Research reportAlcohol drinking produces brain region-selective changes in expression of inducible transcription factors
Introduction
Alcohol consumption and intoxication are widely known to exert actions on several neural systems. The evidence supporting this notion has been confirmed in human and animal brain mapping studies. These studies have detected regional metabolic changes in a number of selective brain structures following intoxication 9, 29, 49, 53. Another approach that provides a useful means of mapping brain activity is the immunohistochemical detection of inducible transcription factors (ITFs) encoded by immediate early genes. This method is advantageous in its cellular resolution and its ability to dissociate changes occurring in neuronal somas vs. changes in synapses and glial cells 23, 37.
This method has been very effective in determining differential activation of neural structures following alcohol administration 4, 11, 34, 44. Two primary effects of alcohol can be observed by mapping ITFs. First, alcohol administration induces ITFs in several brain areas, including the central nucleus of the amygdala, the bed nucleus of stria terminalis, the paraventricular nuclei of hypothalamus and the thalamus 4, 11, 26, 31, 33, 44, 55. Second, alcohol administration is able to suppress ITF expression in hippocampal areas, especially when the hippocampal ITF expression is elevated by environmental stimuli 20, 34.
Changes in ITF expression in these aforementioned studies occurred following involuntary alcohol exposure (i.e., injected by the experimenter). This procedural aspect may contribute in part, to alcohol-induced ITF expression. This suggestion stems from evidence demonstrating increased expression of ITF following the presentation of a stressful stimulus 2, 3, 37, 41. Stress-induced ITF expression overlaps with many structures also showing alcohol-induced ITF expression, thus making it difficult to differentiate stress- and alcohol-induced changes in ITF expression. It has been shown that different routes of alcohol administration (i.e., intraperitoneal injection, intragastric intubation, or alcohol vapor inhalation) produce similar patterns of ITF expression 26, 33. However, these studies only partially overcome this difficulty. Thus, the first alcohol administration is accompanied by a factor of unpredicted novelty, which is stressful and could contribute to increased ITF expression. These concerns are further strengthened by evidence obtained by deoxyglucose mapping, in which alcohol administration shows differential activation of brain areas after alcohol infusion and alcohol self-administration 29, 52, 53.
Given this data, it is likely that the manner of alcohol presentation contributes to differential gene expression. Therefore, we propose that the use of a self-administration paradigm may be useful in eliminating the stress-induced expression of experimenter-administered alcohol and aid in identifying pharmacological effects on the neural substrates involved during voluntary alcohol intoxication. This approach has recently been applied in our lab to show that alcohol consumption can modify restraint stress-induced ITF expression in the nucleus accumbens and hippocampus. Alcohol itself, however, did not produce changes in ITF expression in non-restrained animals [36]. The present approach extends these findings in two ways. First, our previous design compared ITF expression in mice with access to either 10% sucrose or 10% sucrose/10% alcohol. However, the influence of sucrose on ITF expression is not well documented in the higher brain regions which raises a concern about the validity of this group as a control. This concern is addressed in the present analysis by the inclusion of a water control group. Second, ethanol consumption in our previous study (1.32±0.2 g/kg) may have been below the range needed to produce induction of ITFs. However, with a minor change in procedure, we have been able to increase ethanol consumption in C57BL/6J mice to 2 g/kg or higher, see also Ref. [45]. In the present study, we used a self-administration paradigm to identify the alcohol-induced changes in expression of several ITFs including c-Fos, FosB, and Zif268 (also known as NGF-IA, Krox-24, and Egr1) in mouse brain.
Section snippets
Animal procedures
Twenty-eight male, 7-week old C57BL/6J mice were purchased from Jackson Laboratories and placed four per cage on a 12 h light–dark cycle. Food access was ad libitum at all times during experiments. A limited access alcohol/sucrose drinking procedure was used 36, 45. One week after arrival, animals were housed individually in metal hanging racks. Animals were randomly assigned to one of the following three groups: Sucrose (n=10), Alcohol (n=14), and Water (n=4). After a one-week habituation
Behavioral data
Alcohol-exposed animals showed stable alcohol consumption, except one animal that did not drink any alcohol on the day of sacrifice. This animal was excluded from further analysis to avoid potential effects of “craving” on ITF expression [46]. Doses were calculated based on the volumes of consumed alcohol (Fig. 1), and were significantly correlated to BAC in animals sacrificed for immunohistochemistry (r=0.757, p=0.003). Alcohol consumption led to higher doses and BAC than that observed in our
Discussion
In general, the present study confirms our previous finding which shows much smaller changes in ITF expression in response to voluntary alcohol consumption compared with ITF changes seen in animals administered alcohol by an experimenter. Thus, induction of c-Fos was not observed in several brain regions that were previously found to be strongly reactive to alcohol injections including CeC and CeL, BNST, PV, and Pa 4, 11, 26, 31, 33, 44. Our previous study only detected effects of alcohol
Acknowledgements
This work was supported by the National Institute of Alcohol Abuse and Alcoholism (Grants AA10810, AA01760, AA10520 and AA07468).
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