Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Neurochemistry International 37 (2000) 483±496

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Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain P. de Gortari a,b,*, M. MeÂndez c, I. RodrõÂ guez-Keller a, L. PeÂrez-MartõÂ nez d, P. Joseph-Bravo b,d b

a Dept. NutricioÂn, Universidad Iberoamericana, Mexico Dept. Neurociencias, Instituto Mexicano de PsiquiatrõÂa, Calzada Mexico-Xochimiles 101, DF 14370 Col. San Lorenzo Huipulco, Mexico c Dept. Investigaciones ClõÂnicas, Mexico d Dept. de GeneÂtica y FisiologõÂa Molecular, Instituto de BiotecnologõÂa de la Universidad Nacional AutoÂnoma de MeÂxico, Mexico

Abstract Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite e€ects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the e€ects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1±24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied di€erently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quanti®cation by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The e€ect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% ®nal ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quanti®ed by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with di€erent kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral e€ects. 7 2000 Elsevier Science Ltd. All rights reserved.

1. Introduction Acute ingestion of alcohol leads to behavioral manifestations that vary depending on the dose. Low doses cause euphoria and psychomotor activation while high doses produce an intoxication that shows as uncoordi* Corresponding author. Tel.: +52-56-55-28-11 ext 114; fax: +5256-55-99-80. E-mail address: [email protected] (P. de Gortari).

nated, depressed and sedated attitudes. Brain activity is a€ected in mesolimbic and nigrostriatal dopaminergic circuits if low doses are administered. Higher doses depress activity in many brain areas including sensory, motor and limbic areas (Crabbe, 1997). Many of these e€ects have been related to the functioning of ion channels; in particular NMDA±glutamate receptor and certain subtypes of GABAA receptor channels (Chandler et al., 1998). Other neurotransmitter systems (Koob et al., 1998; Koob and Nestler, 1997) as dopa-

0197-0186/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 0 ) 0 0 0 5 9 - 0

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mine (Gessa et al., 1985; Di Chiara and Imperato, 1985; Fadda et al., 1989) and several neuropeptides (Erwin et al., 1994; Hwang et al., 1999; Rivier, 1999) have also been recognized as targets of ethanol e€ects (Herz, 1997; Ulm et al., 1995; Gianoulakis, 1996). The neuroendocrine system is a€ected by acute or chronic ethanol consumption at di€erent levels. Acute administration causes an increase in corticotropin releasing factor (CRF) release from median eminence (Koob, 1990) with a concomitant increase in serum ACTH and corticosterone levels (Rivier, 1999); b-endorphin (Lukas and Mendelson, 1988; North and Walter, 1984) and vasopressin (Ho€man, 1994) are also altered as well as the immune responses (Ogilvie et al., 1998); the thyroid (PorteleÂs et al., 1985; Zoeller and Rudeen, 1992) and gonadal (Sarkola et al., 1999) axis are inhibited. Opioid peptides have received most attention since they share several of the behavioral and pharmacological e€ects produced by ethanol ingestion. Administration of low doses of ethanol or opioids induces locomotor activity through dopaminergic pathway activation in the ventro-tegmental area and high doses activate dopaminergic terminals of n. accumbens (Joyce and Iversen, 1979; Kalivas et al., 1983; Wise and Bozarth, 1987). An important role in alcohol dependence has been proposed for these peptides since low doses of m receptor agonists augment alcohol consumption and preference (Reid and Hunter, 1984; Wild and Reid, 1990) and moderate to high doses reduce it (Volpicelli et al., 1991); non speci®c antagonists (naloxone) (Froehlich, 1995; Reid and Hunter, 1984; Ulm et al., 1995) as well as selective m and d antagonists reduce alcohol consumption and preference (Froehlich et al., 1991; Leà et al., 1993). Neurotensin may also mediate some of the actions of ethanol; its central administration produces the same e€ects as ethanol [hypothermia, locomotor inhibition or activation (high or low doses respectively), analgesia, tolerance as well as an increase in mesolimbic dopamine turnover] (Erwin et al., 1994). Comparison of rat strains that prefer or do not prefer alcohol have shown di€erences in the concentration of several neuropeptides and their receptors (Nylander et al., 1994; Froehlich, 1995; Hwang et al., 1999). In contrast, thyrotropin releasing hormone (TRH) administration produces opposite e€ects to those caused by acute ethanol consumption [hyperthermia (Griths, 1985; O'Leary and O'Cuinn, 1995), increased locomotor activity (French et al., 1993) and arousal (Breese et al., 1985)]. TRH administration to alcohol-preferring rats diminishes alcohol consumption and this e€ect is blocked by D2 dopamine receptor antagonists (Mason et al., 1997). TRH was initially discovered in the hypothalamus as the factor responsible in controlling adenohypophysial-thyroid function [reg-

ulating the synthesis and release of thyrotropin (TSH) (Haisenleder et al., 1992), and also of prolactin (Grosvenor and Mena, 1980)]. In the hypothalamus, TRH is synthesized in various nuclei being the paraventricular nucleus (PVN) the hypophysiotropic one (Lechan et al., 1986). Several other brain areas synthesize TRH (Lechan et al., 1986) where this peptide has been implicated in biological functions such as: the behavior of food ingestion (Vijayan et al., 1997), awakening (Bissette et al., 1976), body temperature maintenance (Rondeel et al., 1991), learning (Khan et al., 1993), memory (Ogasawara et al., 1995) and control of movement (Sharp et al., 1984). TRH is present in most of the limbic areas where antiepileptic (Sato et al., 1985; Kubek et al., 1989) and antidepressive (Sattin et al., 1999) roles have been proposed. Activation of these neurons by amygdala kindling (Meyerho€ et al., 1990; Rosen et al., 1992; de Gortari et al., 1995, 1998) or by electroshocks (Kubek and Sattin, 1989) increase its mRNA and peptide levels; TRH concentration varies according to the region's epileptogenic susceptibility: frontal cortex > amygdala > hippocampus (de Gortari et al., 1998). In combined studies of electroshocks, an antidepressant drug and swim test, the levels of TRH and its immediate precursor (TRH-gly) negatively correlate with swim immobility in hippocampus and forebrain regions leading to the proposal that TRH can serve as an endogenous antidepressant (Sattin et al., 1999). Ethanol and TRH a€ect synaptic transmission by interrelated mechanisms. Acute administration of ethanol (3 g/kg body weight) produces hypothermia and this e€ect is blocked by intracerebroventricular (i.c.v) TRH injection. Intraperitoneal (i.p.) injection of ethanol elevates TRH mRNA levels in the hypothalamus of animals maintained at room temperature after reducing the concentration of circulating triiodothyronine (T3). Cold exposure causes an increase in PVN TRH mRNA (Uribe et al., 1993; Zoeller et al., 1990), which is reduced by ethanol injection (Zoeller and Rudeen, 1992). TRH administration shortens the time of narcosis induced by ethanol and barbiturates (Morzorati et al., 1993; French et al., 1993). Both TRH and ethanol interact with NMDA receptors altering glutamate neurotransmission in hippocampus (Stocca and Nistri, 1994, 1995) and neocortical neurons (Kasparov et al., 1994); TRH also interacts with GABA receptor channels (Barbieri and Nistri, 1997; Crews et al., 1996). TRH activation of the cholinergic pathway improves cognitive function in animals (Itoh et al., 1994; Ogasawara et al., 1995, 1996) a€ected by chronic ethanol administration (Khan et al., 1993). High doses of ethanol suppress locomotor activity and induce sedation while TRH blocks this e€ect (Breese et al., 1985). The aim of this study was to determine if acute ethanol administration modi®es TRH concentration in

P. de Gortari et al. / Neurochemistry International 37 (2000) 483±496

brain regions where the peptide has been implicated in neuromodulatory functions and are known targets of ethanol e€ects. Since the hypothalamic-adenohypophysial-thyroid axis is altered upon ethanol consumption (PorteleÂs et al., 1985) we also quanti®ed TSH serum concentrations. The levels of proTRH mRNA were measured by RT-PCR and by in situ hybridization (ISH) in several brain areas. The levels of proenkephalin (PENK) mRNA were measured by ISH in the same animals to study the speci®city of the e€ect.

2. Experimental procedures 2.1. Materials Ethanol and the kit for its measurement (332-B), cytosine arabinofuranoside and most common reagents were purchased from Sigma; a (35S) dATP (1000 Ci/ mmol) and autoradiography ®lms purchased from Amersham; desoxynucleotidyl transferase from Boheringer. Reagents and materials for cell culture were from Gibco and Costar. Solvents were from Baker. Wistar rats were from the Institute of Psychiatry's animal house, maintained on a 12 h dark/light cycle with water and Purina chow ad libitum. Adult male Wistar rats weighing 300±400 g received ethanol by two di€erent routes (Ogilvie et al., 1997). 2.2. Paradigm A Animals were injected with single (i.p.) injection of ethanol (3 g/kg body weight); ethanol was diluted 1:3 in 0.9% saline solution (Breese et al., 1985; Morzorati and Kubek, 1993). Control animals were injected with an equivalent volume of saline solution. Animals were sacri®ced by decapitation at 11±12 a.m.; injections were performed at di€erent times (stated for each experiment), previous to this hour. Brains were removed and kept at ÿ708C; animal blood was collected for TSH determination. Frozen brains were dissected to obtain left or right: amygdala, n. accumbens, hypothalamus (including median eminence), hippocampus and frontal cortex (Palkovits and Brownstein, 1988). 2.3. Paradigm B A total of 2.5 g/kg of ethanol was administered orally by an intra-gastric cannula (i.g.); controls received equivalent doses of distilled water. Animals were habituated to the cannula by a daily oral administration of distilled water for the previous 7 days. Animals were sacri®ced by decapitation, their brains excised and kept frozen to be later sliced in a cryostat, and their blood collected in heparinized tubes to

485

measure ethanol concentration in plasma (with Sigma kit 332-B). 2.4. Radioimmunoassay TRH was quanti®ed with a speci®c antibody previously characterized (Joseph-Bravo et al., 1979; MeÂndez et al., 1987). Brie¯y, tissues were homogenized in 500 ml of 20% acetic acid and centrifuged for 10 min at 12,000g at 48C. The supernatant was extracted with methanol (90% ®nal concentration), evaporated and treated as described (de Gortari et al., 1995). TSH was determined in serum samples. Aliquots of serum (50 ml) diluted 1:3 with RIA bu€er, were used to quantify TSH using NIDDK-USA (National Hormone and Pituitary Program) materials and protocol. 2.5. TRH mRNA semi-quanti®cation by RT-PCR Total RNA was extracted from frozen tissues, or frozen cultured cells, using 4 M guanidinium chloride (Chomczynsky and Sacchi, 1987) and TRH mRNA semi-quanti®ed by RT-PCR (PeÂrez-Martõ nez et al., 1998), using as internal control an ampli®cation fragment of glyceraldehyde 3-phosphate dehydrogenase cDNA sequence. Densitometry was used to calculate the relative amounts of proTRH versus G3PDH cDNAs. 2.6. In situ hybridization TRH-mRNA was detected using as probe a 50-mer oligonucleotide corresponding to the 317± 367 sequence of TRH cDNA (Lechan et al., 1986). Proenkephalin probe used was a 130±145 fragment of the rat proenkephalin cDNA (Howells et al., 1984). These were synthesized at the Biotechnology Institute (UNAM) on an Applied Biosystem 318A synthesizer. Both probes were labeled at the 3 ' end with a [35S] dATP (1000 Ci/mmol) with desoxynucleotidyl transferase (25 U/ml); probe speci®c activity for TRH: 6±8  108 cpm/mg; PENK: 3:7  108 cpm/mg. Coronal sections (20 mm) were placed in gelatin coated slides, ®xed in 4% paraformaldehyde in phosphate-saline bu€er (PBS) for 20 min, rinsed twice with PBS and treated with 0.25% acetic anyhidride in 0.1 M triethanolamine, 4 SSC (pH 8) for 10 min. After dehydration in increasing concentrations of ethanol, they were delipidated in chloroform for 10 min, rinsed in ethanol, and air-dried. For proTRH mRNA, sections were incubated for 15 h at 428C in hybridization bu€er, treated as described (SaÂnchez et al., 1996) and exposed to b-max autoradiography hyper ®lm at room temperature. For PENK mRNA, slices were preincubated 1 h at 428C and then incubated overnight with 4 SSC, 50% deio-

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nized formamide, 2.5 Denhardt's …100 ˆ 0:5% Ficoll, 0.5% polyvinil pyrrolidone, 0.5% bovine albumin), 250 mg/ml sheared salmon sperm DNA, 10% dextran sulfate, 10 mM DTT, 100 mM Na phosphate bu€er (pH 7.4), 250 mg/ml tRNA and 35S-labeled oligonucleotide probe to get 250,000 cpm/section. Following incubation, sections were washed with: 1  30 min in 4 SSC and 0.02% SDS at 428C; 1  30 min in 2 SSC and 0.02% SDS at 428C; 1  30 min in 1 SSC and 0.02% SDS at 428C; 1  10 min in 0.5 SSC at RT. Sections were exposed to hyper-®lm b-max for autoradiography. Non-speci®c proenkephalin hybridization was tested with an excess of 100 times the oligo concentration and the signal was subtracted from total hybridization. 2.7. Film autoradiograms The signal was digitized and converted to gray values using a NIH Image 1.61. Mean intensity of pixels was registered for circumscribed areas. Values are the mean of ®ve sections/region for each animal ‰n ˆ 3]. 2.8. Hypothalamic primary cell culture Embryonic hypothalamic tissue was dissected from Wistar rats at 17th day of gestation as previously described (PeÂrez-Martõ nez et al., 1998). Brie¯y, 6  105 cells were plated on precoated 16-mm multiwell plates. On the fourth day of culture, 10ÿ5 M citosine arabinofuranoside was added for 48 h to inhibit cell proliferation; at 6 days in vitro (DIV) every second day half of the incubation medium was replaced with 1 ml of fresh serum-supplemented Dulbecco's modi®ed medium (S-DMEM); at 18 DIV, all medium was replaced with fresh S-DMEM for control groups or with SDMEM containing 0.001% ®nal concentration of ethanol and further incubated for one to 3 h; the media was removed, cells rinsed with PBS solution and kept frozen for RNA isolation. 2.9. Statistics Each experimental group had its own control injected with saline solution and was sacri®ced at the same time as the group receiving ethanol. ANOVA was performed between the various controls ®nding di€erence in those of hippocampus and of n. accumbens, probably due to circadian variations (Covarrubias et al., 1994). Therefore, data of each group was compared to its paired control, t-Student test was used for statistic analysis.

3. Results 3.1. In vivo and in vitro e€ects of ethanol on the hypothalamus Two di€erent ethanol injection paradigms were used. In paradigm A, 3 g/kg b.w. of ethanol administered by i.p. injection rapidly (5 min) narcotized the animals for nearly 1 h. They were fully awake by 1.5 h and behaved normal after 4 h. Animals were sacri®ced 1, 6 and 24 h after injection. A drop in serum TSH levels occurred after 1 h, recovering to control values at 6 h (Fig. 1). TRH content in the hypothalamus was decreased at 6 h and recovered at 24 h (Table 1). TRH mRNA levels measured in total hypothalamus by semiquantitative RT-PCR, increased to 24427% after 1 h of i.p. injection …n ˆ 8, controls ˆ 10026%). To evaluate if ethanol could have a direct e€ect on hypothalamic cells, its e€ect was studied in primary cell cultures, measuring TRH mRNA variations by RT-PCR. As observed in Fig. 2, 1 h incubation with 0.001% ®nal ethanol concentration produced a six-fold increase in the relative amounts of TRH mRNA, over 12-fold at 2 h and lowering considerably at 3 h. Animals received a lower dose of ethanol (paradigm B) in order to avoid the narcosis e€ect by oral i.g. administration. Animals were administered water i.g. during 7 days to reduce the stress of novelty of the ethanol administration. Ethanol levels quanti®ed in plasma were highest (45 mg/dl) 1 h after injection decreasing progressively and being undetectable after 8 h injection (results not shown). Behavioral e€ects were observed only during the ®rst 3 h when animals gathered in groups, seemed relaxed (but not asleep) and felt cold to touch (temperature was not measured to avoid stress). Expression of mRNA was determined by in situ hybridization (ISH) for ProTRH and proenkephalin mRNAs in order to compare the site and the kinetics of the response for both neuropeptides. Analysis of proTRH mRNA in the PVN showed an increase of 38% 1 h, and of 71% 4 h after i.g. ethanol administration (Fig. 3). PENK mRNA in this hypothalamic nucleus did not vary during the 1st and 4th hour but increased to 60% at 24 h. Levels of this mRNA were augmented in mammilary bodies at 4 and 24 h while they decreased at 4 h in the arcuate nucleus (Fig. 4). An increase of 30322% …p < 0:005† was measured in the ventromedial region at 24 h (not shown). 3.2. E€ects of ethanol injection in extra hypothalamic brain areas 3.2.1. Paradigm A Analysis of extra hypothalamic brain areas showed signi®cant changes on TRH levels with di€erent kin-

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Fig. 1. Serum TSH levels measured in animals injected with ethanol (3 g/kg body weight); control animals, with saline solution (0.9%). Values are expressed as ng/ml and are the mean2SEM …n ˆ 8), p < 0:05:

(Fig. 3) while PENK mRNA decreased in dentate gyrus, CA1, CA3 at 1 h and in CA1, CA2 and CA3 at 4 h (Fig. 6). At 24 h in contrast, PENK mRNA was signi®cantly increased in dentate gyrus and CA1, CA3 [a similar pattern was observed in CA2 but the increase was not signi®cant (Fig. 6)]. N. accumbens showed altered mRNA levels of both precursors since the ®rst hour post injection (Fig. 7). ProTRH mRNA levels were increased by 64% normalizing afterwards; the signal was scarce but detectable. A smaller increase was measured at this time for PENK mRNA but values remained above controls at 4 and 24 h after injection. Frontal cortex responded transiently, decreasing at 4 h TRH mRNA while increasing that of PENK (Fig. 7). Despite decreased TRH levels in the amygdala 6 h after i.p. injection, no changes were detected in its mRNA levels by ISH determination of this region neither in pyriform cortex (not shown).

etics (Table 1). At 1 h after i.p. injection, the only regions that showed changes in TRH content were hippocampus with a 63% increase and, n. accumbens with a 25% decrease. At longer times (6 h), TRH content was signi®cantly decreased in frontal cortex and amygdala. All values were equal to controls after 24 h. TRH mRNA levels were evaluated by RT-PCR at 0.5, 1, 2 and 6 h post injection to verify if the changes observed in peptide levels corresponded to an e€ect in biosynthesis. In hippocampus, a slight increase was observed at 30 min (33%) and higher at 1 h (67%) returning to control values after 2 h. A decrease of 30% was observed in frontal cortex at 1 h (Fig. 5). 3.2.2. Paradigm B A fast response was observed in the hippocampus for both propeptide mRNAs but in opposite tendencies: proTRH mRNA increased at 1 and 4 h in total hippocampus returning to basal levels at 24 h

Table 1 TRH content measured by RIA in brain regions of animals sacri®ced 1, 6, and 24 h after single i.p. ethanol (3 g/kg) injectiona TRH Content (pg/tissue) 1h

Hypothalamus Frontal cortex Hippocampus N. accumbens Amygdala a

6h

24 h

Control

Ethanol

Control

Ethanol

Control

Ethanol

62952599 237244 1178235 44902454 10232190

66662462 258229  19812207  32952264 966260

76162952 324248 14622158 42822358 943284



55822765 237230 806289 20142579 8602226

51042292 182210 7192130 19382450 691259

4198259  129218 13962192 38382788  6652109

Controls were injected with saline solution. Data are expressed as pg/tissue and are the mean2SEM …n ˆ 8† p < 0:05.

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Fig. 2. Hypothalami primary cell cultures from 17-day rat embryos were incubated with 0.001% ®nal concentration of ethanol at 18 DIV. After 1, 2 or 3 h incubation, cells were rinsed with PBS and frozen. Total RNA was extracted and proTRH mRNA levels semiquanti®ed by RT-PCR by coampli®cation of G3PDH cDNA in the same sample. The ratio of proTRH/G3PDH cDNA (arbitrary units) was calculated …n ˆ 6; p < 0:005).

Fig. 3. ProTRH mRNA levels measured by ISH in paraventricular nucleus of the hypothalamus and in the hippocampus. Animals were sacri®ced 1, 4 and 24 h after an i.g. administration of 2.5 g/kg ethanol. Control animals were given distilled water. Data are expressed as percent of control (100%) and are the mean2SEM …n ˆ 3† p < 0:05:

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Fig. 4. PENK mRNA levels measured by ISH in paraventricular, arcuate nucleus, and mammillary bodies of the hypothalamus. Animals were sacri®ced 1, 4 and 24 h after an i.g. administration of 2.5 g/kg ethanol. Control animals were given distilled water. Data are expressed as percent of control (100%) and are the mean2SEM …n ˆ 3† p < 0:05:

Fig. 5. ProTRH mRNA levels measured by RT-PCR in hippocampus and frontal cortex (for comparison, TRH values from Table 1 are expressed as percent of controls). Animals were sacri®ced 0.5, 1, 2, and 6 h after an i.p. ethanol (3 g/kg) injection. Control animals were injected with saline solution. Data are expressed as percent of control (100%) of proTRH/G3PDH cDNA ratio and are the mean2SEM …n ˆ 8† p < 0:05:

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4. Discussion We studied the acute e€ects of ethanol on the TRHergic neurons of the limbic system in an attempt to contribute to the understanding of the possible role of this peptide. Most of the data implying TRH neuromodulatory role come from studies on e€ects of TRH administration and, as previously mentioned, TRH antagonizes some of the behaviors observed after acute ethanol exposure. Endogenous TRH or its mRNA levels have been measured only in few paradigms (Sattin et al., 1994, 1999; de Gortari et al., 1995, 1998). Although the regional content of any neurotransmitter does not indicate whether the release was modi®ed,

variations point to a possible e€ect either on release and/or synthesis. Measuring the levels of peptide and its mRNA can provide a better index that a particular pathway is activated. Alcohol modi®es the neuroendocrine system at the hypothalamic and hypophysial level activating the adrenal (HPA) axis and inhibiting gonadal and thyroid functions (Sarkola et al., 1999; Zoeller and Rudeen, 1992). The primary action in HPA axis is due to a stimulation of CRH synthesis and release after acute exposure to ethanol; concomitantly, ACTH, b-endorphin and corticosterone levels increase in serum few minutes after exposure (Rivier, 1999; Lukas and Mendelson, 1988; North and Walter, 1984). On the thyroid

Fig. 6. PENK mRNA levels measured by ISH in dentate gyrus, CA1, CA2 and CA3 of the hippocampus. Animals were sacri®ced 1, 4 and 24 h after an i.g. administration of 2.5 g/kg ethanol. Control animals were given distilled water. Data are expressed as percent of control (100%) and are the mean2SEM …n ˆ 3† p < 0:05, p < 0:005, p < 0:0001:

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axis, ethanol decreases TSH and thyroid hormones (PorteleÂs et al., 1985; Zoeller and Rudeen, 1992) and increases TRH mRNA levels in the PVN (Zoeller and Rudeen, 1992). In accordance to published data, we found increased TRH mRNA levels in the PVN 4 h after ethanol administration; peptide levels were decreased in total hypothalamus at 6 h while TSH were diminished only in the ®rst hour. These results suggest that inhibition of TSH release is evident only when ethanol concentrations are high suggesting inhibition of TRH release at this time. A coordinated response of synthesis and release is observed in many paradigms (Joseph-Bravo et al., 1998) however, as pre-

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viously proposed (Zoeller et al., 1995) ethanol seems to act in the PVN by uncoupling these two mechanisms. Due to the alcohol e€ect on ion channels, the possibility of a€ecting calcium induced exocytosis is feasible. The faster response observed in the increase in TRH mRNA levels measured in total hypothalamic cells (in animals injected i.p. the higher dose, or in cell cultures) could be due to a direct e€ect of ethanol on TRHergic neurons in other nuclei besides the PVN. A careful analysis by ISH is in progress since, for example, a modulatory role on thermogenesis has been implied for TRH in the preoptic area (Hori et al., 1988).

Fig. 7. ProTRH and PENK mRNA levels measured by ISH in n. accumbens and frontal cortex. Animals were sacri®ced 1, 4 and 24 h after an i.g. administration of 2.5 g/kg ethanol. Control animals were given distilled water. Data are expressed as percent of control (100%) and are the mean2SEM …n ˆ 3† p < 0:05, p < 0:001:

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Within the hypothalamus, PENK mRNA levels varied depending on the nucleus and the time after injection. Opioid peptides have been proposed to regulate several endocrine systems (Lukas and Mendelson, 1988; North and Walter, 1984; Patel and Pohorecky, 1989), alcohol drinking (Herz, 1997; Ulm et al., 1995; Di Chiara et al., 1996; Gianoulakis, 1996) or feeding behaviors (Olson et al., 1995; Kalra et al., 1999) but most of the work has been centered on POMC precursor (de Waele et al., 1992; Popp and Erickson, 1998; Rasmussen et al., 1998; Ng et al., 1996; Boyadjieva and Sarkar, 1997). Our results suggest that PENK-derived peptides may have a key role in mediating some of the behavioral and pharmacological e€ects of ethanol at the neuroendocrine level. TRHergic neurons present a fast (1 h) and transient response in hippocampus and n. accumbens. Hippocampus is an area that is particularly susceptible to stress (Meaney et al., 1993) and is implicated in memory and learning (Horita et al., 1989). This region showed increased levels of TRH mRNA 1±6 h after administration while the peptide levels increased at 1 h recovering to control values at longer times; it would be interesting to measure released TRH at 1 h to be able to de®ne if the increased peptide levels at this time are due to inhibition of release and relate to the narcosis produced by ethanol. Levels of TRH mRNA are further increased at 4 h but peptide levels at 6 h are not, probably due to an increased release that could relate to the reported analeptic e€ects of TRH. At the times where TRH mRNA levels were increased (1±4 h), the levels of PENK mRNA were decreased; the later rising afterwards above controls, at 24 h. Whether this relates to the opposite behavioral e€ects of these two peptides requires further investigation. TRH mRNA levels were also increased since the ®rst hour after ethanol administration in the n. accumbens. Changes in n. accumbens (increase of mRNA and decrease in peptide content, possibly as result of release) coincide with the animal's awakening from ethanol narcosis (1 h) and several reports have proposed the participation of TRH in locomotion by interacting with dopaminergic neurons in this region (Miyamoto and Nagawa, 1977; Yamamura et al., 1991). In the animals where ethanol was i.g. administered and showed high serum ethanol levels the ®rst 4 h, pro TRH mRNA only increased during the ®rst hour (maximum ethanol levels) while PENK mRNA increase was maintained over 24 h. These results di€er with reported data on alcohol-preferring strain of rats that show an increase in PENK mRNA only when ethanol blood levels are high (non-preferring rats show no alteration); after 8 h of a similar ethanol administration, preferring- or non-preferring rats have diminished levels of this mRNA (Li et al., 1998). Since our animals were from the local animal house, it is dicult

to compare the endogenous levels of opioid peptides in the mesolimbic areas and relate these changes with the reported di€erences in genetic background of those animals selected due to their alcohol preference. If PENK mRNA increase leads to higher levels of opioid peptides and their release, the data would be consistent with the suggested opioid-mediated alcohol-induced release of dopamine (Acquas et al., 1993; Benjamin et al., 1993; Widdowson and Holman, 1992). An increased dopamine turnover in this region has been related with increased locomotion (Imperato and di Chiara, 1986; Wise and Bozarth, 1987; Joyce and Iversen, 1979) as well as increased drive for alcohol consumption (di Chiara et al., 1996). Narcosis induced by systemic administration of ethanol elevates dopamine release in the n. accumbens, blocking the glutamatergic excitatory transmission, and locomotion in consequence. This could be due to an indirect e€ect of ethanol over glutamatergic neurons of the hippocampus because of its n. accumbens innervation (Criado et al., 1995). However, although it is tempting to relate some of the known down regulating e€ects of ethanol on NMDA receptors (Crews et al., 1996) with the positive regulation of TRH in hippocampal postsynaptic neurons (Stocca and Nistri 1994), a careful analysis of neuroanatomical interactions of these di€erent pathways in each region, is ®rst required. As mentioned, TRH neurons of the frontal cortex or the amygdala are very susceptible to amygdaloid kindling stimulation (de Gortari et al., 1995, 1998) but these structures responded di€erently to ethanol. TRH mRNA has not been reported in frontal cortex and we observed by ISH only scarce signal; by RT-PCR we were able to detect TRH messenger in control animals. A small decrease in TRH mRNA was observed in frontal cortex (4 h) and in peptide levels (6 h) suggesting an inhibitory e€ect of ethanol in this region. Amygdala presented no changes in TRH mRNA and only diminished peptide levels at 6 h. A regional speci®city on TRH biosynthesis is thus observed that relates to the regions that are particular targets of ethanol. TRH gene contains consensus sequences for thyroid and glucocorticoid receptors, AP-1 and CREB (cAMP response element) (Lee et al., 1988, 1993; Stevenin and Lee, 1995; Hollenberg et al., 1995). Dexamethasone, phorbol esters or cAMP analogs induce TRH biosynthesis in hypothalamic cell cultures after one hour incubation; however, if cAMP and either dexamethasone or phorbol esters are incubated together, an inhibition is observed (Uribe et al., 1995; PeÂrez-Martõ nez et al., 1998). Therefore, an a€ected synthesis of TRH would depend on the particular e€ectors impinging on the neuron and the levels of transcription factors (Joseph-Bravo et al., 1998) susceptible to ethanol. In this regard, it is interesting to

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note that expressions of glucocorticoid receptors vary depending on the region, being highest in hippocampus, medium in frontal cortex, and lowest in amygdala (Sousa et al., 1989). As in hippocampus, an opposite e€ect to that observed in TRH mRNA was seen for PENK neurons in frontal cortex. PENK mRNA expression is increased in cerebral cortex of alcohol-preferring vs non-preferring rats (Sardinian strain, Fadda et al., 1999) although chronic consumption or withdrawal does not change the level of this opioid precursor messenger (Tajuddin and Druse, 1998; Mathieu-Kia and Besson, 1998; Przewlocka et al., 1997). The di€erence in kinetics of enkephalin synthesis between frontal cortex and n. accumbens in response to acute ethanol administration points to a role of these peptides in the meso-cortical and meso-accumbens pathways involved in alcohol reinforcement mechanisms. The levels of TRH are recovered by 24 h in all the brain regions studied (in n. accumbens recovery happens earlier 6 h p.i.); no changes in its mRNA are detected after 6 h. PENK mRNA levels on the other hand are elevated in most regions studied (n. accumbens, hippocampus, hypothalamus) at 24 h, well after ethanol blood levels are undetectable. Only frontal cortex shows a transient e€ect dependent on alcohol's increased blood concentration. It has been suggested that the reinforcing properties of alcohol are due, at least partially, to the activation of endogenous opioid system (Ulm et al., 1995). It must yet be demonstrated that ethanol induces release of opioids causing a release of dopamine since opioid antagonists block this ethanol-induced dopamine release (Benjamin et al., 1993; Acquas et al., 1993). In conclusion, TRHergic pathways were activated (hippocampus, n. accumbens) or inhibited (f. cortex) during and after the sedative e€ect of a single injection of ethanol. Whether the opposite behavior found for proTRH or PENK mRNA changes relates to the particular interaction these peptides have with ethanol action, requires further study; but it could relate to the antagonist action of TRH with ethanol versus the potentiating e€ect of opioid peptides on ethanol mechanism of action. This model has provided evidence of the activation of these peptidergic neurons in regions involved in learning, memory function, locomotor activity and awakening during and after the sedative condition of animals. Acknowledgements We thank Dr. J.L. Charli for relevant discussions on this work, Miguel Cisneros, Fidelia Romero and Mauricio Badillo (I.M.) for technical assistance and Mario Aguilar for animal care. The photographic

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work of R. Cardoso, the secretarial help of G. Valencia and the gifts of TSH protein and antiserum (National Hormone and Pituitary Program supported by NIDDK (USA)) are gratefully acknowledged. This work was supported by: CONACYT (25386N) (PJB) and (3261P-N9607) (MM), DGAPA (IN217797) (PJB), UIA-412 (PG) and FUNSALUD (MM). References Acquas, E., Meloni, M., Di, Chiara, G., 1993. Blockade of opioid receptors in the nucleus accumbens prevents ethanol-induced stimulation of dopamine release. Eur. J. Pharmacol. 230, 239± 241. Barbieri, M., Nistri, A., 1997. E€ects of the neuropeptide thyrotropin-releasing hormone on GABAergic synaptic transmission of CA1 neurons of the rat hippocampal slice during hypoxia. Peptides 18, 585±591. Benjamin, D., Grant, E.R., Pohorecky, L.A., 1993. Naltrexone reverses ethanol-induced dopamine release in the nucleus accumbens in awake, freely moving rats. Brain Res. 62, 137±140. Bissette, G., Nemero€, C.B., Loosen, P.T., Prange Jr., A.J., Lipton, M.A., 1976. A comparison of the analeptic potency of TRH, ACTH 4±10, LHRH and related peptides. Pharmacol. Biochem. Behav. 5 (Suppl. 1), 135±138. Boyadjieva, N.I., Sarkar, D.K., 1997. E€ects of ethanol on basal and prostaglandin E1-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells. Alcohol Clin. Exp. Res. 21, 1005±1009. Breese, G.R., Coyle, S., Frye, G.D., Mueller, R.A., 1985. E€ects of TRH, ethanol and TRH±ethanol combination on activity in rats with altered monoamine content. Pharmacol. Biochem. Behav. 22, 1013±1018. Chandler, L.J., Harris, R.A., Crews, F.T., 1998. Tolerance and synaptic plasticity. Trends. Pharmacol. Sci. 19, 491±495. Chomczynski, P., Sacchi, P., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate±phenol±chloroform extraction. Anal. Biochem. 162, 156±159. Covarrubias, L., Redondo, J.L., Vargas, M.A., Uribe, R.M., MeÂndez, M., Joseph-Bravo, P., Charli, J.L., 1994. In vitro release from hypothalamus slices varies during the diurnal cycle. Neurochem. Res. 19, 845±850. Crabbe, J.C., 1997. Where does alcohol act in the brain? Molec. Psychiat. 2, 17±20. Crews, F.T., Morrow, L., Criswell, H., Breese, G., 1996. E€ects of ethanol on ion channels. Int. Rev. Neurobiol. 39, 283±367. Criado, J.R., Lee, R.S., Berg, G.I., Henriksen, S.J., 1995. Sensitivity of nucleus accumbens neurons in vivo to intoxicating doses of ethanol. Alcohol Clin. Exp. Res. 19, 164±169. de Gortari, P., FernaÂndez Guardiola, A., Cisneros, M., Martõ nez, A., Joseph-Bravo, P., 1995. Changes in TRH and its degrading enzyme pyroglutamyl peptidase II during the development of amygdaloid kindling. Brain Res. 679, 144±150. de Gortari, P., Joseph-Bravo, P., Monroy-Ruiz, J., Martõ nez, A., Cisneros, M., FernaÂndez-Guardiola, A., 1998. Brain thyrotropinreleasing hormone content varies through amygdaloid kindling development according to after discharge frequency and propagation. Epilepsia 39, 897±903. de Waele, J.-P., Papachristou, D.N., Gianoulakis, C., 1992. The alcohol-preferring C57BL/6 mice present an enhanced sensitivity of the hypothalamic b-endorphin system to ethanol than the alcohol-avoiding DBA/2 mice. J. Pharmacol. Exp. Ther. 261, 788± 794. di Chiara, G., Acquas, E., Tanda, G., 1996. Ethanol as a neuro-

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