Effect of intermittent induced aeration on nitrogen removal and denitrifying-bacterial community structure in Cork and gravel vertical flow pilot-scale treatment wetlands.

In this work, we have evaluated the impact of intermittent induced aeration in total nitrogen (TN), ammonia (NH4-N) and nitrate-nitrogen (NO3-N) removal in four pilot-scale vertical flow constructed wetlands (VFCW) (two aerated two non-aerated) using cork by-product or gravel as the filter material and planted with Phragmites australis. Both aerated and non-aerated systems achieved high COD and BOD5 elimination rates (≥ 90%) at the end of the 5-month test period. However, the aerated systems presented maximal COD and BOD5 removal from the third month of operation onwards since air supply favored the oxidative bioprocesses occurring within the wetlands. Cork and gravel aerated VFCW also proved to be more efficient (p < 0.05) in NO3-N removal than the non-aerated systems and this upgraded performance was correlated with a significant higher relative abundance of the nirS gene. The aerated systems also showed a slightly improved NH4-N removal. Noticeably, cork VFCW showed higher TN removal mean values (∼35%) than gravel wetlands (27-28%) regardless aeration. Moreover, cork VFCW showed higher relative abundance of the nosZ gene. Our results demonstrated a better nitrogen elimination for the aerated cork pilot-scale VFCW, and this behavior was correlated with a higher abundance of both nirS and nosZ, two of the key functional genes involved in nitrogen metabolism.

Effects of neonatal allopregnanolone manipulations and early maternal separation on adult alcohol intake and monoamine levels in ventral striatum of male rats.

Changes in endogenous neonatal levels of the neurosteroid allopregnanolone (AlloP) as well as a single 24h period of early maternal separation (EMS) on postnatal day (PND) 9 affect the development of the central nervous system (CNS), causing adolescent/adult alterations including systems and behavioural traits that could be related to vulnerability to drug abuse. In rats, some behavioural alterations caused by EMS can be neutralised by previous administration of AlloP. Thus, the aim of the present work is to analyse if manipulations of neonatal AlloP could increase adult alcohol consumption, and if EMS could change these effects. We administered AlloP or finasteride, a 5α-reductase inhibitor, from PND5 to PND9, followed by 24h of EMS at PND9. At PND70 we measured alcohol consumption using a two-bottle free-choice model (ethanol 10% (v/v)+glucose 3% (w/v), and glucose 3% (w/v)) for 15days. Ventral striatum samples were obtained to determine monoamine levels. Results revealed that neonatal finasteride increased both ethanol and glucose consumption, and AlloP increased alcohol intake compared with neonatal vehicle-injected animals. The differences between neonatal groups in alcohol consumption were not found in EMS animals. In accordance, both finasteride and AlloP animals that did not suffer EMS showed lower levels of dopamine and serotonin in ventral striatum. Taken together, these results reveal that neonatal neurosteroids alterations affect alcohol intake; an effect which can be modified by subsequent EMS. Thus, these data corroborate the importance of the relationship between neonatal neurosteroids and neonatal stress for the correct CNS development.

Neonatal finasteride administration alters hippocampal α4 and δ GABAAR subunits expression and behavioural responses to progesterone in adult rats.

Allopregnanolone is a neurosteroid that has been reported to fluctuate during early developmental stages. Previous experiments reported the importance of neonatal endogenous allopregnanolone levels for the maturation of the central nervous system and particularly for the hippocampus. Changes in neonatal allopregnanolone levels have been related to altered adult behaviour and with psychopathological susceptibility, including anxiety disorders, schizophrenia and drug abuse. However, the mechanism underlying these changes remains to be elucidated. In the present study we assessed changes in hippocampal expression of α4 and δ GABAA receptor (GABAAR) subunits as a consequence of neonatal finasteride (a 5-α reductase inhibitor) administration during early development (PD6 to PD15) in male rats. We observed that the treatment altered the temporal window of the natural peak in the expression of these subunits during development. Additionally, the level of these subunits were higher than in non-handled and control animals in the adult hippocampus. We observed that in adulthood, neonatal finasteride-treated animals presented an anxiogenic-like profile in response to progesterone administration which was absent in the rest of the groups. In conclusion, these results corroborate the relevance of neonatal maintenance of neurosteroid levels for behavioural anxiety responses in the adult, and point to some of the mechanisms involved in this alterations.

Interaction between neonatal allopregnanolone administration and early maternal separation: effects on adolescent and adult behaviors in male rat.

Endogenous neurosteroid level fluctuations are related to several emotional and behavioral alterations. Neurosteroids also have important roles during neurodevelopment, with there being a relationship between modification of their levels in neurodevelopmental periods and behavioral alterations in adolescence and adulthood. Early maternal separation (EMS) is a stressful event that also alters neurodevelopment and adolescent and adult behaviors. The aim of the present study is to analyze the interaction between the effects of the neonatal alteration of allopregnanolone (AlloP), neurosteroid that increase its levels after acute stress presentation, and EMS on adolescent exploration and adult anxiety and sensorimotor gating in male rats. AlloP (10 mg/kg s.c.) was administrated between postnatal day 5 (PN5) and PN9, and a single 24-hour period of EMS was carried out on PN9. Exploration was analyzed at PN40 and PN60. At adult age (PN85), anxiety was tested by means of the elevated plus-maze test (EPM), and sensorimotor gating by means of prepulse inhibition test (PPI). PPI deterioration has been considered as a reliable indicator of diseases such as schizophrenia. Results showed that the previous neonatal AlloP administration neutralized the effects of EMS in the adolescent exploration (increase of traveled distance and decrease of head-dips). In adult age, an anxiolytic-like profile was observed as a consequence of EMS. Finally, EMS and neonatal AlloP disrupted PPI. Taken together, these data show the important role that physiological neonatal AlloP levels and stressful events play in neural development, adult behavior and vulnerability to neurodevelopmental disorders such as schizophrenia.

Effects of early postnatal allopregnanolone administration on elevated plus maze anxiety scores in adult male Wistar rats.

BACKGROUND/AIMS: Recent findings suggest that neurosteroids are involved in brain development. The present study focused on the long-term effects of developmentally altered allopregnanolone (AlloP) levels on anxiety-like behavior in adulthood. METHOD: We administered AlloP (10 mg/kg) to rat pups once a day from the 5th to the 9th day after birth. A dose-response study on midazolam in the elevated plus maze test was carried out in adulthood (experiment 1) in order to screen GABAA-benzodiazepine function alterations. Given that the anxiety-like responses were not affected by AlloP, we doubled the initial AlloP dose (experiment 2). One group of pups was left undisturbed with their dams in order to control the effects of daily handling. Only males were behaviorally tested. RESULTS: Neonatal AlloP administration (10 mg/kg) did not alter the behavioral response to midazolam in adulthood at the doses tested. Neonatal AlloP administration at the higher dose (20 mg/kg) induced an anxiolytic-like profile in adulthood (increased entries into and time spent in the open arms), without affecting motor activity. The behavioral effects of neonatal AlloP administration were both selective and independent of daily handling. CONCLUSION: Alterations in AlloP levels during maturation could partly explain the interindividual differences shown by adult subjects in response to environmental stress.

Early postnatal neuroactive steroid manipulation differentially affects recognition memory and passive avoidance performance in male rats.

Early postnatal neuroactive steroids (NAS) play a significant role in the neurodevelopment. Their alteration can modify adult behavior, such as anxiety or learning. For this reason, we set out to observe if neonatal NAS levels alteration affects two types of learning implying low or high levels of emotional content, such as recognition memory and aversive learning respectively. Thus, we tested allopregnanolone or finasteride administered from postnatal days 5-9. In adulthood, recognition memory was assessed using the object recognition test, as well as aversive learning throughout the passive avoidance test (PA). Because of the important emotional component of PA, which can be influencing learning, we evaluated anxiety-like behavior by means of the open field test (OF). The results indicated that those animals administered with finasteride showed higher recognition levels of a familiar object. On the other hand, they showed an impairment in a stressful learning, such as PA. However, no effects of finasteride were observed on anxiety-like behavior in OF, despite it has been reported that neonatal finasteride treatment can promote an anxiety-like profile in the elevated plus-maze test in adulthood. Regarding neonatal allopregnanolone, animals showed higher levels in OF exploration only when they were already familiar with the apparatus. Furthermore, neonatal allopregnanolone did not affect recognition memory or aversive learning. In conclusion, the neonatal NAS manipulation by means of finasteride differently affected two types of learning implying distinct stress levels. Altogether, the results show the importance of the emotional content to explain the effects of neonatal NAS manipulation on learning.

Early postnatal allopregnanolone levels alteration and adult behavioral disruption in rats: Implication for drug abuse.

Several studies have highlighted the role that early postnatal levels of allopregnanolone play in the development of the CNS and adult behavior. Changes in allopregnanolone levels related to stress have been observed during early postnatal periods, and perinatal stress has been linked to neuropsychiatric disorders. The alteration of early postnatal allopregnanolone levels in the first weeks of life has been proven to affect adult behaviors, such as anxiety-related behaviors and the processing of sensory inputs. This review focuses on the first studies about the possible relationship between the early postnatal allopregnanolone levels and the vulnerability to abuse of drugs such as alcohol in adulthood, given that (1) changes in neonatal allopregnanolone levels affect novelty exploration and novelty seeking has been linked to vulnerability to drug abuse; (2) early postnatal administration of progesterone, the main allopregnanolone precursor, affects the maturation of dopaminergic meso-striatal systems, which have been related to novelty seeking and drug abuse; and (3) alcohol consumption increases plasma and brain allopregnanolone levels in animals and humans. Manipulating neonatal allopregnanolone by administering finasteride, an inhibitor of the 5α-reductase enzyme that participates in allopregnanolone synthesis, increases alcohol consumption and decreases the locomotor stimulant effects of low alcohol doses. At a molecular level, finasteride decreases dopamine and serotonin in ventral striatum and dopamine release in nucleus accumbens. Preliminary results suggest that serotonin 5HT3 receptors could also be affected. Although an in-depth study is necessary, evidence suggests that there is a relation between early postnatal allopregnanolone and vulnerability to drug use/abuse.

Early post-natal neuroactive steroid manipulation modulates ondansetron effects on initial periods of alcohol consumption in rats.

Neuroactive steroids (NS) such as allopregnanolone are crucial for brain development and adult behaviour. Early post-natal alterations of NS by administering finasteride induce a decrease in the sensitivity to stimulant effects of low alcohol doses, an increase in alcohol consumption, and a decrease in ventrostriatal dopamine and serotonin levels. The aim of the present study is to observe if the effects of the 5HT3 receptor antagonist ondansetron on initial alcohol consumption are modulated by post-natal NS manipulation. For this purpose, allopregnanolone, finasteride, or vehicle was injected from day 5 to 9. In adulthood, a novel object preference test was carried out in order to detect a possible novelty-seeking pattern in our animals, which has been related to vulnerability to drug abuse. The subjects then had access to two bottles (alcohol or control solutions) one hour daily for two consecutive weeks. Ondansetron (0.01 mg/kg, 0.1 mg/kg or vehicle) was administered before the hour of consumption in the initial phase (days 1, 2, 3) of the procedure, and after prolonged alcohol intake (days 11, 12, 13). Results indicated that finasteride animals showed a higher preference to explore the new object, as well as a higher alcohol consumption than the rest of the groups. Moreover, 0.1 mg/kg of ondansetron decreased alcohol consumption, but only in the post-natal finasteride group, suggesting a possible increase in 5HT3 receptor sensitivity in these animals. In conclusion, NS manipulation in crucial stages of development, such as early post-natal periods, seems to play an important role on the effects of ondansetron on alcohol intake and in the vulnerability to develop drug use or abuse.

Intrahippocampal allopregnanolone decreases voluntary chronic alcohol consumption in non-selected rats.

We have recently shown that 0.2 microg of the neurosteroid allopregnanolone (AlloP) administered to the hippocampus induced an anxiolytic-like profile and also reduced alcohol withdrawal symptoms in voluntary and chronic alcohol-drinking rats. The aim of the present work was to study whether the administration of this dose of AlloP could affect alcohol consumption in non-selected rats that have been voluntarily ingesting high doses of alcohol for long periods of time in a limited access procedure. We used a free-choice drinking procedure that involved providing the rats with an alcoholic solution (10% ethanol) at an early age. Alcohol and control rats were assigned randomly to three groups that received an intrahippocampal (dorsal CA1) injection before the period of alcohol consumption after a long history of chronic alcohol intake. The injection groups were AlloP (0.2 microg, 1.26 microM), pregnenolone sulfate (PregS) (5 ng, 24 microM) or vehicle. Blood alcohol concentrations (BAC) were assessed before testing the effects of injections on alcohol consumption. Although AlloP did not eliminate alcohol ingestion, it significantly decreased alcohol consumption. The intrahippocampal administration of PregS, at the dose tested, did not effectively modify alcohol consumption levels. These results indicate that the positive modulation of hippocampal GABA(A) receptors induced by neurosteroids can be an important neurobiological target for reducing chronic alcohol consumption.

Effects of neonatal and adolescent neuroactive steroid manipulation on locomotor activity induced by ethanol in male wistar rats.

Neonatal neuroactive steroids levels are crucial for brain development. Alterations of neonatal neuroactive steroids levels induce anxiolytic-like effects and improve exploration in novel environments in adulthood. These behavioural traits, i.e. sensation/novelty seeking, anxiety or impulsivity, are associated with vulnerability to drug use and abuse. Adolescence is also recognized as a particularly critical developmental phase to contribute to vulnerable phenotype. However, the influence of neuroactive steroids during development in the vulnerability to drug addiction has been poorly studied. The aim of the present experiment is to study the effect of early neonatal and adolescent manipulations of neuroactive steroids on the sensitivity to the stimulant effects of ethanol in adult male rats. Therefore, allopregnanolone or finasteride, an allopregnanolone synthesis inhibitor, were injected from postnatal day 5-9. In early adolescence, half of the subjects were injected with progesterone, the main allopregnanolone precursor, and the elevated plus-maze anxiety test was performed. Results indicated that early adolescent progesterone induced anxiolytic-like effects (increase in the percentage of entries and time in open arms). Neonatal finasteride administration decreased locomotor activity induced by ethanol in adolescent vehicle subjects. Interestingly, differences induced by neonatal treatments were not present in the animals that received progesterone in the early adolescence. In conclusion, neuroactive steroid manipulations in crucial stages of development could be playing an important role in behavioural effects of alcohol such as the sensitivity to locomotor stimulation.

Individual differences in cognitive aging: implication of pregnenolone sulfate.

In humans and animals, individual differences in aging of cognitive functions are classically reported. Some old individuals exhibit performances similar to those of young subjects while others are severely impaired. In senescent animals, we have previously demonstrated a significant correlation between the cognitive performance and the cerebral concentration of a neurosteroid, the pregnenolone sulfate (PREG-S). Neurotransmitter systems modulated by this neurosteroid were unknown until our recent report of an enhancement of acetylcholine (ACh) release in basolateral amygdala, cortex and hippocampus induced by intracerebroventricular (i.c.v.) or intracerebral administrations of PREG-S. Central ACh neurotransmission is known to be involved in the regulation of memory processes and is affected in normal aging and severely altered in human neurodegenerative pathologies like Alzheimer's disease. In the central nervous system, ACh neurotransmission is also involved in the modulation of sleep-wakefulness cycle, and particularly the paradoxical sleep (PS). Relationships between paradoxical sleep and memory are documented in the literature in old animals in which the spatial memory performance positively correlates with the basal amounts of paradoxical sleep. PREG-S infused at the level of ACh cell bodies (nucleus basalis magnocellularis, NBM, or pedunculopontine nucleus, PPT) increases paradoxical sleep in young animals.Finally, aging related cognitive dysfunctions, particularly those observed in Alzheimer's disease, have also been related to alterations of mechanisms underlying cerebral plasticity. Amongst these mechanisms, neurogenesis has been extensively studied recently. Our data demonstrate that PREG-S central infusions dramatically increase neurogenesis, this effect could be related to the negative modulator properties of this steroid at the GABA(A) receptor level. Taken together these data suggest that neurosteroids can influence cognitive processes, particularly in senescent subjects, through a modulation of ACh neurotransmission associated with paradoxical sleep modifications; furthermore, our recent data suggest a critical role for neurosteroids in the modulation of cerebral plasticity, mainly on hippocampal neurogenesis.

Neonatal finasteride administration decreases dopamine release in nucleus accumbens after alcohol and food presentation in adult male rats.

Endogenous levels of the neurosteroid (NS) allopregnanolone (AlloP) during neonatal stages are crucial for the correct development of the central nervous system (CNS). In a recent work we reported that the neonatal administration of AlloP or finasteride (Finas), an inhibitor of the enzyme 5α-reductase needed for AlloP synthesis, altered the voluntary consumption of ethanol and the ventrostriatal dopamine (DA) levels in adulthood, suggesting that neonatal NS manipulations can increase alcohol abuse vulnerability in adulthood. Moreover, other authors have associated neonatal NS alterations with diverse dopaminergic (DAergic) alterations. Thus, the aim of the present work is to analyse if manipulations of neonatal AlloP alter the DAergic response in the nucleus accumbens (NAcc) during alcohol intake in rats. We administered AlloP or Finas from postnatal day (PND) 5 to PND9. At PND98, we measured alcohol consumption using a two-bottle free-choice model (ethanol 10% (v/v)+glucose 3% (w/v), and glucose 3% (w/v)) for 12 days. On the last day of consumption, we measured the DA and 3,4-dihydroxyphenylacetic acid (DOPAC) release in NAcc in response to ethanol intake. The samples were obtained by means of in vivo microdialysis in freely moving rats, and DA and DOPAC levels were determined by means of high-performance liquid chromatography analysis (HPLC). The results revealed that neonatal Finas increased ethanol consumption in some days of the consumption phase, and decreased the DA release in the NAcc in response to solutions (ethanol+glucose) and food presentation. Taken together, these results suggest that neonatal NS alterations can affect alcohol rewarding properties.

Intrahippocampal nicotine and neurosteroids effects on the anxiety-like behaviour in voluntary and chronic alcohol-drinking rats.

Considerable evidence suggests that the anxiolytic effects of ethanol may be one of the factors that promotes alcohol consumption. The present study aimed to characterize the effects of intrahippocampal administrations of nicotine and the two neurosteroids pregnenolone sulphate (PregS) and allopregnanolone (AlloP) on anxiety-like behaviours in alcohol-drinking rats. A long-lasting free-choice drinking procedure with an early availability (from weaning) of an alcoholic solution (10% (v/v) ethanol, 3% (w/v) glucose in distilled water) was used. After 80 days of consumption, alcohol-drinking and control rats were deprived of food and assigned at random to six groups. After 100 days of consumption, each group received two consecutive intrahippocampal (dorsal CA1) injections. First injection: nicotine (4.6 microg, 20 mM) or saline; second injection: PregS (5 ng, 24 microM), AlloP (0.2 microg, 1.26 microM) or saline. Following the injections, novelty-directed activity (open field, OF), and motor coordination (80 degrees inclined screen) were tested. Blood alcohol concentrations (BACs) were assessed. Anxiolytic-like effects of voluntary ethanol consumption and intrahippocampal AlloP administration were observed. Alcohol intake increased the novelty-induced ambulation and exploration of central areas, and decreased defecation. The high exploration levels induced by AlloP decreased significantly over sessions, indicating a rapid habituation to the environmental conditions. Motor coordination was deteriorated by ethanol consumption. These results demonstrate the effects of chronic alcohol intake and neurosteroid administration on anxiety-related behaviours, and suggest an important role of the hippocampal GABA(A) receptor in these behaviours.

The intrahippocampal administration of the neurosteroid allopregnanolone blocks the audiogenic seizures induced by nicotine.

Allopregnanolone (AlloP), GABA(A) positive modulator, has efficacy as anticonvulsant. In contrast, nicotine and pregnenolone sulfate (PregS) act as potent convulsants. The present study aims to evaluate whether a promnesic dose of PregS and/or an anxiolytic dose of AlloP administered in the hippocampus can affect the audiogenic seizures induced by nicotine administration. Rats were assigned at random to six groups that received two consecutive intrahippocampal (dorsal CA1) injections once a week during three consecutive weeks. First injection: nicotine (4.6 microg, 20 mM) or saline, second injection: PregS (5 ng, 24 microM), AlloP (0.2 microg, 1.26 microM) or saline. After the last injections, locomotor activity and audiogenic seizures were tested. AlloP decreased the horizontal and vertical activity, suggesting sedative effects. Nicotine induced behavioral convulsions and AlloP acted as an anticonvulsant. AlloP reversed the seizures induced by nicotine and decreased the audiogenic convulsions in comparison with the controls. PregS also reversed the nicotine-induced audiogenic seizures in the nicotine group but not in the control group. These results suggest that anticonvulsive effects of AlloP and PregS are mediated by different action mechanisms such as GABA(A) positive modulation, or negative modulatory action on neural nicotinic acetylcholine receptors. Even though several brain structures could be involved, these results highlight the important role played by hippocampal cholinergic and GABAergic activities, as well as neurosteroids, especially AlloP, in preventing convulsive behavior.

The neurosteroid pregnenolone sulfate infused into the medial septum nucleus increases hippocampal acetylcholine and spatial memory in rats.

The effects of an infusion of the neurosteroid pregnenolone sulfate into the medial septum on acetylcholine release in the hippocampus and on spatial memory were evaluated in two experiments. Results show that pregnenolone sulfate enhanced acetylcholine release by more than 50% of baseline and improved recognition memory of a familiar environment. Therefore, our results suggest that the septo-hippocampal pathway could be involved in the promnesic properties of this neurosteroid.

Tolerance and sensitization to the hypnotic effects of alcohol induced by chronic voluntary alcohol intake in rats.

The effect of a chronic alcohol exposure on the development of tolerance to the depressive effects of alcohol were examined in male Wistar rats that voluntary self-administered alcohol. A free-choice drinking procedure based on the limited access paradigm and the addition of glucose that implies an early availability of the alcoholic solution was used (Alcoholism Primary Praecox procedure). Alcohol induced sleep time (3.5 g alcohol per kg i.p.) was measured at 90 days (after 2 months of alcohol consumption) or at 60 + 90 days old (1 or 2 months of alcohol consumption). The psychomotor performance was also evaluated by means of an 80 degrees inclined screen test. Subjects that had been tested for the hypnotic effects at both 60 and 90 days showed a higher intake of alcoholic solution than the animals only tested at 90 days. The same consumption increase was observed in the glucose group. No significant differences between groups were observed in the inclined screen test. Tolerance to the hypnotic effects of alcohol was observed at 90 days. On the other hand, no significant differences between alcohol and control groups (glucose or water) were observed in the sleep time at 60 days. In the alcohol-drinking rats tested for two trials (60 and 90 days), sensitization instead of tolerance to the second hypnotic alcohol injection was seen. Tolerance to the hypnotic effects of alcohol observed after chronic voluntary alcohol consumption may provide animal models of alcoholism based on limited access to sweetened alcoholic solutions with construct validity.

Opposite effects of ethanol and ketamine in the elevated plus-maze test in Wistar rats undergoing a chronic oral voluntary consumption procedure.

The anxiolytic effects of ethanol (EtOH) have been involved in the vulnerability to EtOH drinking in humans. However, the role of the anxiolytic effects of EtOH during a chronic ingestion of the drug has not been extensively addressed, either in humans or in animal models. Since it was first shown that EtOH interacts with the N-methyl-d-aspartate (NMDA) receptor, a growing body of evidence demonstrating the involvement of this receptor in a wide range of EtOH effects has been reported. The present study aimed to investigate the ability of a voluntary consumption of EtOH to exert its putative anxiolytic-like activity in non-selected male Wistar rats held under a voluntary chronic oral consumption procedure using the elevated plus-maze (EPM) test. The effects of EtOH were compared with those of the noncompetitive NMDA receptor antagonist ketamine (KET), and with a mixture of both drugs. Rats were provided with 1-h limited access to one of the following sweetened (10% w/v glucose) solutions: (i) control; (ii) EtOH (ethanol, 10% v/v); (iii) KET (ketamine HCl, 0.28 mg/ml); or (iv) mixed (EtOH 10% v/v plus ketamine HCl 0.28 mg/ml) for 35 consecutive days. At the end of this period, and immediately after the last 1-h access to the respective solution, animals were independently tested in either EPM or open field tests. Previously, rats were tested on the inclined screen test during 15 consecutive days. The opposite effects were observed with EtOH and KET consumption in the EPM test, with EtOH decreasing and KET increasing the percentage of time spent in the open arms of the EPM, which was shown to be independent of any locomotor impairment, whereas consumption of the mixed solution did not significantly affect any test. Since the EtOH did not exhibit anxiolytic-like effects after its chronic oral consumption, it might be hypothesized that the anxiolytic activity of the EtOH is not critically involved in the maintenance of a voluntary EtOH consumption in non-selected rats. On the other hand, the lack of effects from mixed solution consumption suggests that EtOH and KET may interact in such a way that their effects are neutralized.

Effects of voluntary alcohol intake on nicotine-induced behavioural sensitisation in rats.

Behavioural sensitisation has been suggested to play a role in the acquisition and maintenance of addictive behaviour. The aim of the present study was to assess nicotine-induced behavioural sensitisation in chronic voluntary alcohol drinking rats. Subjects had free access to alcohol/water or glucose/water solutions since weaning. Rats were pretreated after 2 months of voluntary alcohol drinking. Pretreatment consisted of once-daily intraperitoneal injection of nicotine (0.5 mg/kg) or saline administered for five consecutive days. The nicotine-induced behavioural sensitisation of locomotor activity was tested 3 weeks latter. Horizontal motor activity was monitored for 30 min and expressed as distance travelled (in centimetres). During all the experimental procedure, the animals were maintained under 1-h limited access to alcohol. In glucose-drinking animals, results indicated that nicotine induced locomotor activity sensitization: The locomotor effects of nicotine challenge in the nicotine-pretreated group of rats were significantly enhanced as compared with the saline-pretreated group (Duncan, P<.01). Instead, in the alcohol-drinking animals, no significant differences were observed between the nicotine- and saline-pretreated groups. Thus, chronic alcohol consumption at mild doses prevented the development and/or the long-term expression of the nicotine-induced sensitisation at the doses tested.

Neonatal allopregnanolone or finasteride administration modifies hippocampal K(+) Cl(-) co-transporter expression during early development in male rats.

The maintenance of levels of endogenous neurosteroids (NS) across early postnatal development of the brain, particularly to the hippocampus, is crucial for their maturation. Allopregnanolone (Allop) is a NS that exerts its effect mainly through the modulation of the GABAA receptor (GABAAR). During early development, GABA, acting through GABAAR, that predominantly produces depolarization shifts to hyperpolarization in mature neurons, around the second postnatal week in rats. Several factors contribute to this change including the progressive increase of the neuron-specific K(+)/Cl(-) co-transporter 2 (KCC2) (a chloride exporter) levels. Thus, we aimed to analyze whether a different profile of NS levels during development is critical and can alter this natural progression of KCC2 stages. We administrated sustained Allop (20mg/kg) or Finasteride (5α-reductase inhibitor, 50mg/kg) from the 5th postnatal day (PD5) to PD9 and assessed changes in the hippocampal expression of KCC2 at transcript and protein levels as well as its active phosphorylated state in male rats. Taken together data indicated that manipulation of NS levels during early development influence KCC2 levels and point out the importance of neonatal NS levels for the hippocampal development.

Neonatal neurosteroid levels are determinant in shaping adult prepulse inhibition response to hippocampal allopregnanolone in rats.

Diverse studies indicate that the alteration of the physiological levels of neurosteroids in early neonatal phases provokes alterations in the maturation of certain cerebral structures. Allopregnanolone (ALLO) has important modulatory effects in the hippocampus during the postnatal period where the adult pattern of inhibitory transmission is being established. In order to study whether endogenous neonatal ALLO levels would be a determinant parameter involved in mediating adult hippocampal GABAA system maturation, we investigated the effects of neonatal finasteride (50mg/kg, SC) treatment and ALLO (ALLO; 20mg/kg, SC) supplementation on an animal behavioural model with relevance to neurodevelopmental disorder, such as schizophrenia. Two sets of experiments were conducted. Neonatal treatment (from postnatal day (pnd) 5 to pnd9) was performed in 23 male Wistar rats and steroid quantification was performed in hippocampal homogenates at pnd9. A second group (n=127) underwent neonatal treatment (pnd5-pnd9) and were submitted to hippocampal surgery at 80d. The behavioural response to bilateral intrahippocampal neurosteroid administration (ALLO, 0.2µg/0.5µl per side or pregnenolone sulphate 5ng/0.5µl per side) on novelty-induced exploration activity and prepulse inhibition (PPI) was assessed at 95d. Results showed that neonatal ALLO and finasteride administration decreased novelty directed exploratory behaviour and impaired the prepulse inhibition of the acoustic startle response at 95 days of age. Moreover, intrahippocampal ALLO increased head-dipping behaviour independently of the neonatal treatment, while intrahippocampal ALLO decreased PPI only in finasteride and ALLO groups. The results obtained in the present study indicate the importance of neonatal neurosteroid levels in the development of hippocampal function and their relevance in a behavioural phenotype that some have likened to that present in schizophrenia.