David Rotllant

Enduring effects of environmental enrichment from weaning to adulthood on pituitary-adrenal function, pre-pulse inhibition and learning in male and female rats

Environmental enrichment (EE) increases stimulation and provides richer sensory, cognitive and motor opportunities through the interaction with the social and physical environment. EE produces a wide range of neuroanatomical, neurochemical and behavioural effects in several animal species. However, the effects of EE have mainly been studied shortly after the treatment, so its long-lasting effects remain to be elucidated. Thus, we studied in male and female Sprague-Dawley rats the enduring effects of EE on tasks that measured emotional reactivity, social exploration and memory, sensorimotor gating and learning. After weaning, rats reared in EE were housed in single-sex groups of 12-14 in enriched cages during 12 weeks, whereas control rats were housed in single-sex groups of 2-3 animals in standard cages. Then, all rats were housed in pairs and successively exposed to different tests between 4 and 60 weeks post-EE. The results indicated that animals of both sexes reared in EE gained less weight during the enrichment period; differences disappeared in females during the post-EE period, but were maintained intact in males. Rats reared in EE showed an altered daily pattern of corticosterone and a lower hormone response to a novel environment (hole board, HB), although no differences in ACTH were found. EE resulted in more exploratory behaviour in the HB and higher number of entries in the open arms of the elevated plus maze (with no changes in the time spent in the open arms), suggesting a greater motivation to explore. Unexpectedly, rats reared in EE showed reduced pre-pulse inhibition (PPI), a measure of sensorimotor gating, suggesting lower capability to filter non-relevant information compared with control rats. EE increased social exploratory behaviour towards juvenile rats and social discrimination in males, but decreased social discrimination in females. Finally, in the Hebb-Williams maze, rats reared in EE showed better performance in terms of reduced number of errors and shorter distances travelled in the mazes. It is concluded that EE exposure from weaning to adulthood has important and long-lasting consequences on physiological and behavioural variables, most of them similar in both sexes, although sex differences in response to the EE are also reported.

Evidence that metyrapone can act as a stressor: effect on pituitary-adrenal hormones, plasma glucose and brain c-fos induction.

Metyrapone, a 11‐β steroid hydroxylase inhibitor that blocks stress‐induced glucocorticoid release, is extensively used to study the physiological and behavioural roles of glucocorticoids. However, there is circumstancial evidence suggesting that metyrapone could act as a pharmacological stressor. Thus, the effects of various doses of metyrapone on two well‐characterized stress markers (ACTH and glucose) were studied in male rats. Metyrapone administration, while exerting a modest effect on plasma corticosterone levels, dose‐dependently increased plasma ACTH and glucose levels. Using the highest doses previously tested (200 mg/kg) we further observed, as evaluated by fos‐like immunoreactivity (FLI), a strong activation of a wide range of brain areas, including the parvocellular region of the hypothalamic paraventricular nucleus (PVNp), the origin of the main ACTH secretagogues. Metyrapone‐induced FLI was observed in neocortical and allocortical areas, in several limbic, thalamic and hypothalamic nuclei and, to a lesser extent, in the brainstem. In a final experiment, a dose–response study of metyrapone‐induced FLI was carried out focusing on selected brain areas. The study revealed that the paraventricular thalamic nucleus and central amygdala were the areas most sensitive to metyrapone as they responded even to the lowest dose of the drug. Most areas, among them the PVNp, only showed enhanced FLI with the two highest doses, i.e. when it was associated with ACTH and glucose responses. These data suggest that some of the effects of metyrapone could be due to its stressful properties rather than its ability to inhibit glucocorticoid synthesis. The exact mechanisms involved remain to be established.

Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation

Stress exposure resulted in brain induction of immediate‐early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c‐fos and arc (activity‐regulated cytoskeleton‐associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c‐fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos‐like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO‐induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double‐labelling in the LS of IMO‐exposed rats revealed that arc was expressed in only one‐third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c‐fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

The brain pattern of c-fos induction by two doses of amphetamine suggests different brain processing pathways and minor contribution of behavioural traits.

Previous studies have shown that amphetamine (AMPH) markedly activates dopaminergic projection areas, together with some important limbic nuclei. However, a global picture of the brain areas activated is lacking and the contribution of the dose of the drug and individual differences to this global brain activation is not known. In the present experiment, we studied in adult male rats the c-fos expression induced by two doses of AMPH (1.5 and 5 mg/kg sc) in a wide range of brain areas, and investigated the possible contribution of novelty-induced activity and anxiety traits. AMPH administration increased Fos+ neurons in an important number of telencephalic, diencephalic and brainstem areas. Interestingly, the ventral tegmental area (VTA) and the dorsal raphe nucleus were activated by the drug, but c-fos expression was restricted to non-dopaminergic and non-serotoninergic neurons, those activated in the VTA being predominantly GABAergic. The use of the factorial analysis, which grouped the areas in function of the correlation between the number of Fos+ neurons observed in each area, revealed three main factors, probably reflecting activation of various relatively independent brain circuits: the first included medial prefrontal cortex regions, most dorsal and ventral striatal subregions and VTA; the second, raphe nuclei; and the third, the different subdivisions of the paraventricular nucleus of the hypothalamus. Other areas such as the central amygdala did not group around any factor. The finding that an important number of activated areas grouped around specific factors is suggestive of activation of partially independent brain circuits. Surprisingly, a minor contribution of novelty-induced activity and anxiety traits on brain activation induced by AMPH was found. It is possible that normal variability in these traits is poorly related to the effects of AMPH or that c-fos expression is not a good tool to reveal such differences.

Exposure to Severe Stressors Causes Long-lasting Dysregulation of Resting and Stress-induced Activation of the Hypothalamic-Pituitary-Adrenal Axis

Exposure to some predominantly emotional (electric shock) and systemic (interleukin‐1β) stressors has been found to induce long‐term sensitization of the hypothalamic‐pituitary‐adrenal (HPA) responsiveness to further superimposed stressors. Since exposure to immobilization on wooden boards (IMO) is a severe stressor and may have interest regarding putative animal models of post‐traumatic stress disorders (PTSD), we have characterized long‐lasting effects of a single exposure to IMO and other stressors on the HPA response to the same (homotypic) and to novel (heterotypic) stressors and the putative mechanisms involved. A single exposure to IMO caused a long‐lasting reduction of peripheral and central responses of the HPA axis, likely to be mediated by some brain areas, such as the lateral septum and the medial amygdala. This desensitization is not explained by changes in negative glucocorticoid feedback, and, surprisingly, it is positively related to the intensity of the stressors. In contrast, the HPA response to heterotypic stressors (novel environments) was enhanced, with maximal sensitization on the day after IMO. Sensitization progressively vanished over the course of 1–2 weeks and was not modulated by IMO‐induced corticosterone release. Moreover, it could not be explained by changes in the sensitivity of the HPA axis to fast or intermediate/delayed negative feedback, as evaluated 1 week after exposure to IMO, using shock as the heterotypic stressor. Long‐lasting stress‐induced behavioral changes reminiscent of enhanced anxiety and HPA sensitization are likely to be parallel but partially independent phenomena, the former being apparently not related to the intensity of stressors.

Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin

Early mother-infant relationships exert important long-term effects in offspring and are disturbed by factors such as postpartum depression. We aimed to clarify if lack of vasopressin influences maternal behavior paralleled by the development of a depressive-like phenotype. We compared vasopressin-deficient Brattleboro mothers with heterozygous and homozygous normal ones. The following parameters were measured: maternal behavior (undisturbed and separation-induced); anxiety by the elevated plus maze; sucrose and saccharin preference and forced swim behavior. Underlying brain areas were examined by c-fos immunocytochemistry among rest and after swim-stress. In another group of rats, vasopressin 2 receptor agonist was used peripherally to exclude secondary changes due to diabetes insipidus. Results showed that vasopressin-deficient rats spend less time licking-grooming their pups through a centrally driven mechanism. There was no difference between genotypes during the pup retrieval test. Vasopressin-deficient mothers tended to explore more the open arms of the plus maze, showed more preference for sucrose and saccharin and struggled more in the forced swim test, suggesting that they act as less depressive. Under basal conditions, vasopressin-deficient mothers had more c-fos expression in the medial preoptic area, shell of nucleus accumbens, paraventricular nucleus of the hypothalamus and amygdala, but not in other structures. In these areas the swim-stress-induced activation was smaller. In conclusion, vasopressin-deficiency resulted in maternal neglect due to a central effect and was protective against depressive-like behavior probably as a consequence of reduced activation of some stress-related brain structures. The conflicting behavioral data underscores the need for more sex specific studies.

Perseverance of exploration in novel environments predicts morphine place conditioning in rats

Reactivity to novelty has been related to operant drug self-administration but does not seem involved in the conditioned place preference (CPP). To further assess this issue our aims were to investigate: (1) the importance of the initial versus delayed activity in the novel environment to predict the CPP induced by morphine; (2) the separate contribution of trait anxiety in morphine CPP. Male Sprague-Dawley rats were exposed to a circular corridor for 30 min to assess reactivity to novelty and to the elevated plus-maze and the light-dark tests as measures of anxiety and morphine CPP was then studied (three pairings with 5 mg/kg s.c. morphine and three with saline). Delayed activity in the corridor (16-30 min) correlated positively with CPP score, whereas the initial activity (0-15 min) did not. High-responders (HR), those more active during the second half of the corridor, developed morphine CPP in contrast to low-responders (LR). Also, HR and LR did not differ in anxiety nor any plus-maze or light-dark test measure correlated with CPP behaviour. Enhanced vulnerability to develop morphine CPP is predicted by a higher delayed activity in a novel environment, regardless of anxiety.

A single dose of metyrapone caused long-term dysregulation of the hypothalamic–pituitary–adrenal axis in the rat

There is evidence that metyrapone (MET), apart from its inhibition of 11-beta steroid hydroxylation, may exert some stress-like effects in the brain, including the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the induction of c-fos. Since a single exposure to some stressors has been found to exert long-term effects on the HPA axis, we hypothesized that a single dose of MET (200 mg/kg, s.c.) could exert even stronger effects, due to the combination of its stressful properties with the lack of constrain of the HPA axis by glucocorticoids. Whereas the inhibitory effect of the drug on corticosterone secretion lasted less than 24 h, its stimulatory effect on the HPA axis could be seen for at least 2 days after the injection. Surprisingly, on day 8, an exacerbated HPA response to immobilization stress was observed in MET rats, despite complete normalization of resting levels of HPA hormones. At this time it was also observed, under basal conditions, increased levels of mRNA for CRH and arginin-vasopressin in the parvocellular region of the paraventricular nucleus of the hypothalamus (pPVN), along with reduced mRNA for glucocorticoid receptors in dentate gyrus and hippocampus CA1, but not in pPVN or medial prefrontal cortex. These data suggest that a single MET administration can exert a marked and long-lasting dysregulation of both resting and stress-induced activity of the HPA axis. Thus, attention should be paid to these properties when using the drug to study the functional role of glucocorticoids.

Dynamics of immediate early gene and neuropeptide gene response to prolonged immobilization stress: evidence against a critical role of the termination of exposure to the stressor.

Stress‐induced expression of immediate early genes (IEGs) appears to be transient even if the exposure to the stressor persists. However, there are some exceptions which suggest that particular characteristics of stressors can affect the dynamics of IEG expression. We studied in selected telencephalic, diencephalic and brainstem regions the mRNA levels of two clearly distinct IEGs (c‐fos and arc) during prolonged exposure to a severe stressor such as immobilization (IMO) and after releasing the rats from the situation. Although regional differences were observed with the two IEGs, overall, c‐fos mRNA levels progressively declined over the course of 4 h of continuous exposure to IMO, whereas arc mRNA levels were maintained at high levels in the brain regions that express this gene under stress (telencephalon). Levels of CRF hnRNA in the hypothalamus paraventricular nucleus only slightly declined during prolonged exposure to IMO. Surprisingly, termination of exposure to IMO did not modify CRF gene expression in the paraventricular nucleus or the pattern of IEGs expression, with the exception of c‐fos in the lateral septum. Thus, putative signals associated to the termination of exposure to IMO were unable to modify either IEG expression in most brain areas or CRF gene expression in the paraventricular nucleus.

Depressive- and anxiety-like behaviors and stress-related neuronal activation in vasopressin-deficient female Brattleboro rats.

Vasopressin can contribute to the development of stress-related psychiatric disorders, anxiety and depression. Although these disturbances are more common in females, most of the preclinical studies have been done in males. We compared female vasopressin-deficient and +/+ Brattleboro rats. To test anxiety we used open-field, elevated plus maze (EPM), marble burying, novelty-induced hypophagia, and social avoidance tests. Object and social recognition were used to assess short term memory. To test depression-like behavior consumption of sweet solutions (sucrose and saccharin) and forced swim test (FST) were studied. The stress-hormone levels were followed by radioimmunoassay and underlying brain areas were studied by c-Fos immunohistochemistry. In the EPM the vasopressin-deficient females showed more entries towards the open arms and less stretch attend posture, drank more sweet fluids and struggled more (in FST) than the +/+ rats. The EPM-induced stress-hormone elevations were smaller in vasopressin-deficient females without basal as well as open-field and FST-induced genotype-differences. On most studied brain areas the resting c-Fos levels were higher in vasopressin-deficient rats, but the FST-induced elevations were smaller than in the +/+ ones. Similarly to males, female vasopressin-deficient animals presented diminished depression- and partly anxiety-like behavior with significant contribution of stress-hormones. In contrast to males, vasopressin deficiency in females had no effect on object and social memory, and stressor-induced c-Fos elevations were diminished only in females. Thus, vasopressin has similar effect on anxiety- and depression-like behavior in males and females, while only in females behavioral alterations are associated with reduced neuronal reactivity in several brain areas.