Chronic ingestion of ethanol or glucose solutions affects hypothalamic and limbic TRH metabolism in dams and their pups

Neurochemistry International 41 (2002) 237–249

Chronic ingestion of ethanol or glucose solutions affects hypothalamic and limbic TRH metabolism in dams and their pups P. de Gortari a,∗ , M. Cisneros b , M.A. Medell´ın a , P. Joseph-Bravo b a

b

Depart. of Neuroscience, Instituto Nacional de Psiquiatr´ıa Ramón de la Fuente Muñiz, Calzada México-Xochimilco #101, San Lorenzo Huipulco, Mexico D.F. 14370, Mexico Depart. Genetics and Molecular Physiology, Instituto de Biotecnolog´ıa de la Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca, Mor 62271, Mexico Received 6 September 2001; received in revised form 16 November 2001; accepted 8 January 2002

Abstract The effect of chronic ethanol consumption during pregnancy and lactation on thyrotropin releasing hormone (TRH) metabolism was investigated in the hypothalamus and limbic areas of female rats and their weaned pups. Pregnant female rats received ethanol or isocaloric glucose solution during pregnancy either alone, or also during the 3 weeks of lactation. Thyrotropin (TSH) and corticosterone levels were measured in serum; TRH and TRH-gly concentrations were determined in hypothalamus, hippocampus, n.accumbens, frontal cortex and amygdala of dams and pups at 21 days after parturition. Ethanol or glucose consumption during pregnancy and lactation produced a decrease in TSH levels compared with control animals fed at libitum; water replacement during lactation normalized TSH levels only in glucose-fed dams. Pups from ethanol or pair-fed dams showed low weight and increased TSH levels compared with normal rats. Variations in TRH metabolism were detected in limbic areas. Chronic ethanol caused a decrease in the levels of TRH in the hippocampus and frontal cortex of dams. In contrast, glucose chronic ingestion increased TRH content specifically in n.accumbens and amygdala of dams. Most of the variations in TRH content of limbic areas of pups were not specific for glucose or ethanol treatment and correlated with the deleterious effect of the mother’s thyroid condition, although some differences were observed depending on pup’s gender. These results support the involvement of TRHergic neurons in the limbic system of the female rat exposed to alcohol or glucose during pregnancy and lactation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: TRH; TSH; TRH-gly; Prenatal ethanol exposure; Hypothalamus; Limbic areas; Hippocampus; n.accumbens; Hypothyroidism; Amygdala; Malnutrition

1. Introduction Prenatal ethanol exposure produces a set of symptoms in the offspring, including low birth weight, growth retardation and, impaired brain development as a consequence of abnormalities in different neuromodulatory systems (Driscoll et al., 1990; Tran and Kelly, 1999). Infants of women with chronic ethanol consumption during pregnancy also have deficits in learning and memory processes, motor dysfunction, as well as alterations in hypothalamic adrenal and thyroid axis, that could be correlated with associated malnutrition and/or hypothyroidism of the alcoholic mothers Abbreviations: TRH, thyrotropin releasing hormone; TSH, thyrotropin; T3 , triiodothyronine; NMDA, N-methyl-d-aspartate; HPT, hypothalamus-pituitary-thyroid; PVN, paraventricular nucleus; THR, thyroid hormone receptor ∗ Corresponding author. Tel.: +52-5-6-55-28-11; fax: +52-5-6-55-99-80. E-mail address: [email protected] (P. de Gortari).

(Streissguth et al., 1994; Rivier and Lee, 1996; Kim et al., 1999; Portolés et al., 1985, 1988; Baumgarten et al., 1994). Thyrotropin releasing hormone (TRH, pyroglu-his-proNH2 ), has hypophysiotropic and neuromodulatory functions. The hypophysiotropic role of TRH is confined to the neurons of the hypothalamic paraventricular nucleus (PVN) that project to the median eminence and release TRH to control the synthesis and release of thyrotropin and prolactin in the pituitary (Burgus et al., 1969; Haisenleder et al., 1992; Lechan and Toni, 1992). TRH neuromodulatory role has been inferred by its presence in several brain regions (Lechan and Toni, 1992), together with its receptors (TRH-R1 and TRH-R2; Calzá et al., 1992; Cao et al., 1998; Itadani et al., 1998) and TRH specific degrading ectoenzyme (Charli et al., 1998; Heuer et al., 1998). Pharmacological evidence supports the role of the peptide in biological functions such as arousal, feeding behavior, cognitive and memory functions as well as locomotion activities (Breese et al., 1985; O’Leary and O’Connor, 1995; Vijayan and McCann, 1977;

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Kahn et al., 1993; Ogasawara et al., 1996). TRH administration attenuates ethanol-induced narcosis and hypothermia (Morzorati and Kubek, 1993; French et al., 1993). An acute administration of alcohol induces sedation and hypothermia in male adult rats and disrupts endocrine functions involving the adrenal (Rivier et al., 1984) and the thyroid axis. The hypothalamus-pituitary-thyroid (HPT) axis function is diminished after a single ethanol injection: TSH and T3 serum levels are decreased while proTRH mRNA levels in the PVN are increased after 1 h of administration (Zoeller et al., 1995; de Gortari et al., 2000a). The reduced TSH levels are proposed to be due to decreased TRH release (Zoeller and Rudeen, 1992). Levels of TRH and its mRNA are also altered in hippocampus, n.accumbens and frontal cortex during the period of sedation; after 24 h levels return to basal values (de Gortari et al., 2000a). Chronic ethanol ingestion in humans as well as in rats causes lower levels of T3 and T4 compared with control groups (Singh et al., 1979; Portolés et al., 1985). TRH mRNA levels in the PVN are increased and, as a result of a blunted response of the adenohypophysis to TRH, TSH levels decreased (Loosen et al., 1983). Central stimulation of TRH hypothalamic neurons, such as that induced by cold stress, which normally increases TRH and TSH release, is impaired by chronic ethanol consumption (Zoeller et al., 1996). Reports of the effects of prenatal exposure to ethanol on serum thyroid hormones are still controversial (Hannigan and Bellisario, 1990; Lee and Wakabayashi, 1986). The nutritional status is known to affect the HPT axis; malnutrition (whether caloric or proteic) or fasting cause centrally mediated hypothyroidism (Mori et al., 1988; Van Haasteren et al., 1995, 1996). During fasting, TRH mRNA levels are also reduced in the PVN of adult animals (Blake et al., 1991). In contrast, recently weaned animals (21 days of age) do not show changes in hypothalamic TRH or serum TSH after 48 h fasting (de Gortari et al., 2000b). Age differences in the response to fasting of male animals are also observed in the levels of TRH in extrahypothalamic areas: decreased in n.accumbens and increased in hippocampus of weaned animals while decreased only in amygdala of adults (de Gortari et al., 2000b). TRH content in septum is increased in animals under a total caloric restricted diet during 4 weeks (Nikodémová et al., 1998). Ethanol or malnutrition exposure in utero also impairs adrenal axis functioning. While food restriction and an acute intraperitoneal injection of alcohol elevates corticosterone and ACTH levels in male animals’ serum, chronic ingestion of ethanol or glucose does not (Ogilvie et al., 1997). However, hypophysiotropic adrenal responses of animals prenatally exposed to ethanol are altered only when submitted to stress in adulthood (Kim et al., 1999). Regional variations in the response of TRHergic neurons after acute ethanol administration (de Gortari et al., 2000a), or after fasting (that differed depending on the age of the animal; de Gortari et al., 2000b), prompted us to study the effect of chronic ethanol ingestion in dams consuming ethanol

during pregnancy and lactation, as well as in their offspring. As prenatal ethanol exposure and undernutrition retards development in animals and humans, we compared the effect of ethanol with equivalent caloric intake of glucose solution. We measured the levels of TRH and its immediate precursor TRH-gly since variations in TRH content could reflect changes in its biosynthesis or its release, TRH-gly levels could represent effects in the biosynthetic pathway prior to release events (Joseph-Bravo et al., 1998; Sattin et al., 1999). The response of HPT axis was evaluated by measuring TSH serum concentration, and corticosterone levels, for possible stress effects. We present data that show a differential regional response of TRH neurons. 2. Experimental procedures Wistar adult male and female rats from the institute’s colony were used. Four virgin females were caged with a male for 5 days. Female rats were considered pregnant if their weight increased 10% after 1 week. Pregnant rats were then placed in individual cages (12 h light–dark cycles 06:00–18:00) and started an ethanol or glucose regime. Controls were fed ad libitum Lab rodent diet #5008 (PMI feeds; Brentwood, MO, USA) and water. Delivery was at day 21 ± 2. Animal procedures were approved by the institute’s ethical committee and followed the guidelines of the Neuroscience Society (USA). 2.1. Ethanol administration A group of eight rats received food ad libitum and 100 ml 20% ethanol (Baker, México). The amount of food and liquid consumed was daily registered. After parturition, four animals continued to receive ethanol solution through lactation (group EE) and four animals received food and water at libitum (group EW). 2.2. Pair fed Another group of eight rats received 30% glucose solution in quantity calculated for isoenergetical substitution of the amount of alcohol solution consumed the previous day by EE animals; solid food was adjusted to the amount consumed by EE group. After parturition, half of dams continued to receive glucose solution pair fed with the EE group (group GG). The other half received water ad libitum and the amount of food consumed by EW group. Body weight of dams was recorded every week and of pups, at the end of the lactation period (21 days old). Three days after birth, the offspring were reduced aleatorily to six pups for each dam, to homogenize energy requirements of the mothers. At the end of lactation period, the dams and their pups were sacrificed by decapitation (1000–1200 h). Brains were removed and kept at −70 ◦ C; blood was collected for serum TSH and corticosterone determination. It was not possible to get enough blood from all pups and the number of samples

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analyzed is stated in the Fig. 3. Frozen brains were freehand dissected to obtain amygdala, frontal cortex, n.accumbens, hippocampus, and hypothalamus (including median eminence; Palkovits and Brownstein, 1988). Sections from left hemisphere were processed to quantify TRH, and from the right, to quantify TRH-gly. Two independent experiments were performed using different lot of animals.

Hormone and Pituitary Program) materials and protocol and for verifying parallelism with a standard curve. Dilutions (1:1000) of an aliquot of serum were used to quantify corticosterone using ICN Biomedicals kit (Costa Mesa, CA, USA). Intra- and inter-assay variation coefficients were 3 and 4%, respectively.

2.3. Radioimmunoassays (RIA)

Two independent experiments were performed (each experimental group consisted of four dams with six pups each). Data was calculated as percent of control (100%) in order to pool both experiments, since basal content differs between different lots of animals introducing additional errors. Data represent the mean ± S.E.M. One-way ANOVA analysis was made to compare food and liquid consumption, or body weight of animals, through the pregnancy and lactation periods; also, to compare TRH and TRH-gly content between the different experimental groups. ANOVA was followed by Fisher’s or by Student’s t-test to specify the differences, considered significant at P < 0.05 or less. Each individual experiment showed similar tendencies using untransformed data to the analysis performed pooling both expressed as % of control.

Tissues were homogenized in 500 ␮l of 20% acetic acid and centrifuged for 10 min at 12,000 × g at 4 ◦ C. The supernatant was extracted with methanol (90% final concentration), evaporated, resuspended in 0.25% bovine serum albumin (Sigma, RIA grade), 0.15 M NaCl, 0.05 phosphate buffer at pH 7.4 (RIA buffer), and treated as described (de Gortari et al., 1995; all reagents are from Sigma, St. Louis, MO, USA). TRH was quantified with a specific antibody previously characterized (Joseph-Bravo et al., 1979). TRH-gly was quantified using an antibody obtained in our laboratory using as immunogen pglu-his-pro-gly (Peninsula, Belmont, CA, USA) conjugated with thyroglobulin as described for TRH (Joseph-Bravo et al., 1979). Antibody titer was followed after several immunizations obtaining 30% binding with 1/4000 final dilution, using RIA buffer. Lowest amount of TRH-gly detected was 2.5 pg/tube. Specificity of assay was verified using several TRH related peptides: TRH, pglu-his-pro (TRH-OH), pglu-his-glyNH2 , pglu-his-gly, pglu-phe-pro NH2 , and also LHRH (Pen´ınsula). Only TRH and TRH-OH presented 10% cross-reactivity at amount of 20 ng and 40% at 200 ng. Specificity of the antibody was further verified by analyzing the chromatographic properties of endogenous TRH-gly from rat hypothalamus or olfactory bulb. Five hypothalami or five olfactory bulbs were homogenized in 20% acetic acid, centrifuged, and the supernatant treated with an equal volume of ether; the acetic acid fraction was extracted with methanol (60% final; Sigma, St. Louis, MO, USA), centrifuged and the supernatant evaporated. The residue was suspended in 20% acetic acid, passed through a Sep Pak C18 cartridge (Waters Corp., Milford, MA, USA), and subjected to HPLC C18 column eluted with a linear gradient 0–45% B (A: 0.1% trifluoroacetic acid (TFA)/H2 O, B: 0.1% TFA–acetonitrile 70%; HPLC grade, Merk, Darmstadt FR, Germany) at 1 ml/min flux. Fractions of 1 ml were collected and evaporated. They were resuspended in 250 ␮l of RIA buffer and aliquots of 25–100 ␮l assayed. The only fraction that showed immunoreactivity corresponded to the retention time of TRH-gly. Parallelism with standard curve was verified. 2.4. Serum hormone quantitation Aliquots of serum (25 and 50 ␮l), diluted 1:3 with RIA buffer, were used to quantify TSH using NIDDK (National

2.5. Statistics

3. Results 3.1. Liquid and food ingestion Although ethanol solution and solid food were available ad libitum for the ethanol group, they reduced their intake during pregnancy to 40% of that consumed by the control group. The pair-fed group had limited amount of glucose solution and solid food set by the amount consumed by the ethanol group of animals (Fig. 1A and B). The group that had water replacement at parturition consumed the same amount of food as controls; GW greatly increased their water intake compared with EW or controls. The EE and GG groups continued, during lactation, with reduced liquid and solid food ingestion (−70%) compared with controls and water-replaced groups (Fig. 1A and B). This led to a reduction of the total metabolizing energy in EE and GG groups of 28% during pregnancy and 33% in the last week of lactation; a protein-deficient diet of 56% for both periods. Energy requirements from carbohydrates were covered by the consumed ethanol or glucose solutions (12 g/day = 48 kcal/day during pregnancy and 24 g/day = 96 kcal/day at the end of lactation; calculations are based on PMI, Lab Diet Product Reference Manual). Pregnant and lactating dams exposed to the ethanol solution voluntarily reduced their ingestion of liquid and solid food, as previously reported in males, producing a decreased caloric intake and decreased body weight. Food was adjusted in the pair-fed group (GG) to that consumed by the lactating ethanol-fed animals, they showed a similar weight loss. There were no apparent signs of dehydration.

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Fig. 1. Liquid and food consumption and changes in body weight during pregnancy and lactation. (A) Liquid consumption. Pregnant rats fed ad libitum (C: control); chronic ingestion of ethanol (20% of ethanol in drinking water) during pregnancy and lactation: EE; animals received ethanol only during pregnancy and water during lactation: EW; animals pair fed with an isoenergetically solution of glucose (30% of glucose) during pregnancy and lactation: GG; animals consumed glucose solution only during pregnancy: GW. All values are the mean (ml) ± S.E.M. of the average of daily measures (n = 8) during each week; n = 8; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 with Student’s t-test comparing experimental vs. control data. (B) Food ingestion. A measured amount of food was placed in the cage and the left over was daily recorded. All values are the mean (g consumed) ± S.E.M. of the average of daily measures during each week; n = 8; ∗ P < 0.05, ∗∗ P < 0.02, ∗∗∗ P < 0.01, ∗∗∗∗ P < 0.001 with Student’s t-test comparing experimental vs. control data. (C) Weekly body weight. Weight was recorded each week, values are expressed as % of weight recorded at day that female rats were exposed to males; mean ± S.E.M.; n = 8 in each group. One-way ANOVA between the treatments showed significant differences in EE and GG against control group values; (a) P < 0.001, (b) P < 0.01, (c) P < 0.02, (d) P < 0.05 in Student’s t-test against control data. During pregnancy, one-way ANOVA showed no difference in weight gain between the treatments.

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Animals fed the alcohol-containing diet consumed more alcohol as pregnancy advanced but, since they increased weight, the daily ingestion did not differ (average consumption/day over the 3 weeks of pregnancy: 13.9 ± 0.24 g of ethanol/kg of body weight). During lactation, the animals of the EE group did not show any change in the amount of ethanol consumed, probably due to their stability in body weight. 3.2. Body weight Weight loss after delivery was 5% in controls, 11% in GG, and 19% in EE group with no difference at the end

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of pregnancy between all treatments (Fig 1C). In contrast, over the course of lactation, EE and GG differed by −28% from control group. Water replacement at parturition allowed full weight recovery as they improved their food and liquid consumption, but not of those that remained on ethanol or glucose regime (Fig. 1C). The weights of pups in the EE, GG and GW groups at sacrifice (21 days old) was decreased compared with controls (49.8 ± 1.4 g), no differences were observed by gender (not shown). Pups from mothers receiving ethanol during pregnancy and lactation showed the lowest weight (25.7±1.5∗ g); water replacement during lactation allowed full recovery (54.9±1.4∗ g). Although the effect of glucose ingestion dur-

Fig. 2. Endocrine changes in dams. (A) Serum TSH concentration. C: control group; EE: dams drinking 20% of ethanol during pregnancy and lactation; EW: dams consumed ethanol only during pregnancy; GG: dams pair fed with an isoenergetically solution of glucose as consuming water (30% of glucose) during pregnancy and lactation; GW: dams consumed glucose only during pregnancy. All values are the mean ± S.E.M. of ng/ml of serum TSH, expressed as % of control (control = 100%; 2.16 ± 0.35 ng/ml). One-way ANOVA followed by post-hoc Fisher test, revealed significant differences between the groups against the control values when P < 0.05. (B) Hypothalamic TRH and TRH-gly content. C: control group; EE: dams consuming ethanol during pregnancy and lactation; EW: dams received ethanol only during pregnancy; GG: dams pair fed with an isoenergetically solution of glucose as consuming water (30% of glucose) during pregnancy and lactation; GW: dams consumed glucose only during pregnancy. All values are the mean ± S.E.M. of pg/tissue, expressed as % of control (control = 100%); n = 8 in all groups. TRH content in controls: 2228 ± 337 pg/tissue; TRH-gly 206 ± 16 pg/tissue. One-way ANOVA followed by post-hoc Fisher analysis revealed significant differences between the groups comparing to control values of (∗ TRH and & TRH-gly) when P < 0.05.

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ing pregnancy and lactation was less drastic than EE group (significant difference between EE versus control group, P < 0.0005), these animals had lower weight than controls even when water-replaced glucose solution during lactation (GG (31.4 ± 0.3∗ g), GW (38.7 ± 1.8∗ g; ∗ P < 0.01)).

3.3. Serum hormone levels No variation was observed in serum corticosterone concentration in dams or their pups in any of the studied paradigms (not shown).

Fig. 3. Endocrine changes in pups. (A) Serum TSH concentration. C: control group (n = 10); EE: pups of dams consuming ethanol during pregnancy and lactation (n = 8); EW: pups of dams consuming ethanol only during pregnancy (n = 7); GG: pups of dams pair fed with an isoenergetically solution of glucose as consuming water (30% of glucose) during pregnancy and lactation (n = 9); GW: pups of dams consuming glucose only during pregnancy (n = 10). All values are the mean ± S.E.M. of ng/ml of serum TSH, expressed as % of control (control: 2.05 ± 0.19 ng/ml = 100%). One-way ANOVA analysis revealed significant differences between the groups. Significant differences against the control values in Student’s t-test specifiy the difference between experimental groups comparing to control values; ∗ P < 0.05, ∗∗ P < 0.01. (B) Hypothalamic TRH and TRH-gly content. C: control group (male n = 22, female n = 19); EE: pups of dams receiving ethanol during pregnancy and lactation (male or female, n = 12); EW: pups of dams receiving ethanol only during pregnancy (male or female n = 10); GG: pups of dams pair fed with an isoenergetically solution of glucose during pregnancy and lactation (male n = 12, female n = 13); GW: pups of dams receiving glucose only during pregnancy (male n = 17, female n = 13). Results are expressed as % of control group (TRH of male pups 3706 ± 1267 pg/tissue; TRH of female pups 3846 ± 603 pg/tissue; TRH-gly of male pups 150 ± 26 pg/tissue; female pups 103 ± 18 pg/tissue). One-way ANOVA analysis revealed significant differences between the groups. Significant differences against the control values in Student’s t-test specifiy the difference between experimental groups comparing to control values; ∗, & P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 TRH (bars), TRH-gly (line).

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TSH concentration was decreased in sera of dams receiving either ethanol or glucose during pregnancy and lactation; water replacement during lactation recovered normal values only in the glucose group (GW; Fig. 2A). In pups, TSH serum concentration increased in those whose mothers consumed alcohol or glucose through lactation, the effect being more pronounced in female pups; their TSH values were not normalized if water-replaced alcohol solution at parturition (EW) but they did if water-replaced glucose. The

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highest TSH values were detected in pups from mothers consuming glucose during pregnancy and lactation (GG; Fig. 3A). 3.4. TRH and TRH-gly content 3.4.1. Hypothalamus TRH hypothalamic content was not affected by ethanol consumption but was increased in GG group of dams

Fig. 4. TRH and TRH-gly in brain areas of dams. C: control group; EE: dams consuming ethanol during pregnancy and lactation; EW: dams receiving ethanol only during pregnancy; GG: dams pair fed with an isoenergetically solution of glucose during pregnancy and lactation; GW: dams receiving glucose only during pregnancy. TRH and TRH-gly values are expressed as % of control group (=100%) mean ±S.E.M. (hippocampus TRH 664±76 pg/tissue; TRH-gly 179 ± 10.4 pg/tissue; n.accumbens TRH 183 ± 29 pg/tissue; TRH-gly 83 ± 36 pg/tissue; frontal cortex TRH 326 ± 42 pg/tissue; TRH-gly 235 ± 20 pg/tissue; amygdala TRH 213 ± 22 pg/tissue; TRH-gly 197 ± 48 pg/tissue); n = 8 in all groups. One-way ANOVA showed significant differences between the groups. Significant differences against the control values in a Fisher post-hoc analysis (∗ TRH and & TRH-gly) & or ∗ P < 0.05, ∗∗ P < 0.01. TRH (bars), TRH (line). TRH-gly content is not shown in those brain regions with any significant difference against control values (frontal cortex and amygdala).

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receiving glucose (Fig. 2B); a significant decrease in TRH-gly content was detected in GG group that had the highest concentration of TRH. In contrast, pups from EE mothers showed increased TRH content and only females

presented decreased levels in GG group while males had a small decrease in GW. The decrement in TRH levels of female GG group was accompanied by an increase in TRH-gly (Fig. 3B).

Fig. 5. TRH and TRH-gly in brain areas of pups. C: control group (male n = 22, female n = 19); EE: pups of dams consuming 20% of ethanol during pregnancy and lactation (male or female, n = 12); EW: pups of dams receiving ethanol only during pregnancy (male or female, n = 10); GG: pups of dams pair fed with an isoenergetically solution of glucose during pregnancy and lactation (male n = 12, female n = 13); GW: pups of dams receiving glucose only during pregnancy (male n = 13, female n = 17). TRH and TRH-gly values are the mean ± S.E.M. of % of control (control = 100%; hippocampus TRH of male pups 781±56 pg/tissue, female pups 923±56 pg/tissue, TRH-gly of male pups 213±34 pg/tissue, female pups 136±19 pg/tissue; n.accumbens TRH of male pups 1720 ± 318 pg/tissue, female pups 1338 ± 244 pg/tissue; TRH-gly of male pups 110 ± 8 pg/tissue, female pups 83 ± 23 pg/tissue; frontal cortex TRH of male pups 1875 ± 249 pg/tissue, female pups 1565 ± 218 pg/tissue; TRH-gly of male pups 242 ± 40 pg/tissue, female pups 222 ± 36 pg/tissue; amygdala TRH male pups 1681 ± 216 pg/tissue, female pups 975 ± 193 pg/tissue; TRH-gly of male pups 163 ± 28 pg/tissue, female pups 140 ± 16 pg/tissue). One-way ANOVA showed significant differences between the groups. Significant differences against the control values in Student’s t-test ∗ for TRH and & for TRH-gly content; & or ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. TRH (bars), TRH-gly (line).

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3.5. Extrahypothalamic brain regions 3.5.1. Dams Ethanol consumption during pregnancy and lactation (EE) decreased TRH content in hippocampus and frontal cortex; water replacement during lactation (EW) reversed these changes only in frontal cortex (Fig. 4). Variations in TRH-gly levels were opposite to those observed for TRH in hippocampus. Ethanol administration did not change TRH levels in amygdala or n.accumbens and were increased only in glucose-fed groups; a small increase in TRH-gly was found in n.accumbens of all groups (Fig. 4). 3.5.2. Pups No difference in total TRH or TRH-gly content (as pg of TRH/ brain region, or per mg protein) were observed between male or female pups in any of the regions analyzed. TRH levels were decreased in hippocampus of male and female pups, and in n.accumbens and amygdala of male pups whose mothers had ingested ethanol or glucose during pregnancy (Fig. 5). Water replacement allowed recovery to control values only in the GW group (except in n.accumbens where values recovered also in EW group). Gender differences were observed in the response of amygdala (a decrease in EE and EW males, and an increase in E females) and of frontal cortex (decreased values in G males and increased in E females; Fig. 5). TRH-gly content augmented in several conditions where there was a decrement in TRH content: hypothalamus (GG and GW females), hippocampus of EE male and female; male frontal cortex of GG group; female amygdala of EE and EW group. In female n.accumbens, EW group had increased TRH and decreased TRH-gly levels (Fig. 5).

4. Discussion The hypophysiotropic role of TRH has been well characterized and the hypothalamic neurons responsive to metabolic alterations as fasting and malnutrition or to neural stimulation in response to cold or suckling stimulus have been identified (Joseph-Bravo et al., 1998; Fekete et al., 2000; Sánchez et al., 2001). Less is known about the involvement of TRHergic neurons in other brain areas (Pekary and Sattin, 2001). Chronic exposure to alcohol affected the HPT axis of pregnant dams, as reported in human and experimental rats (Portolés et al., 1985, 1988) as well as in glucose pair-fed animals (isocaloric to ethanol diet) supporting an effect due to malnutrition (Mason et al., 1992; Shi et al., 1993; Van Haasteren et al., 1996; de Gortari et al., 2000b). The lack of variations in corticosterone levels in the different groups suggest that malnutrition was not due to caloric restriction but most probably, protein deficiency (Van Haasteren et al., 1995, 1996; de Gortari et al., 2000b; Ogilvie et al., 1997). Glucose regime during pregnancy and lactation induced a

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greater weight reduction than that reported for males (Strbak et al., 1998) but it should be taken into account that the lactation period elevates energy requirements. Our results suggest that deficient nutrition is enough to alter TSH values but ethanol ingestion during pregnancy produced long lasting effects since values were not recovered after water replacement. Inadequate availability of micronutrients could contribute to these effects as for example, thiamine deficiency provoked by ethanol although remains to be defined if the high glucose intake combined with low protein ingestion can induce thiamine deficiency to similar pathological levels (Greenwood et al., 1985; Ahmed et al., 1988; Bakker et al., 2000; Todd and Butterworth, 1999). The mechanism of hypothyroid condition might differ at the hypothalamic level. The normal levels of TRH or its immediate precursor TRH-gly in ethanol groups could imply that if TRH biosynthesis was increased as previously suggested, the increase in TRH was compensated by increased release (Zoeller et al., 1996; Zoeller and Rudeen, 1992). Desensitization of the hypophysis to TRH in chronic ethanol consuming male rats has been proposed to explain the low TSH levels (Winokur et al., 1984; Zoeller et al., 1996). In contrast, the increased TRH levels found in the glucose-fed lactating dams coincides with the proposed inhibition of TRH release caused by malnutrition (Van Haasteren et al., 1996; de Gortari et al., 2000b). Pups were not refractory to the malnutrition of their mothers during pregnancy and lactation, they had considerable weight reduction and increased TSH levels, probably due to primary hypothyroidism as a consequence of the induced secondary hypothyroid condition of their mothers. Offspring of hypothyroid mothers have low body weight and increased TSH levels that can be overcome by thyroid hormone replacement (Bonet and Herrera, 1988; Porterfield and Hendrich, 1993). The deleterious effect of ethanol on brain development can be reversed by thyroid hormone treatment (Gottesfeld and Silverman, 1990). Possible deleterious effects of the high glucose diet on the metabolism of pregnant dams are worth studying further since a higher water consumption was evident in lactating dams after parturition and their pups did not recover the weight of controls even though TSH levels were normal. 4.1. Effects on TRH neurons of the limbic system TRH metabolism was differentially affected by ethanol or glucose ingestion in pregnant-lactating dams, depending on the limbic region studied. In contrast, their pups did not show specific alterations due to prenatal exposure of ethanol. Ethanol ingestion caused specific changes in TRH metabolism in hippocampus and cortex of dams, regions defined as ethanol specific targets. Chronic consumption induces sprouting of the dentate gyrus, increases sensitivity to seizures, alters cfos and NMDA subunit expression, and impairs memory and learning functions (Bond, 1981; Trevisan et al., 1995; Follesa and Ticku, 1995; Fadda and Rossetti,

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1998). The hippocampus is the most sensitive, followed by cortical areas (Randoll et al., 1996). The changes observed in TRH and TRH-gly levels in hippocampus and cortex of pregnant dams after chronic ethanol consumption are coincident with increased or decreased synthesis (respectively) previously observed after acute ethanol ingestion of adult male rats (de Gortari et al., 2000a). The effect is long lasting in hippocampus and supports TRH participation in some of the hippocampal related and altered conducts due to ethanol consumption. Other limbic regions as amygdala and n.accumbens were affected but only in pair-fed dams. This latter region is important in locomotion activity where TRH may participate; it is important in reward mechanisms, responds to stress-induced metabolic alterations as changes in brain glucose levels to regulate energy homeostasis (Carelli et al., 2000; Tzchentke and Schmidt, 2000; Hajnal et al., 2000). A fast and transient increase in TRH mRNA levels of n.accumbens is produced by an acute ethanol injection (de Gortari et al., 2000a) and TRH levels increase after fasting in 21 days old animals but not in adults (de Gortari et al., 2000b). This region showed significant changes in TRH metabolism in ethanol or glucose pair-fed dams, compared with naive controls, suggesting that TRH neurons of n.accumbens were activated in response to the metabolic alterations produced. Amygdala has been implicated in the regulation of feeding behavior, body weight control and hunger, and it is activated by pleasant and unpleasant tastes (Morris and Dolan, 2001; O’Doherty et al., 2000). Neurons in this region respond to a direct effect of glucose altering their firing rate in response to changes in ambient glucose levels. This effect is modulated by a K+ channel that is sensitive to ATP (KATP channel) proposed to be potentially capable of monitoring and integrating a multitude of metabolic and neural signals (Trapp and Ashcroft, 1997; Levin et al., 1999). Amygdala is the only region in adults where TRH levels are modified by fasting and also after 6 and 24 h of acute ethanol administration supporting their susceptibility to metabolic alterations (de Gortari et al., 2000a,b). TRH content increased in the amygdala of lactating dams pair-fed with glucose; however, a direct effect of glucose cannot explain the increased levels of TRH in dams that had water replacement at parturition since they recovered their normal food ingestion. As mentioned, higher consumption of water was detected; therefore, it remains to be studied whether these rats had altered insulin levels and if TRH neurons in these regions can be modulated through changes in insulin concentration. The distribution of insulin receptor differs depending on the region and direct effects of insulin on brain activity have been reported (Schulingkramp et al., 2000; Park, 2001); alternatively, the observed changes could be due to an altered glucose–insulin balance produced by a high carbohydrate diet during pregnancy. It is tempting to speculate that TRH neurons in the amygdala are involved in responses to nutrient availability to regulate body weight and metabolism, as

shown for hypophysiotropic TRH neurons (Van Haasteren et al., 1995, 1996; Fekete et al., 2000). As mentioned, several cellular functions and brain development are affected by prenatal ethanol, protein malnutrition and vitamin deficiencies (Zoeller et al., 1994; Gressens et al., 1997; Osborn et al., 1998; Costa et al., 2000; Ramakrishna, 1999). The expression of thyroid hormone receptor (␣2 TR) is affected in hippocampus and in cortex of fetus at 21 days of gestation from glucose pair-fed compared with the specific effect of ethanol on ␣1 TR (Scott et al., 1998). Very little is known regarding TRH neurons of the limbic system of infants. Upon fasting, they respond differently to adults (de Gortari et al., 2000b). There were no consistent variations in either TRH or TRH-gly in the different regions studied in pups brain except for hippocampus that was, as in their mothers, the most affected region but changes were equal whether mothers received alcohol or glucose solutions. The effects of ethanol were long lasting or, were due to the hypothyroid condition of their mothers since TSH was normalized only in GW dams. Gender differences were observed, probably related to the neonatal elevation of androgen concentration that induces organizational effects and leads to sexual dimorphisms, as well as differential neuropeptide expression within some areas of the limbic system (Simerly et al., 1988; Wang et al., 1993; Osborn et al., 1998). We conclude that although chronic ethanol ingestion and pair-fed glucose diets led to a protein-deficient consumption, the HPT alterations in dams consuming alcohol were long lasting. Ethanol affected selectively dams’ TRHergic neurons in brain regions involved in cognitive functions (hippocampus and cortex) and add up to the list of other neurotransmitter systems altered by ethanol chronic consumption (Fadda and Rossetti, 1998; Hwang et al., 1999; Weiss et al., 1996). It is interesting, albeit speculative for the moment, to relate the coincidences of TRH variations with behaviors promoted by particular treatments and the regions involved: cognitive and task performance (hippocampus and frontal cortex; Ogasawara et al., 1996), areas proposed to participate in metabolic regulation, appetite regulation and food reward mechanisms affected by glucose regime as n.accumbens and amygdala (Tzchentke and Schmidt, 2000; Hajnal et al., 2000; O’Doherty et al., 2000; Morris and Dolan, 2001). The role of TRH in these behaviors has been inferred mainly by pharmacological studies with administration of TRH or its analogs (Vijayan and McCann, 1977; Kahn et al., 1993; O’Leary and O’Connor, 1995). Changes in TRH metabolism in areas relevant to these conducts imply the involvement of this peptide although it remains to be studied the particular circuits in which TRH neurons participate.

Acknowledgements We thank the technical aid of Fidelia Romero, Lakshmi Charl´ı, and Alejandro Rubio, as well as the secretarial assistance of Graciela Valencia. The aid of M.V.Z. Mario

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