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Perinatal hypothyroidism effects on neuromotor competence, novelty-directed exploratory and anxiety-related behaviour and learning in rats

Thyroid hormone is essential for proper development of the mammalian CNS. Previous studies have documented a decrease in the ability of neonatal hypothyroid animals to learn and to habituate to maze tests and an increase in spontaneous activity. However, there is little information about the effects of perinatal (i.e. perinatal and postnatal) hypothyroidism on behaviour. The aim of the present work was to investigate the longitudinal effects of perinatal hypothyroidism on certain aspects of the behaviour in rats. Neuromotor competence was tested at 21, 40 and 60 days, novelty-directed exploratory behaviour and anxiety-related behaviour were evaluated at 40 and 60 days by means of the Boissier tests and associative learning ability was tested at 80 days by means of a step-through passive avoidance task. The persistence of the effects of perinatal hypothyroidism on psychomotor performance was highly dependent on the task examined. Perinatal hypothyroidism caused an increase of locomotor activity as revealed by the total distance travelled in the Boissier test and this increase also comprised a component of decreased anxiety-related behaviour. Methimazole-treated subjects also had higher head-dip scores than controls at 40 days while no differences were observed at 60 days. Finally, our results showed that methimazole-treated rats performed poorly in a passive avoidance learning task.

Neurosteroids infusion into the CA1 hippocampal region on exploration, anxiety-like behaviour and aversive learning.

Neurosteroids (NS) are substances synthesised de novo in the brain that have rapid modulatory effects on ionotropic receptors. Specifically, NS can act as positive allosteric modulators of GABAA receptors as pregnanolone or allopregnanolone (Allop), or GABAA negative modulators and NMDA positive modulators as pregnenolone (PREG) or dehydroepiandrosterone (DHEA) and their sulphate esters (PREGS and DHEAS). Given this, their role in anxiety and emotional disturbances has been suggested. In addition, NS such as PREGS or DHEAS have demonstrated a promnesic role in several learning tests. The aim of the present work is to highlight the role that the dorsal (CA1) hippocampus plays in the behavioural profile of NS such as Allop and PREGS in tests assessing exploration, anxiety and aversive learning in rats. For this purpose, animals were administered intrahippocampally with Allop (0.2μg/0.5μl), PREGS (5ng/0.5μl) or vehicle in each hippocampus, and tested in the Boissier and elevated plus maze (EPM) tests. For learning test we have chosen the passive avoidance paradigm. Results indicate that intrahippocampal administration of Allop enhances exploration, reflected in an increase in the total and the inner number of head-dips. Allop-injected animals also showed an increase in the percentage of entries into the open arms of the EPM, suggesting an anxiolytic-like profile. In addition, post-acquisition PREGS administration enhanced passive avoidance retention, while post-acquisition Allop administration had no effects on aversive learning retention. These results point out the important role of the dorsal (CA1) hippocampus in several NS behavioural effects, such as exploration, anxiety, learning and memory.

Alterations in neonatal neurosteroids affect exploration during adolescence and prepulse inhibition in adulthood

Allopregnanolone (AlloP) is a neurosteroid that plays an important role during neural development. Alterations of endogenous neonatal allopregnanolone levels alter the localisation and function of GABA neurons in the adult brain and affect behaviour in adulthood. We have carried out research into the effects of an increase (AlloP administration) or a decrease (administration of finasteride, inhibitor of the AlloP synthesis) of neonatal AlloP levels during the fifth to ninth postnatal days in male Wistar rats on the novelty exploration (Boissier test) at adolescent ages (40 and 60 days old), and on the prepulse inhibition achievement in adulthood (85 days). We also investigated the role of a GABA(A) modulator (midazolam, 1, 1.75 or 2.5mg/kg body weight) in the long-lasting behavioural changes in adulthood (85 days). Results indicate that neonatal finasteride decreases both novelty-exploration (head-dipping and locomotion) and anxiety-relevant scores (the distance travelled in and the number of entries into the central zone) at adolescent age, along with a reduction in body weight and general locomotion. Also, neonatal AlloP administration decreases prepulse inhibition in adulthood. Prepulse inhibition disruption was only partially reproduced decreasing the neonatal AlloP levels by means of finasteride administration. Although there was no interaction between neonatal neurosteroid manipulation and adult benzodiazepine treatments, the effects of midazolam were dose-dependent: the lowest dose of midazolam increased whereas the highest disrupted the expected progressive reduction of the startle response (and the consequent improvement of the PPI percentage) after the gradual increase in prepulse intensity. Reduced prepulse inhibition of startle provides evidence of deficient sensorimotor gating in several disorders, including schizophrenia. Alterations of AlloP levels during maturation could partly explain the inter-individual differences shown by adult subjects in response to novelty (exploration) and in the sensorimotor gating and prepulse inhibition. Also, abrupt changes in neonatal levels of AlloP could be related to a susceptibility to neurodevelopmental disorders.

Effects of dysthyroidism in plus maze and social interaction tests

The aim of the present study was to determine the influence of thyroid hormones on the anxiety of male Wistar rats. Dysthyroidism was induced by adding 20 mg of methimazole (100 ml) to their drinking water or by adding 0.3 mg of L-thyroxine (100 ml) to their drinking water from the ninth day of gestation. After weaning, the drugs were administered to young rats until the end of the experiment. Anxious behavior was measured using the elevated plus maze and social interaction tests when the animals were 85 days old. Chronic methimazole administration produced a significant anxiolytic pattern in both tests. In the plus maze test, the methimazole-treated animals entered and remained more time in the open arms than the control animals. In the social interaction test, they spent more time in bodily contact, and did this more frequently than those in the control group did. Results from this experiment suggest that chronic thyroid deficiency produces an anxiolytic-like effect in both tests.

Perinatal alterations of thyroid hormones and behaviour in adult rats

Several studies have shown the relevance of the neuroendocrinological system in the development and function of the nervous system. In order to observe the influence of thyroid hormones during development on the behaviour of adult rats we induced dysthyroid states during the perinatal period. Results indicate that some behaviours are more susceptible to the action of thyroid hormones than others. We observed that the thyroid hormone deficiency causes an increase of activity in animals in spite of a large period of rehabilitation. Thyroxine-treated rats showed an anxiogenic behavioural pattern in the elevated plus-maze, while animals rehabilitated from perinatal deficit of thyroid hormones showed an anxiolitic pattern. These findings suggest that an excess of thyroid hormones has less effect on behaviour than a deficiency of these hormones.

Neonatal finasteride induces anxiogenic-like profile and deteriorates passive avoidance in adulthood after intrahippocampal neurosteroid administration.

Recent findings indicate that neurosteroids could act as important keys during the brain development. Fluctuations in neonatal allopregnanolone (AlloP) could result in altered pharmacological properties of the GABA(A) receptor system in adulthood. Recent studies demonstrated that neurosteroids play a critical role in regulating normal neurodevelopment in the hippocampus. The aim of the present work is to screen whether developmentally altered neurosteroid levels influence the behavioral response to adult intrahippocampal administration of AlloP, a GABA(A) positive modulating neurosteroid, and pregnenolone sulfate (PregS), a GABA(A) negative modulator in rats. For this purpose, pups received AlloP (10 mg/kg, s.c.), a 5alpha-reductase inhibitor (finasteride, 50 mg/kg, s.c.) or vehicle from the fifth to the ninth postnatal day. At maturity (i.e. 90 days old) a bilateral cannula was implanted into the hippocampus. After recovery from surgery, animals received an administration of AlloP (0.2 microg/0.5 microl), PregS (5 ng/0.5 microl) or vehicle in each hippocampus 5 min before they were tested in the elevated plus maze (EPM) and immediately after the passive avoidance training session, and retention was tested 24 h later. Results indicated that neonatal finasteride treatment deteriorated passive avoidance retention and elicited an anxiogenic-like effect in the EPM test in adulthood, as seen by the reduction of open arm entries and in the time spent in the open arms. Intrahippocampal PregS administration also disrupted passive avoidance, possibly related to its anxiogenic profile. Fluctuations in neonatal AlloP affect the aversive learning and the anxiety-related behavior in adulthood, and this effect could be in part mediated by alterations of the mature functions of the hippocampus, possibly via the GABA(A) receptor. These data point to the role of GABAergic neurosteroids in critical periods of vulnerability that influence normal development of GABAergic pathways in the CNS.

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.

Neonatal allopregnanolone increases novelty-directed locomotion and disrupts behavioural responses to GABAA receptor modulators in adulthood

Recent findings indicate that neurosteroids could act as important modulators during brain development. The aim of the present work is to screen whether developmentally altered AlloP levels may have long‐lasting effects on behaviour and influence the emotional response to several GABAA receptor modulating drugs in adulthood. Acute allopregnanolone administration (10 mg/kg) in the fifth postnatal day: (1) provoked long‐term effects, as an increase of the novelty‐directed locomotor activity and a decrease of its habituation in the open field in adult rats; (2) altered GABAA receptor response in adulthood, as reflected by the disruption of the effects of midazolam (1 mg/kg) and flumazenil (10 mg/kg) on the locomotor habituation in adulthood. Whereas the behavioural responses to 0.75 mg/kg of lorazepam or 3 mg/kg of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) were not affected, although lorazepam decreased locomotor activity in both neonatal AlloP and control rats, probably related to the sedative properties of the dose tested. Also, in the elevated plus‐maze, the anxiolytic effects of lorazepam were observed in controls, but not in neonatal allopregnanolone‐treated rats. This suggests that neonatal allopregnanolone decreases sensitivity to the anxiolytic effects of lorazepam at a dose of 0.75 mg/kg. Results suggest that alterations in neonatal allopregnanolone could result in an altered GABAA receptor response in adulthood that is evident behaviourally. These results point out the importance of the maturation of the endogenous neurosteroid mechanisms in the brain related to locomotor response to novelty and the responses to GABAA modulators in adulthood. This work opens future directions focused on the effects of acute and long‐lasting neonatal alterations of AlloP levels on vulnerability to psychopathology in adulthood.

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° 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.

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.