The neurosteroid pregnenolone sulfate neutralized the learning impairment induced by intrahippocampal nicotine in alcohol-drinking rats

Neuroscience 136 (2005) 1109 –1119

THE NEUROSTEROID PREGNENOLONE SULFATE NEUTRALIZED THE LEARNING IMPAIRMENT INDUCED BY INTRAHIPPOCAMPAL NICOTINE IN ALCOHOL-DRINKING RATS E. MARTÍN-GARCÍA AND M. PALLARÈS*

the GABAA receptor complex (Baulieu and Robel, 1990), and a positive modulator of glutamate N-methyl-D-aspartate (NMDA) receptors (Bowlby, 1993). Conversely, the neurosteroid allopregnanolone (AlloP) is a positive modulator of the GABAA receptor and may have sedative, hypnotic, anticonvulsant and anxiolytic effects (Lambert et al., 1995). Behavioral studies on rodents have demonstrated the promnesic effect of PregS in passive (s.c.) and active (i.c.v. or locally into the amygdala, hippocampus, septum or mammillary bodies) avoidance tests (Frye and Sturgis, 1995) and on spatial memory (i.c.v.) (Ladurelle et al., 2000). Furthermore, the intrahippocampal administration of PregS to old cognitively-impaired rats reverses the previous memory dysfunction tested in mazes (Vallee et al., 1997; Mayo et al., 2003). Indeed, PregS infused into the nucleus basalis magnocellularis (NBM) enhances: acetylcholine release in the cortex (Pallares et al., 1998); paradoxical sleep (Darnaudery et al., 1999); spatial memory performance in Y-mazes (Mayo et al., 1993); and two-trial spatial memory tasks (Pallares et al., 1998). Conversely, AlloP injected into the NBM disturbed memory performance in Y-mazes (Mayo et al., 1993), and i.c.v. (Ladurelle et al., 2000), and spatial learning in Morris water mazes when injected i.v. (Johansson et al., 2002). Ethanol, which induces memory and learning impairment in several paradigms like shock avoidance (File and Mabbutt, 1990), win-shift foraging (Melchior et al., 1993), trace fear conditioning (Weitemier and Ryabinin, 2003), Morris water mazes (Markwiese et al., 1998; Cain et al., 2002), elevated radial arm mazes (Matthews et al., 1999, 2001), operant signal detection (Givens and McMahon, 1997), operant position discrimination and timing behavior (Popke et al., 2000), may affect the physiological levels of neurosteroids. In this way, alcohol increased AlloP and allotetrahydrodeoxycorticosterone (THDOC) levels in the cerebral cortex and hippocampus in Sardinian alcoholpreferring rats (Barbaccia et al., 1999), the cortical levels of AlloP and THDOC in non-selected rats (Morrow et al., 2001), and the plasma levels of AlloP in human female adolescents (Torres and Ortega, 2004). The tendency for ethanol and some neurosteroids to act as positive modulators of GABAA receptors suggests that their effects on cognitive tasks can be caused by similar mechanisms (Silvers et al., 2003). There is little information available about the effects of voluntary alcohol consumption on learning tasks in nonselected rats. Prolonged voluntary ethanol intake induces a specific impairment of the subject’s capability to inhibit responses that were previously reinforced (Beracochea et

Institut de Neurociències, Departament de Psicobiologia i Metodologia de les Ciències de la Salut, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Abstract—The effects of intrahippocampal administration of nicotine and the neurosteroids pregnenolone sulfate and allopregnanolone on acquiring the lever-press response and extinction in a Skinner box were examined using voluntary alcohol-drinking rats. A free-choice drinking procedure that implies early availability of the alcoholic solution (10% ethanol v/vⴙ3% glucose w/v in distilled water) was used. Alcohol and control rats were deprived of food and assigned at random to six groups. Each group received two consecutive intrahippocampal (dorsal CA1) injections immediately after 1-h of drinking ethanol and before the free lever-press response shaping and extinction session. The groups were: saline–saline; saline–pregnenolone sulfate (5 ng, 24 ␮M); saline–allopregnanolone (0.2 ␮g, 1.26 ␮M); nicotine (4.6 ␮g, 20 mM)–saline; nicotine–pregnenolone sulfate; nicotine–allopregnanolone. Blood alcohol concentrations were assessed the day before conditioning. The combination of the oral self-administration of ethanol and the intrahippocampal injection of nicotine deteriorated the ability to acquire the lever-press response. This effect was neutralized by intrahippocampal pregnenolone sulfate (negative modulator of the GABAA receptor complex), and it was not affected by intrahippocampal allopregnanolone (positive GABA receptor complex A modulator). Pregnenolone sulfate and allopregnanolone had no effects per se on lever-press acquisition, neither in alcohol-drinking rats nor in controls. Alcohol consumption facilitated operant extinction just as anxiolytics that act as positive modulators of the GABA receptor complex A receptors do, possibly reducing the anxiety or aversion related to non-reinforcement. This effect was increased by intrahippocampal nicotine. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: operant conditioning, hippocampus, chronic voluntary alcohol intake, allopregnanolone, extinction.

Neurosteroids are a subclass of steroids that can be synthesized in the CNS independently from peripheral sources (Baulieu and Robel, 1990). The neurosteroid pregnenolone sulfate (PregS) is a negative modulator of *Corresponding author. Tel: ⫹34-93-581-2542; fax: ⫹34-93-581-2001. E-mail address: [email protected] (M. Pallarès). Abbreviations: AlloP, allopregnanolone; BAC, blood alcohol concentration; CA1, hippocampal area CA1; CR, continuous reinforcement schedule; EXT, operant extinction; FS, free shaping; GLM, general lineal model; NBM, nucleus basalis magnocellularis; NMDA, N-methylD-aspartate; nnAChRs, neuronal nicotinic acetylcholine receptors; PregS, pregnenolone sulfate; S.E.M., standard error of the mean; THDOC, allotetrahydrodeoxycorticosterone. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.08.036

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al., 1987; Lobaugh et al., 1991). It has been suggested that alcohol reduces the capability of the working memory to modulate response inhibition in humans (Finn et al., 2002), and that the mechanisms that underlie response inhibition may be more sensitive to the effects of ethanol than mechanisms that underlie other abilities (Popke et al., 2000). Nicotine, like alcohol, affects the cognitive function. It has been reported that it can improve cognitive abilities in rodents and humans (for a review see Rezvani and Levin, 2001). For instance, systemic intake of nicotine enhances the working memory (Rezvani and Levin, 2002). Moreover, intrahippocampal or intra-amygdala nicotine antagonists produce working memory deficits (Addy et al., 2003). Nevertheless, the interaction between nicotine and ethanol, both administered systemically, impaired the radial arm maze performance whereas when they were administered independently neither nicotine nor ethanol affected the memory (Rezvani and Levin, 2002). In the present experiment, we have analyzed the interaction between voluntary alcohol consumption and intrahippocampal nicotine administration on learning acquisition, and its possible modulation through neurosteroids. For this purpose we have injected nicotine (salt weight) (4.6 ␮g, 20 mM), PregS (5 ng, 24 ␮M) and AlloP (0.2 ␮g, 1.26 ␮M) into the hippocampus (CA1) of chronic voluntary alcohol-drinking rats. The doses of nicotine (Garcia-Rebollo et al., 2005) or neurosteroids (Vallee et al., 1997; Pallares et al., 1998; Darnaudery et al., 1999) were chosen because they had previously been found to be effective in altering learning and/or paradoxical sleep in rats. In order to obtain alcohol-drinking rats we used a free-choice drinking procedure that makes the alcoholic solution available (Darbra et al., 2002, 2004; Garcia-Rebollo et al., 2005), then progressively reduces alcohol availability. Taste factors, such as sweetness, are not the primary factors in controlling alcohol consumption in rats (Samson et al., 1996; Heyman, 1997; Goodwin and Amit, 1998). Thus, we used sweet alcohol solutions to avoid taste aversion. In order to encourage a high degree of drug consumption, we restricted access to food to three hours, since food restriction has been shown to be effective in increasing the self-administration of drugs with rewarding properties in animals, including not only those agents with caloric content, such as ethanol (Koob and Bloom, 1988), but also those agents without a caloric content, such as phencyclidine, Ketamine and d-amphetamine (Carroll, 1982; Carroll and Stotz, 1983; Meisch, 1987). Moreover, restricted access to the drug increases self-administration in sufficient quantities to produce intoxication, even for alcohol (Heyser et al., 1997). In addition, repeated cycles of alcohol withdrawal and alcohol access were used because it increased the reinforced effects of ethanol (Rodd et al., 2003).

EXPERIMENTAL PROCEDURES Animals A hundred thirty-five male Wistar rats (Universitat Autònoma de Barcelona, Barcelona, Spain) were used. The rats were 21 days at the beginning of the experiment and they were housed in a temperature-controlled animal room (22–24 °C) on a 12-h light/dark (08:00 – 20:00 h) cycle. All the experimental sessions were run during the light

portion of the cycle. The experimental protocol was approved by the Committee of Ethics of the Universitat Autònoma de Barcelona and was carried out in accordance with the European Council Directive guidelines (86/609/EEC). Attempts were made to introduce the minimum suffering of animals and to use the minimum number of animals compatible with our objectives.

Drugs, solutions and reagents Nicotine (hydrogen tartrate salt, Sigma, St. Louis, MO, USA), PregS (5-pregnen-3␤-ol-20-one sulfate; Sigma), and AlloP (3␣hydroxy-5␣-pregnan-20-one; Sigma) were dissolved in 0.9% NaCl. Also neurosteroids were dissolved by sonication for 10 min and AlloP was suspended in 10% 2-hydroxypropyl-␤-cyclodextrin (Sigma). Alcoholic-sweetened solution (ethanol 10% v/v and glucose 3% w/v) was prepared from 99.9% ethanol (Normasolv, Barcelona, Spain) and D(⫹)-glucose anhydrous (Panreac, Barcelona, Spain) diluted in distilled water. The control solution was prepared from D(⫹)-glucose anhydrous (3% w/v) diluted in distilled water. For the determination of blood alcohol concentration (BAC), a Sigma Diagnostics® alcohol kit and trichloroacetic acid solution (6.25% w/v) were used.

Apparatus Experiments were conducted in eight operant chambers (Lafayette Instruments, IN, USA) encased in sound-attenuation cubicles. Each chamber was equipped with one response lever placed at 7.5 cm from the floor and one pellet dispenser that delivered 45 mg food pellets (J.C. Noyes Company, Inc., Lancaster, UK). A 10-W lamp was situated 3 cm above the lever. The operant chambers were interfaced to a computer that was programmed with MED-PC®IV software package (St. Albans, GA, USA). SoftCr™ for Windows (St. Albans, GA, USA) was used to display cumulative records. For the intracerebral injections, a multiple micro syringedriven pump (Harvard 22, Harvard Apparatus, Holliston, MA, USA) was used.

Chronic alcohol consumption This procedure has been previously described (Darbra et al., 2002, 2004; Garcia-Rebollo et al., 2005), and is based in prior work carried out in our laboratory (Pallares et al., 1992; Nadal et al., 1996a,b; Pallares et al., 1997, 2001; Silvestre et al., 2002). Free access phase. At the age of 21 days, rats were housed in groups of two to five per cage. Animals were randomly distributed into two groups: control (n⫽70) and ethanol (n⫽65). The ethanol group had one bottle with the alcoholic solution (10% ethanol v/v, 3% glucose w/v in distilled water) and the other one with water. The control group had one bottle with a sweetened solution (3% glucose w/v in distilled water) and the other one with water. The position of the bottles was changed daily at random until the end of the study, in order to avoid positional preferences. All animals had access to food ad libitum. This phase lasted for three weeks. Limited access phase. Over the following two weeks, the animals had two-bottle choices, but also restriction of the treatment solution at the weekends. Thus, rats consumed ethanol five days a week (not on weekends). In the previous phase and in this one, the alcohol intake was recorded daily for each housed group and the individual consumption was estimated for each rat; dividing the ethanol intake (ml) by the number of rats per cage. For this measure a randomly selected group of rats was used (ethanol n⫽22, and control n⫽20). One-hour limited access phase. At the age of 60 days old rats were housed individually. This phase lasted for four weeks

E. Martín-García and M. Pallarès / Neuroscience 136 (2005) 1109 –1119 and the conditions of two-bottle choices and restriction of the treatment solution at the weekends were maintained. The twobottle choice procedure consisted of the solution being available only 1h/day (with free water) at 15:00 –16:00 h and a food restriction of 3 h/day (15:00 –18:00 h) both conditions coinciding in the first hour. On weekends, rats had access to food and water ad libitum. Their weight was registered every Monday and Friday before the 1-h of treatment and the solution intake (ml) was recorded daily. This procedure was maintained during the whole experiment for the solution intake. The food availability was changed in post-surgical recovery, in food deprivation phase and in conditioning phase as described below.

Surgery Surgery was carried out at 90 days. Animals were anesthetized (i.p.) with ketamine (100 mg/kg) and xylazine (10 mg/kg). A guide cannula (Plastics One, VA, USA) was stereotactically implanted (right hemisphere) 1 mm above the hippocampus at the following coordinates relative to Bregma, according to the atlas of Paxinos and Watson (1998): (anterior–posterior to bregma: ⫺3.6 mm, lateral to bregma: 1.8 mm and ventral from the skull surface: 2.8 mm from the skull surface; incisor bar ⫺3.3 mm). The guide cannula (26-gauge, 15 mm long) was mounted on the skull with stainless-steel screws and dental cement. Alcohol and control solutions were not administered the day in which surgery was carried out. Rats were allowed 14 days of recovery after surgery to regain their pre-surgical alcohol intake level and their preoperative body weight. In this recovery phase, they had access to food ad libitum the first three days after surgery. The next 11 days food was restricted for 3 h per day. Body weight and solution intake were recorded daily.

Food deprivation Following two weeks of post-surgical recovery, rats were fooddeprived to maintain their body weight at 80% of the free-feeding levels, adjusted for growth. The food deprivation method is the standard protocol used in our laboratory (Pallares et al., 1992, 1995, 2001; Nadal et al., 1996b). Food deprivation was necessary to the subsequent conditioning phase in which food was used as a positive reinforcer. This phase lasted for three weeks.

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of infusion. Injection needles were removed from the guide cannula 2 min after infusions in order to prevent drug reflux.

Conditioning The conditioning schedule consisted of free shaping (FS: one session), continuous reinforcement schedule (CR: four sessions), and operant extinction (EXT: one session) conducted on sequential days. FS was performed using an automatic procedure previously described (Pallares et al., 1992, 1995, 2001). This consisted of placing the animal in the Skinner box (the pellet dispenser containing three pellets). The light remained on for the entire session. When the program started, the pellet dispenser delivered one gratuitous pellet every 50 s throughout the first 20 min of the session. After this period, the interval of time for free pellets increased from 50 s to 2 min until the end of the session. When the animal acquired the operant conditioning the pellet dispenser stopped of delivering free pellets. Then a CR schedule was executed automatically. The acquisition criterion was the time (in seconds) to perform the first 10 responses minus the time to perform the first response (Pallares et al., 1995, 2001). This session finished after 50 min or when rats had obtained 120 pellets. The apparatus was cleaned at the end of each trial to prevent behavioral modifications in presence of odor. For the animals that did not acquire the acquisition criterion the next session (CR1) started with a gratuitous schedule of free reinforcement given every 2 min. CR sessions stopped after 30 min or after animals obtained 120 pellets. CR was performed with the light on. EXT (30 min) was conducted with the light off. In addition, the time the animal passed without responding within the 30 min of the session was considered as a responding pause, so the sum of pauses and the latency of the first responding pause (minutes) was registered in the EXT session. The criterion to consider a responding pause as significant was a minimum of 3 min (10% time of session) without pressing the lever. Additionally, the index of the response rate weighed in function of the time the animal spends on responding was calculated to quantify the EXT level acquired by the animal. This index was obtained according with the following formula: I⫽(RREXT⫻PTR)/100. RREXT was the rate of responding (response/min) in the EXT session; PTR was the percent time the animal spends on responding. Lower scores in this index indicate higher EXT levels.

Determination of BAC One day before the beginning of the conditioning phase, BACs were assessed in a group of 16 randomly selected rats (ethanol n⫽13, and control n⫽3). Blood samples were collected from the tip of the animals’ tails immediately after 1-h of limited access to the solutions. Subsequently, BACs were determined by spectrophotometry following the procedure of Sigma Diagnostics®.

Intrahippocampal infusion Ethanol and control rats were assigned at random to six groups. Each group received two consecutive (5 min between injections) intrahippocampal (dorsal CA1 region) injections immediately after the 1-h of ethanol drinking. First injection: nicotine or saline; second injection: PregS, AlloP or saline. The infusions were administered 5 min before: (1) the free lever-press response shaping; and (2) the extinction session. PregS, AlloP and nicotine were infused at, respectively, 5 ng/0.5 ␮l, 0.2 ␮g/0.5 ␮l, and 4.6 ␮g/0.5 ␮l (salt weight) into the hippocampus of freely moving rats via 33-gauge needles cut (16 mm long) to extend 1 mm beyond the ventral tip of the guide cannula. The needles were connected with polyethylene tubing to a micro syringe (10 ␮l) driven by the infusion pump. Solutions were infused at a constant rate of 0.5 ␮l/1 min. Control rats received the same volume (0.5 ␮l) of 0.9% NaCl at the same rate

Histological control Animals were killed by deep anesthesia, sodium pentobarbital, and brains were removed and stored in 10% formalin. Brains were sectioned in 100 ␮m coronal sections on a vibroslice, mounted, and Cresyl-Violet stained. Localization of the guide cannula and the infusion site was confirmed histologically for each rat. Only animals in which histology confirmed that the infusion cannula was located within the CA1 area of the hippocampus were included in the analysis. Thus, 11 animals were excluded from statistical analysis because of placement errors: saline–saline (ethanol n⫽1); saline–PregS (ethanol n⫽2); saline–AlloP (control n⫽1; ethanol n⫽1); nicotine–PregS (ethanol n⫽2); nicotine–AlloP (control n⫽3; ethanol n⫽1).

Data analysis The statistical analysis was performed using the Statistical Package for Social Science program SPSS® 12.0 (SPSS Inc, Chicago, USA). For the analysis of variance, a general lineal model (GLM) was used to analyze: the time in seconds of the acquisition of the lever press response (FS); the rate of responding (response/min) (CR; EXT); the total time without responding (sum of pauses) (EXT); the latency of the first responding pause (EXT); and the index of the responding rate

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weighed in function of the time the animal spends on responding (EXT). A design that consisted of three between-factors was used: Treatment solution (two levels: Control/Ethanol), Nicotine injection (two levels: Saline/Nicotine) and Neurosteroids infusion (three levels: Saline/PregS/AlloP). Body weight, ethanol consumption and the rate of response in CR (four sessions) were analyzed with GLM and a mixed design with repeated measures in the last factor (Treatment⫻Nicotine⫻Neurosteroids⫻sessions/ days). Newman-Keuls (NK) tests were used for the post hoc analysis. Differences were considered significant at P⬍0.05. All measures are expressed in mean⫾S.E.M. Pearson’s chi-square test was used to analyze the qualitative variable: percentage of subjects reaching criterion in the FS session.

RESULTS Body weight The average body weight of the animals the day of individualization housing at 60 days was (mean⫾S.E.M) 322.59⫾4.02 g in control rats and 319.46⫾4.21 g in ethanol group, and there were no significant differences between groups of treatment [F(1,112)⫽0.29; P⬎0.05]. Also, the analysis of the global averaged body weight over the whole experiment showed no significant differences between groups of treatment [F(1,112)⫽0.1; P⬎0.05]. Alcohol consumption and BAC Concerning ethanol intake in the limited access phase, the results showed that there was no difference in the average ethanol consumption by individual rats (estimated) independently of the number of rats in the cage [F(3,18)⫽0.90; P⬎0.05]. The last week of limited access phase the average of alcohol intake (g ethanol/kg body weight per day) recorded was (mean⫾S.E.M) 1.96⫾0.87. Table 1 shows the pattern of consumption throughout the different experimental phases. In the first four weeks of 1-h limited access, the average dose of alcohol consumption computed was 2.36⫾ 0.06 g alcohol/kg body weight in 1-h. The day after surgery, the mean alcohol dose registered was 1.98⫾0.12. The last day of the post-surgical recovery phase ethanol intake reach an average dose of 2.19⫾0.13 g alcohol/kg Table 1. Average dose of the alcohol intake (g ethanol/kg body weight in 1 h) throughout the experimental phases and the conditioning sessions Pre-conditioning phases

Dose

Sessions of conditioning phase

Dose

Free access Limited access 1-h Limited access 1st Week 2nd Week 3rd Week 4th Week Post-surgical recovery Food deprivation

2.01⫾0.12 1.98⫾0.18

FS CR1 CR2 CR3 CR4 EXT

2.90⫾0.14 2.66⫾0.12 2.71⫾0.12 3.09⫾0.13 2.88⫾0.10 2.67⫾0.12

2.35⫾0.08 2.42⫾0.09 2.30⫾0.09 2.36⫾0.10 2.25⫾0.13 2.89⫾0.07***

Data are presented as means⫾S.E.M. *** P⬍0.001: food deprivation⬎ mean of previous phases.

body weight in 1-h. Ethanol intake increased significantly in the food deprivation phase [polynomial (linear), F(1,52)⫽ 33.94; P⬍0.001]. The average BAC obtained the day before the behavioral testing phase was 0.095⫾0.013 g ethanol/dl blood with average doses recorded of 3.05⫾0.2 g ethanol/kg body weight in 1-h. In the conditioning phase, the mean of alcohol intake was 2.8⫾0.07 g/kg. During this phase, the ethanol intake remained steady throughout the sessions. There were no significant differences in alcohol intake related to nicotine [F(1,52)⫽0.00; P⬎0.05] or neurosteroids administration [F(2,52)⫽1.79; P⬎0.05] or their interaction [F(2,52)⫽0.14; P⬎0.05]. During the whole experiment, the minimum dose of ethanol ingested was 0.20 g ethanol/kg body weight (postsurgical recovery phase) and the maximum dose of ethanol registered was 6.30 g ethanol/kg body weight (conditioning phase). Taken into account the whole experimental phases, the mean of dose ingested was 2.53⫾0.06 g ethanol/kg body weight in 1-h. Over the whole experiment, the control group consumed an average of 41.14⫾0.76 ml/kg of sweetened solution in 1-h, with significant increase observed from the postoperative period to the food deprivation phase [polynomial (linear), F(1,60)⫽971.51; P⬍0.001]. In the same way, no significant main effects of Nicotine [F(1,60)⫽0.41; P⬎0.05] or Neurosteroids [F(2,60)⫽0.57; P⬎0.05] factors or their interaction [F(2,60)⫽0.06; P⬎0.05] were found on the sweetened solution intake during the conditioning phase. Conditioning FS. In the learning acquisition criterion, the main effect of Treatment [F(1,112)⫽6.10; P⬍0.05], Nicotine [F(1,112)⫽ 12.01; P⬍0.001] and Neurosteroids [F(1,112)⫽3.70; P⬍ 0.05] was significant. Also, results showed a significant interaction of these three factors: Treatment⫻Nicotine⫻ Neurosteroids [F(2,112)⫽5.45; P⬍0.01]. Post hoc comparisons revealed that the time to acquire the operant learning conditioning was significantly higher in the ethanol groups that were injected with nicotine–saline (P⬍0.05) and nicotine–AlloP (P⬍0.001) than in the rest of the groups. See Fig. 1 for the means of the experimental groups and for the rest of post hoc comparisons (Newman-Keuls). Concerning the number of animals reaching the criterion in the FS session of training we have found that there was a significant effect of treatment [Chi-square⫽27.8; P⬍0.001]. In the ethanol group, 11 (19%) animals failed to reach the criterion in the FS session of training within the 50 min test time, whereas only three (4.5%) animals of the control group did not reach the criterion. In addition, there were no differences between groups of injection in the control treatment group. Furthermore, the proportion of animals that failed to reach the criterion within the 50 min of test time was significantly greater in the nicotine [Chisquare⫽6.14; P⬍0.05] and nicotine–AlloP [Chi-square⫽ 30.75; P⬍0.001] groups that belonged to alcohol treatment compared with controls. The percentage of control–saline rats that did not reach the criterion (8.3%) is taken as a model to make comparisons with the Pearson’s Chi-

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Fig. 1. Learning acquisition criterion (time to perform 10 responses minus the time to perform the first response). Groups of injection abbreviated: saline (saline–saline), PregS (saline–PregS), AlloP (saline–AlloP), nicotine (nicotine–saline). Post hoc Newman-Keuls: * P⬍0.05: (ethanol; nicotine)⬎(ethanol; saline), (control; AlloP, nicotine–AlloP). ** P⬍0.01: (ethanol; nicotine)⬎(ethanol; PregS, AlloP, nicotine–PregS), (control; PregS, nicotine, nicotine–PregS). *** P⬍0.001: (ethanol; nicotine–AlloP)⬎(ethanol; saline, PregS, AlloP, nicotine–PregS), (control; saline, PregS, AlloP, nicotine–PregS). Data are presented as mean⫾S.E.M. Final sample sizes for each group: saline–saline (control n⫽12; ethanol n⫽9); saline–PregS (control n⫽12; ethanol n⫽10); saline–AlloP (control n⫽11; ethanol n⫽10); nicotine–saline (control n⫽10; ethanol n⫽10); nicotine–PregS (control n⫽14; ethanol n⫽8); nicotine–AlloP (control n⫽7; ethanol n⫽11).

square test. With respect to the CR first session, all the animals reached the criterion. See Fig. 2 for the percentage of animals reaching learning criterion in the FS session. In the analysis of the time to finish the FS session, results showed that the main effect of Treatment [F(1,112)⫽ 11.89; P⬍0.001] and Nicotine [F(1,112)⫽5.08; P⬍0.05] was significant. The interaction of Treatment⫻Nicotine⫻ Neurosteroids was also significant [F(2,112)⫽3.95; P⬍ 0.05]. Individual comparisons (Newman-Keuls post hoc test) showed that the co-administration of nicotine and AlloP in the ethanol group increased the time to finish the shaping session compared with the rest of the groups (P⬍0.01). There were no significant differences between nicotine–AlloP and nicotine–saline in the ethanol group. Fig. 3 illustrates the means of the experimental groups and the rest of post hoc comparisons (Newman-Keuls). CR. No significant main effects in the response rate between groups of Treatment [F(1,112)⫽3.36; P⬎0.05], Nicotine factor [F(1,112)⫽0.21; P⬎0.05], Neurosteroids factor [F(2,112)⫽0.97; P⬎0.05] or their interaction [Treatment⫻ Nicotine⫻Neurosteroids: F(2,112)⫽0.26; P⬎0.05] were observed. Global mean of response rate increased from 4.04⫾0.15 (first session) to 4.9⫾0.17 (fourth session) [F(1,112)⫽30.49; P⬍0.001].

Extinction. The average rate of responding of the ethanol group was lower (2.61⫾0.17) than the response rate of the control group (3.06⫾0.17), although the main effect of Treatment did not reach statistical significance [F(1,112)⫽3.38; P⬍0.07]. In addition, no significant main effects of Nicotine [F(1,112)⫽2; P⬎0.05], Neurosteroids [F(2,112)⫽1.44; P⬎0.05] or their interaction [Nicotine⫻ Neurosteroids: F(2,112)⫽0.29; P⬎0.05] were seen. In the time of responding pause, significant differences were obtained between Treatment groups [F(1,112)⫽13.75; P⬍ 0.001]. Alcohol-drinking rats rested more time in pause (without responding) (14.04⫾0.79 min) compared with control rats (9.99⫾0.75 min). No significant effects were found in the nicotine [F(1,112)⫽2.44; P⬎0.05] or neurosteroids administration [F(2,112)⫽1.57; P⬎0.05] or in their interaction [Nicotine⫻Neurosteroids: F(2,112)⫽0.004; P⬎ 0.05]. The analysis of the latency of the first responding pause revealed, also, a significant main effect of the Treatment factor [F(1,112)⫽9.37; P⬍0.01]. Ethanol group presented a significantly lower latency of the first responding pause (10.33⫾0.89 min) compared with the control group (14.12⫾0.85 min). No significant differences were found in the Nicotine [F(1,112)⫽3.04; P⬎0.05] and Neurosteroids factors [F(2,112)⫽1.11; P⬎0.05] or in their interaction [Nicotine⫻Neurosteroids: F(2,112)⫽0.45; P⬎0.05]. Fur-

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Fig. 2. Percentage of animals reaching learning criterion in the FS session. Groups of injection abbreviated: saline (saline–saline), PregS (saline–PregS), AlloP (saline–AlloP), nicotine (nicotine–saline). Chi-square test: * P⬍0.05: (ethanol; nicotine)⬍(control; saline). *** P⬍0.001: (ethanol; nicotine–AlloP)⬍(control; saline). Data are presented in percentages. Final sample sizes for each group: saline–saline (control n⫽12; ethanol n⫽9); saline–PregS (control n⫽12; ethanol n⫽10); saline–AlloP (control n⫽11; ethanol n⫽10); nicotine–saline (control n⫽10; ethanol n⫽10); nicotine– PregS (control n⫽14; ethanol n⫽8); nicotine–AlloP (control n⫽7; ethanol n⫽11).

thermore, results revealed a significant main effect of the Treatment factor in the analysis of the index of the response rate weighed in function of the time the animal spends on responding [F(1,112)⫽8.89; P⬍0.01]. The ethanol group performed a lower EXT score than controls. In addition, an interaction between Treatment and Nicotine factors was seen [F(2,112)⫽4.15; P⬍0.05]. Post hoc analysis showed that ethanol groups injected with nicotine performed a better EXT than the ethanol groups not injected with nicotine (P⬍0.05), better than control groups not injected with nicotine (P⬍0.01) and better than control groups injected with nicotine (P⬍0.01). No significant differences were found in the Nicotine [F(1,112)⫽1.89; P⬎0.05] and in the Neurosteroids [F(2,112)⫽1.93; P⬎0.05] factors or in their interaction [F(2,112)⫽0.16; P⬎0.05]. See Fig. 4 for the means of the experimental groups.

DISCUSSION The principal findings in this study suggest that the neurosteroid PregS neutralizes the deterioration of learning acquisition induced by the intrahippocampal administration of nicotine in alcohol-drinking rats, and that the hippocampus could be an important target to explain the effects on learning produced by the co-administration of alcohol and nicotine. Ethanol drinking remained stable throughout the limited access phase, increasing during the food deprivation

period. In this way, it has been recognized that deprivation of food enhances drug consumption independent of its caloric value (Carroll and Stotz, 1983; Meisch, 1987). The ethanol doses registered in this experiment were similar to those found in previous studies conducted in our laboratory (Pallares et al., 1992, 1997, 2001; Nadal et al., 1996a; Darbra et al., 2002, 2004). Previous studies carried out in our laboratory described average BACs of 1.051⫾0.11 g ethanol/l blood and an ingested dose of 3.55⫾0.18 g ethanol/kg body weight after nine weeks of food deprivation (Robles et al., 2003), or 0.25 g ethanol/l blood, after a consumption of 1.26 g ethanol/kg body weight (Pallares et al., 1997). This last result was obtained after 15 days of inducing alcohol consumption and a condition of 1 h of simultaneous access to alcohol and free food, therefore, without total deprivation of food and with the presence of food in the gastric cavity (Pallares et al., 1997). However, blood alcohol levels in rodents are influenced by several factors including strain, age, gender, administration route, concentration of alcohol solution, length of treatment, and dose (Bielawski and Abel, 2002). In relation to learning, the learning acquisition impairment induced by intrahippocampal nicotine in alcoholdrinking rats, that was reversed by PregS administration, was increased (not significantly) by administering AlloP: from 1671.94⫾1323.2 s in alcohol–nicotine group to 2137⫾1507.98 s in alcohol–nicotine–AlloP group. This im-

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Fig. 3. Time to finish the FS session. Groups of injection abbreviated: saline (saline–saline), PregS (saline–PregS), AlloP (saline–AlloP), nicotine (nicotine–saline). Post hoc Newman-Keuls: ** P⬍0.01: (ethanol; nicotine–AlloP)⬎(ethanol; saline, PregS, AlloP, nicotine–PregS), (control; saline, nicotine, nicotine–PregS). *** P⬍0.001: (ethanol; nicotine–AlloP)⬎(control; PregS, AlloP, nicotine–AlloP). Data are presented as mean⫾S.E.M. Final sample sizes for each group: saline–saline (control n⫽12; ethanol n⫽9); saline–PregS (control n⫽12; ethanol n⫽10); saline–AlloP (control n⫽11; ethanol n⫽10); nicotine–saline (control n⫽10; ethanol n⫽10); nicotine–PregS (control n⫽14; ethanol n⫽8); nicotine–AlloP (control n⫽7; ethanol n⫽11).

pairment in learning acquisition cannot be attributed to the positive modulating actions of alcohol on GABAA receptors (Morrow et al., 1990; Morrisett and Swartzwelder, 1993), because it appears after the co-administration of nicotine and ethanol, but not after ethanol alone. The documented agonistic effects of alcohol on the neuronal nicotinic acetylcholine receptors (nnAChRs) (Collins et al., 1990; Butt et al., 2004) suggest that the additive actions of nicotine and alcohol on the hippocampal nnAChRs could provoke an excessive activation of these receptors that could be based on learning impairments. Moreover, this fact could explain why nicotine alone did not affect learning acquisition. On the other hand, neither PregS nor AlloP had significant effects per se on the rats acquiring the leverpress response. This fact could be due to different factors such as: a possible floor effect (low levels of difficulty in the lever-press response task); the kind of learning paradigm (spatial learning could be more sensitive to the effects of neurosteroids); or the administration route (systemic administration could be more effective, as other brain structures could be involved in the effects of neurosteroids on learning). The opposite effects of PregS and AlloP in relation to neutralizing the impairment of learning acquisition due to the co-administration of alcohol and nicotine could be related to their different profiles in the GABAA receptor. It is important to note that not only cholinergic inputs from the

medial septum (Lewis and Shute, 1967), but also gabaergic interneurons (Toth et al., 1997) and gabaergic inputs from the medial septum (Kohler et al., 1984; Freund and Antal, 1988) modulate pyramidal neurons in the hippocampus. It seems plausible therefore that the reversal of the nicotine-impaired learning acquisition of the free response caused by PregS can be attributed to the inhibition of GABAA receptor activity coupled with potentiation of NMDA receptor activity, rather than any direct interaction with nicotinic receptor. In this way, PregS have been reported to block retention deficits induced by the competitive NMDA receptor antagonists CPP in passive avoidance memory task (Mathis et al., 1994), D-AP5 in Y-maze active avoidance and lever-press tasks (Mathis et al., 1996). It also reversed the learning deficits induced by the noncompetitive NMDA receptor antagonist MK801 in passive avoidance memory task (Cheney et al., 1995). Otherwise, we cannot rule out the possible role of nnAChRs, as neurosteroids are potential endogenous regulators of the activity of different nnAChRs (Pereira et al., 2002) acting as allosteric inhibitors of brain nicotinic receptors (Bullock et al., 1997). Recent studies have shown that PregS non-competitively and reversibly inhibits the nicotinic receptoroperated ion channels through a nongenomic mechanism in cultured bovine adrenal chromaffin cells (Kudo et al., 2002).

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Fig. 4. Extinction index (response rate weighed in function of the time the animal spends on responding). Groups of injection abbreviated: saline (saline–saline), PregS (saline–PregS), AlloP (saline–AlloP), nicotine (nicotine–saline). Post hoc Newman-Keuls: # P⬍0.05: (ethanol; nicotine, nicotine–PregS, and nicotine–AlloP)⬍(ethanol; saline, PregS, AlloP). ** P⬍0.01: (ethanol; nicotine, nicotine–PregS, Nicotine–AlloP)⬍(control; saline, PregS, AlloP, nicotine, nicotine–PregS, Nicotine–AlloP). Data are presented as mean⫾S.E.M. Final sample sizes for each group: saline–saline (control n⫽12; ethanol n⫽9); saline–PregS (control n⫽12; ethanol n⫽10); saline–AlloP (control n⫽11; ethanol n⫽10); nicotine–saline (control n⫽10; ethanol n⫽10); nicotine–PregS (control n⫽14; ethanol n⫽8); nicotine–AlloP (control n⫽7; ethanol n⫽11).

EXT was facilitated by alcohol consumption, with a decrease in the response rate and in the latency of the first pause, and an increase in the total time of the pause. The extinction process of a previously-reinforced operant behavior is particularly long and never reaches a zeroresponse level (Tarpy, 1997), so the animals alternate periods of responding with response pauses. At the beginning, there is normally a continuous pattern of responding without pauses, but as the extinction process continues, the pause periods increase and then dominate when the response level is near zero. Thus, analyzing the pause patterns in the extinction processes is very important (Tarpy, 1997). The present results are in accordance with previous work in our laboratory that has shown that EXT is facilitated by chronic ethanol consumption (Pallares et al., 1992, 2001). Indeed, it has been proposed that anxiolytic compounds that potentate GABA, especially benzodiazepines, facilitate the extinction of positively reinforced operant behavior (for a review see Leslie et al., 2004). It seems paradoxical that positive GABAA modulators that generally impair learning and memory (Mtchedlishvili and Kapur, 2003; Maren and Holt, 2004) could facilitate EXT. However, it has been proposed that a range of anxietyreducing drugs may facilitate the EXT reducing the aversive effects of the non-reinforcement or the anxiety asso-

ciated with these effects (for a review see Leslie et al., 2004). In order to make an in depth study of performance during EXT we computed an EXT index that takes into account the combination of the two main EXT variables: response rate and the time the animal spends on responding. Results showed that intrahippocampal nicotine facilitated EXT in alcohol-drinking rats. There is considerable evidence of cross-tolerance between nicotine and ethanol (Collins et al., 1988; de Fiebre and Collins, 1993; Luo et al., 1994). In addition, nicotine increases GABA release in the hippocampus (Alkondon et al., 1997; Lu et al., 1998), and excites septohippocampal GABAergic neurons (Wu et al., 2003). Nicotine has bimodal effects on anxiety, with low doses having anxiolytic effects (File et al., 1998; Genn et al., 2003). Moreover, the combination of alcohol and nicotine can elicit a reduction in anxiety in ethanol-resistant short-sleep (SS) mouse lines (Cao et al., 1993). Thus, the extinction facilitation induced by the co-administration of alcohol and nicotine could be related to its anxiolytic-like profile. AlloP had no effects on EXT. It is logical to expect that AlloP, an allosteric positive modulator of the GABAA receptor that has anxiolytic properties (Zimmerberg et al., 1994), facilitates EXT in the same way as anxiolytics that modulate GABAA receptors do. However, systemic administration would be necessary in order to rule out EXT

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facilitation induced by AlloP. In previous studies it has been shown that voluntary ethanol consumption and intrahippocampal AlloP administration in a dose of AlloP (0.2 ␮g) have anxiolytic-like effects on the initial stress that takes place in the confrontation behavior to new situations (neophobia) measured in the open field test (Martín-García et al., 2004; Martin-Garcia and Pallares, 2005). Otherwise, the anxiety profile measured in extinction seems to have a phenomenological relationship with the conflict tests, where frustration is generated by punishment or the absence of reinforcement. AlloP has shown to be effective in reversing this kind of profile, when it is administered into the amygdala and at a dose of 8 ␮g (Akwa et al., 1999), thus 40-fold higher. PregS, which reversed the deterioration of learning induced by the combination of ethanol and nicotine, did not affect this interaction in extinction. This suggests that different neural substrates may be involved in these two behaviors.

CONCLUSION In conclusion, PregS neutralized the deterioration of the lever-press response acquisition induced by the interaction between intrahippocampal nicotine administration and chronic alcohol consumption, whereas it was not affected by AlloP administration. Furthermore, alcohol consumption facilitated EXT in the same way as anxiolytics that act as positive modulators of the GABAA receptors do, possibly reducing the anxiety or aversion related to the non-reinforcement situation. In addition, this effect could be increased by the intrahippocampal administration of nicotine. The hippocampal nnAChRs seem to be an important target to explain these effects. Acknowledgments—This work was supported by a pre-doctoral fellowship from the “Generalitat de Catalunya” and a grant from the “Dirección General de Investigación” (BSO2001-1899), Ministerio de Ciencia y Tecnologia, Spain.

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(Accepted 15 August 2005) (Available online 3 October 2005)