Current Neurobiology 2010; 1 (1): 40-45

Alcohol ingestion has adverse effects on the central nervous system (CNS). The hippocampus is one of the target sites of ethanol neurotoxicity. We hypothesized that short-term ethanol exposure alters the expression of neurotrophins and their receptors, leading to functional disruption in the CNS. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. Pair-fed control mice were maintained on an identical liquid diet, except that ethanol was isocalorically substituted with sucrose. The hippocampus of mice exhibiting stages 1-2 of ethanol intoxication signs were used in the present study. Short-term ethanol exposure did not alter the mRNA expression of neurotrophin ligand/receptor (nerve growth factor [NGF]/TrkA and brain-derived neurotrophic factor [BDNF]/TrkB) systems in the hippocampus. Similarly, the expression of the glial-derived neurotrophic factor (GDNF), which is known to be a first-acting agent against ethanol neurotoxicity, and its receptor GFRα1 was not affected by short-term ethanol exposure. The mechanisms involved in the hippocampal neurotrophin responses against ethanol neurotoxicity remain unknown. However, our findings could provide a basis for further studies on the possible alterations in the expression of various neurotrophins related to hippocampal functions. Introduction Alcohol ingestion has various adverse effects on the mind and body, depending on the dose ingested. Of these effects, impairment of the central nervous system (CNS) is one of the most serious alcohol-related conditions. Impairment in learning, memory, and cognitive functions are well-known signs related to alcohol consumption in humans and laboratory animals. Such impairment can be noted even in the absence of obvious organic lesions in the CNS, such as those associated with Wernicke-Korsakoff’s syndrome. In fact, basic and clinical studies have revealed that ethanol exposure alters neurotrophin expression, neuronal excitability, or synaptic transmission, without causing any histological alterations in brain regions such as the hippocampus, cerebral cortex, and cerebellum, which are closely related to functions of cognition, learning, and memory [1-3]. Neurotrophins are known to play key roles in normal brain functions, neural regulation, and the differentiation and survival of neurons in specific brains regions. The neurotrophin family includes various trophic factors such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT-4/5. These factors initiate signaling by binding to receptors with high (TrkA and TrkB) or low (p75) affinity. The high-affinity receptor isoforms TrkA and TrkB preferentially bind to NGF and BDNF, respectively [4-6]. Moreover, the uptake and internalization of neurotrophins are predominantly mediated by the high-affinity receptors, not the low-affinity ones [7, 8], and these processes seem sufficient for neurotrophins to elicit biological effects such as enhanced neuronal survival and neurite outgrowth [9-11]. Many studies have examined the effects of acute or chronic ethanol exposure on the responsiveness of various neurotrophic factor ligand/receptor systems in the CNS [12]. Surprisingly, no reports have addressed the effects of shortterm exposure to alcohol on the brain. Moreover, we have previously demonstrated that short-term ethanol exposure for 3–5 days can disrupt neuron-astrocyte interactions in the hippocampus of mice [13]. More recently, we reported that long-term (19 weeks) ethanol exposure decreased the mRNA expression of oligodendrocyte-myelin glycoprotein (OMgp) but did not affect that of BDNF or the glial-derived neurotrophic factor (GDNF) in the rat hippocampus [14]. The results of our previous studies on the hippocampus [13, 14] raise the question of whether short-term ethanol exposure affects the mRNA expression of neurotrophins in the hippocampus, which is profoundly involved in learning and memory functions. These hippocamal functions are known to be disturbed by alcohol ingestion. In the present study, therefore, we evaluated the effects of short-term ethanol exposure on the hippocampus with respect to alterations in the neurotrophin ligand/receptor system by using real-time reverse transcription-polymerase chain reaction (RT-PCR). Materials and Methods Animals and ethanol administration Adult (7–8 weeks old, male) BALB/C mice (SLC Japan, Shizuoka), weighing 22–25 g, were used in the present study. The animals were housed in separate cages in a room with strictly controlled temperature (21–23 °C) and humidity (50– 65%) conditions. Lighting was programmed according to a regular light/dark (12 h/12 h) cycle. The ethanol exposure paradigm used in the present study was identical to the one we have previously described [13]. In brief, the mice were divided into two groups—the ethanol-fed group and the pair-fed control group. Both groups were allowed to acclimatize to the housing environment and were fed a normal liquid diet for 7 days prior to ethanol administration. Subsequently, the ethanol-fed group (n = 5) was provided unrestricted access to a liquid diet (Oriental Yeast, Tokyo, Japan) containing 5% (v/v) ethanol (99.5% ethyl alcohol; Wako, Osaka, Japan) as the sole fluid source. The pair-fed control group (n = 5) was maintained on an identical liquid diet, except that ethanol was isocalorically substituted with sucrose. The mice that exhibited stages 1-2 of ethanol intoxication signs, as per the classification proposed by Freund [15], were selected for use in subsequent experiments. Freund [15] classified intoxication into four stages characterized by hyperreactivity and tremor (stage 1); episodes of rapid tail beating, a slow broad-based gait, rapid backward movements (retropulsion), stereotype or repetitive movement (stage 2); generalized tonic convulsions (stage 3); and death during a convulsion (stage 4). This study was carried out in compliance with the guidelines for the experimental use and care of laboratory animals, issued by the European Communities Council Directive of November 24, 1986 (86/609/EEC), and was approved by the Kagawa University Animal Ethics Committee. Real-time RT-PCR analysis The mice showing ethanol intoxication signs of stages 1-2 were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally) and subjected to intracardiac perfusion with medical-grade physiological saline. Blood was collected from the left ventricle prior to the perfusion, and its ethanol concentration was determined by gas chromatography (Shimadzu GC-8A, Tokyo, Japan). The brains were sectioned into 1-mm-thick slices in the horizontal plane by using a vibratome. The hippocampus was dissected from the brain in chilled physiological saline, with the aid of a dissection microscope. These specimens were processed for RT-PCR by using RNAlater (Ambion, Austin, USA). Total RNA was isolated from the processed hippocampal slices by using TRIzol reagent (Invitrogen, Carlsbad, USA), according to the manufacturer’s instructions. cDNA synthesis and quantitative detection were performed using a LightCycler rapid thermal cycler system (Roche Diagnostics, Lewes, UK) and the LightCycler-FastStart DNA Master SYBR Green I mix (Roche Diagnostics). The forward and reverse primers used in the present study are shown in Table 1. β-actin was used as an internal control. To confirm the specificity of the amplification, the PCR products obtained with each primer pair were subjected to melting-curve analysis and subsequent sequencing. To prevent genomic contamination, the PCR products that were amplified from cDNA by using each primer were electrophoresed on 2% agarose gel and stained with ethidium bromide. Similarly, the PCR products obtained without reverse transcription were electrophoresed and used as a negative control. The quantification data were analyzed using the LightCycler analysis software. The mRNA expression of each gene was determined as the ratio of this expression to that of the housekeeping gene β-actin. Each sample was analyzed in duplicate to ensure consistent results. Statistic analysis Data are presented as the mean ± standard error of the mean (S.E.M.). All statistical analyses were carried out by Student’s t-test, using the SigmaStat software (Systat, version 3.0).