during the entire period of the tracer uptake. Nine rats were implanted with mini-pumps filled with distilled water and served as controls. The pumps were calibrated by the manufacturer to deliver 5 µl/h for 14 days, which translates into a delivery of 2.5 µl of ethanol per hour. All implantation procedures were carried out under halothane (1.02.0%) anaesthesia and a sterile operating room technique.
Experimental procedure
The autoradiographic experimental procedure used in the present study was previously described in detail (Diksic et al., 1990; Nagahiro et al., 1990
; Mück-
eler and Diksic, 1995
). We will provide a short summary here. Before the autradiographic studies, the animals were starved overnight with water provided ad libitum. Starvation was necessary to obtain a stable concentration of plasma amino acids (Diksic et al., 1990
). All rats were killed between 14.00 and 16.00. The physiological state of the animals was assessed by monitoring their rectal temperature and blood gases to ensure that these parameters were within normal limits established for the laboratory (Nagahiro et al., 1990
). Blood gases were determined by using a micro blood gas analyser (model 274; Chiron Medical, Montreal, Quebec, Canada). Under light halothane (1.02.0%) anaesthesia, plastic catheters were inserted into the femoral artery (for blood sampling) and vein (for tracer injection). The rats were attached to lead bricks with a masking tape to prevent them from moving. The rats were then allowed to awaken before the tracer injection.
-[14C]MTrp (30 µCi; specific activity of about 55 mCi/mmol), synthesized by us using the procedure described by Mzengeza et al.(1993)
, was injected intravenously in 1 ml of saline over 2 min, using constant infusion with an injection pump. With the beginning of the tracer injection, arterial blood samples were taken at progressively increased time intervals up to the killing time. Rats were killed at 1 or 2.5 h after the beginning of tracer injection. Usually 1416 blood samples were taken during the procedure. The blood samples were centrifuged for 3 min at 12 500 g, and 20 µl of plasma was taken for liquid scintillation counting to measure the plasma 14C-radioactivity (input function) (Diksic et al., 1990
). The brains were removed, frozen in isopentane and cut into 30 µm slices in a cryostat at about -20°C. The brain sections were mounted on glass slides, dried on a warm plate (about 60°C), and exposed to an X-ray film along with a 14C-polymer standard (American Radiolabel; calibrated to the dry tissue equivalent) for 3 weeks to obtain autoradiograms.
At the beginning, midpoint, and end of each experiment, an additional 50 µl of plasma was deprotenized with 25 µl of 20% (w/v) trichloroacetic acid and used for the determination of plasma total Trp concentration. An additional 50 µl of plasma was filtered through an Ultrafree-MC filter spinning at 12 500 g for 10 min for free (non-albumin-bound) Trp. Total and free Trp concentrations were measured using the HPLC method described previously (Takada et al., 1993).
Calculation of the 5-HT synthesis rate
The images were digitized using a microcomputer-based image analysis system (MCID; Imaging Research Inc., St Catharine, Ontario, Canada). Optical densities were converted into tissue radioactivity concentration (nCi/g) using a calibration curve. Usually a third-order polynomial was utilized as a calibration curve for conversion of the optical density into the tissue tracer concentration. The outlining regions of interest were placed on each structure. The tissue concentration (nCi/g) of the tracer was measured separately in 31 brain structures. The average of six values in each brain structure in three consecutive sections was obtained by the aid of the rat brain atlas (Paxinos and Watson, 1986).
The tissue radioactivity concentrations (nCi/g) were converted into the volume of distribution (VD; ml/g) by dividing tissue concentrations (nCi/g) with the plasma tracer concentration [nCi/ml; C*p(T)] at the end of experiment (where T is the time of the end of an experiment). The VD and the exposure time (; min) [
=
0T C*p(T)dt / C*p(T)] have a linear relationship (Diksic et al., 1990
, 1995
; Nagahiro et al., 1990
). The slope of this relationship is equal to K* (constant of tracer trapping in the brain) (ml/g/min) and the intercept is Vapp (ml/g; an apparent volume of distribution of the tracer in the precursor pool; Diksic et al., 1990
, 1995
). The rate of 5-HT synthesis, R (pmol/g/min), was calculated as: R = K* Cp / LC (conversion constant), where Cp (nmol/ml) is the concentration of the plasma non-protein-bound Trp. The use of plasma-free Trp in this calculation was discussed in detail elsewhere (Diksic, 2001
). The LC was measured in vivo and found to be constant throughout the brain. It has a value of 0.42 ± 0.07 (Vanier et al., 1995
). In the present experiments, all of the linear relationships between VD and
had a significant (P < 0.05; F-statistics) and positive slope. SD values were calculated from the SD values in K* reported by the variancecovariance matrix of the least-squares fit. For the pictorial presentation given in Fig. 1
, an average value of Vapp was calculated from the values in the respective sets of experiments (Diksic et al., 1995
); however, the values provided in Table 1
were calculated with the individual values of the Vapp (Nagahiro et al., 1990
).
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RESULTS |
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A set of representative autoradiograms obtained in the brains of the control and alcohol-treated rats is shown in Fig. 1. A visual evaluation of these images suggests a non-uniform 5-HT synthesis throughout the rat brain. It can be seen that 5-HT synthesis is not homogeneous throughout the cortex (e.g. layer VI has the greatest synthesis of 5-HT), as well as some other structures (e.g. caudate). The greatest synthesis of 5-HT was observed in the cell body structures (raphe, pineal body). The rates of 5-HT synthesis measured in 22 representative structures are provided in Table 1
.
The rate of 5-HT synthesis was significantly increased in the majority of structures in the treated group, when compared with the controls (Table 1). The greatest increases were observed in the descending serotonergic nuclei [(given as a percentage increase over the rates in the control animals): raphe pallidum, 84%; raphe obscurus, 41%; raphe magnus, 40%], the nigrostriatal structures (caudate-putamen, 30%; medial caudate, 52%; substantia nigra reticulosa, 22%), the hippocampus (ventral hippocampus, 37%; dorsal hippocampus, 34%), the locus coeruleus (76%), the superior olive (37%), the inferior colliculus (41%), and the cortices (auditory cortex, 39%; visual cortex, 33%; somato-sensory cortex, 126%). No significant changes were observed in the rate of 5-HT synthesis in the dorsal and median raphe nuclei, pineal body, parietal or frontal cortex, as well as some other deep brain structures (Table 1
).
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DISCUSSION |
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In the present study, we found an increase in 5-HT synthesis in the caudate nucleus and nucleus accumbens. The nigrostriatal structures are well known as a main part of the dopaminergic projections, but these structures also receive very important serotonergic innervation from the raphe nuclei (Azmitia and Segal, 1978). The nucleus accumbens has been recently identified as a structure in which dopamine (DA) release is correlated with reward (Laruelle et al., 1995
; Drevets et al., 2001
). There are also important projections from the dorsal raphe to the extrapyramidal and baso-limbic ganglia (Azmitia and Segal, 1978
). The present results could also be related to serotoninDA interactions in the brain, which is likely to be linked to alcohol-related behaviour. The extracellular levels of serotonin and glutamate were low in the prefrontal cortex and the nucleus accumbens, using in vivo microdialysis, and were proposed to be associated with a predisposition to alcohol-drinking behaviour (Selim and Bradberry, 1996
). This low level of 5-HT could be one of the reasons that serotonergic neurons attempt to compensate for this alcohol-induced reduction in 5-HT content by an activation of the tryptophan hydroxylase. However, we should note that in the present experimental set-up, alcohol was continuously delivered at all times.
An increase in 5-HT synthesis was also observed in the cortices (visual, auditory and somatosensory); however, no significant change was observed in the frontal cortex. The latter is an unexpected finding, as it has been reported that the dorsal prefrontal cortex is involved in the regulation of alcohol intake (Deckel et al., 1996), and also because lesion of the prefrontal cortex increases the release of DA in the nucleus accumbens in response to a variety of reinforced behaviours, such as intracranial self stimulation, feeding and olfactory stimulation.
An important finding in the present study was the absence of significant changes in serotonin synthesis in the dorsal and medial raphe nuclei. It seems that a chronic alcohol treatment, like the one used in this study, has an effect on 5-HT synthesis in the serotonergic terminals, but not in these cell bodies. Lower levels of 5-HT and its metabolite occur in different regions of the brain of alcohol-preferring (P) rats, in contrast to the alcohol-non-preferring (NP) rats (Murphy et al., 1982; McMillan et al., 1998
) The concentration of 5-HT in the dorsal raphe is lower in the P rat compared with the NP rat (Zhou et al., 1994
).
Autoradiographic experiments after chronic alcohol treatment revealed the up-regulation of the somato-dendritic 5-HT1A autoreceptors in the dorsal raphe, and the down-regulation of the postsynaptic 5-HT1A receptors in some projection areas (frontal cortex, entorhinal cortex and hippocampus) (Nevo et al., 1995). The selective destruction of the midbrain raphe nuclei by 5,7-dihydroxytryptamine exerts no influence on the pattern of alcohol drinking in SpragueDawley rats (Adell and Myers, 1995
). Modifications in certain characteristics of various 5-HT receptor types upon chronic alcoholization, and in genetically alcohol-preferring rat lines, have been reported (McMillan et al., 1998
). However, in other studies, chronic ethanol treatment did not down-regulate 5-HT1A receptor functions in the CA1 (Lau and Frye, 1996
). A relatively low concentration of 5-HT in the central nervous system is associated with a high voluntary intake of alcohol. Drinking alcohol is reduced by drugs known to stimulate 5-HT transmission, or by 5-HT itself, its precursors, as well as selective 5-HT uptake inhibitors (McMillan et al., 1998
). An increase in 5-HT synthesis in the hippocampus could be related to the hippocampal increase in the concentration of 5-HT found in the alcohol-preferring rats, in comparison to that in the alcohol-avoiding rats (Korpi et al., 1988
).
Discordant results have been reported on the effects of chronic alcohol treatments on the rates of 5-HT synthesis (Frankel et al., 1974). However, many of these differences could be explained by differences in the doses of ethanol, the duration of its administration, the species and/or strains of animals used, as well as the route of ethanol administration. Since in the present study the alcohol mixture was delivered by mini-pumps, the alcohol concentration in the brain was expected to be constant throughout a large portion of the 14 days of the experimental protocol, as well as until the rats were killed. In this respect, no other study is comparable with this one. It should also be noted that no other study had a similar anatomical resolution. The present study indicated that 2.5 µl/h ethanol treatment by osmotic mini-pumps affects, in a region-specific manner, 5-HT synthesis in the rat brain. This study confirms the increases in 5-HT biosynthesis following long-term treatment with alcohol (Kuriyama et al., 1971
; Palaic et al., 1971
); however, it should be noted that the alcohol administration schedule was different from that used in the present study. This would suggest that, when there is no development of the withdrawal symptoms between doses in a chronic treatment, the effect of the treatment on the 5-HT synthesis could be similar to that observed in a continuous delivery experiment. The data reported here clearly show that a continuous ethanol treatment delivered by osmotic mini-pumps has a remarkable rate-enhancing effect on 5-HT synthesis in some brain regions. However, the rate-enhancing effect was not uniform throughout the brain. The results suggest that there are differences in the influences of ethanol on 5-HT synthesis between the different brain structures, especially between the cell bodies and terminal areas. Certainly, the alcohol delivery protocol used in the present study is not directly related to alcohol consumption in humans; however, it should give information on the changes in brain 5-HT synthesis produced by a constant exposure to alcohol. Further research is necessary to obtain a better understanding of the above-mentioned possibilities by which ethanol affects brain serotonergic neurotransmission. In conclusion, we can state that a continuous exposure to alcohol results in an increase in the brain 5-HT synthesis in rat in many projection areas.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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2 Permanent address: Department of Neurosurgery, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo, Hokkaido, 060-8638 Japan.
* Author to whom correspondence should be addressed.
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