Nélida M. Conejo

Effects of maternal separation, early handling, and standard facility rearing on orienting and impulsive behavior of adolescent rats

Effects of maternal separation in rats have been extensively investigated, but no studies have examined its effects in rat adolescence. We examined the effects of neonatal infant-mother separation (MS) for 6h/day and early handling (EH) for 10 days during the first 2 weeks of life by comparing MS and EH groups to standard facility reared (SFR) controls. At adolescence, the animals were evaluated in a novel and familiar open-field, the light-dark box, and the sucrose consumption test. Behavioral indices included orienting behavior (rearing frequency and duration), impulsive behavior (movement velocity and risk taking by entering the center of the open field or the light compartment of the light-dark box), hyperactivity (ambulatory distance and stereotypic movement), and reward-seeking behavior (sucrose drinking time). The prolonged MS during the first 2 weeks of life resulted in decreased orienting behavior and increased impulsive behavior in adolescence. Measures of ambulatory and stereotypic movements showed that MS rats were hyperactive in the novel environment whereas EH rats were less active overall. The impulsive/hyperactive phenotype produced by this MS protocol may provide a useful animal model to investigate the neurological basis for the similar behavioral phenotype found in attention deficit/hyperactivity disorder.

Spatial learning of the water maze: progression of brain circuits mapped with cytochrome oxidase histochemistry.

The progression of brain circuits involved in spatial learning tasks is still a matter of debate. In addition, the participation of individual regions at different stages of spatial learning remains a controversial issue. In order to address these questions, we used quantitative cytochrome oxidase histochemistry as a metabolic brain mapping method applied to rats (Rattus norvegicus) trained in a water maze for 1, 3 or 5 days of training. Sustained changes throughout training were found in the lateral septal nucleus and anteroventral thalamic nucleus. As compared to naïve or habituation groups, rats with 1 day of training in the spatial learning task showed involvement of the lateral mammillary nucleus, basolateral amygdala and anterodorsal thalamic nucleus. By 5 days of training, there were mean changes in the hippocampal CA3 field and the prefrontal cortex. The regions involved and their pattern of network interactions changed progressively over days of training. At 1-day there was an open serial network of pairwise correlations. At 3-days there was a more closed reciprocal network of intercorrelations. At 5-days there were three separate parallel networks. In addition, brain-behavior correlations showed that CA1 and CA3 hippocampal fields together with the parietal cortex are related to the mastery of the spatial learning task. The present study extends previous findings on the progressive contribution of neural networks to spatial learning.

Changes in brain oxidative metabolism induced by water maze training.

Although the hippocampus has been shown to be essential for spatial memory, the contribution of associated brain regions is not well established. Wistar rats were trained to find a hidden escape platform in the water maze during eight days. Following training, the oxidative metabolism in different brain regions was evaluated using cytochrome oxidase histochemistry. Metabolic activations were found in the prelimbic cortex, cornu ammonis (CA) 1 subfield of the dorsal hippocampus and the anterior thalamic nuclei, relative to yoked swim controls and naïve rats. In addition, many cross-correlations in brain metabolism were observed among the latter regions. These results support the implication of a hippocampal-prefrontal-thalamic system to spatial memory in rats.

Acute effects of 17β-estradiol and genistein on insulin sensitivity and spatial memory in aged ovariectomized female rats

Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.

Influence of gonadal steroids on the glial fibrillary acidic protein‐immunoreactive astrocyte population in young rat hippocampus

It is known that expression of glial fibrillary acidic protein (GFAP) as an astrocyte‐specific marker can be regulated by levels of circulating gonadal steroids during postnatal development. In addition, astrocytes play an important role in the physiology of the hippocampus, a brain region considered sexually dimorphic at the neuronal level in rodents. To evaluate the contribution of glial cells to gender‐related differences in the hippocampus, we estimated the number of GFAP‐immunoreactive (GFAP‐IR) astrocytes in the hippocampus (CA1 and CA3 areas, dorsal and ventral regions) of male and female rats aged 30 days. Groups of 30‐day‐old masculinized females (TP‐females; injected with testosterone propionate at birth) and feminized males (FLU‐males, castrated and treated with flutamide, an androgen receptor antagonist) were included to assess the effects of gonadal hormones on these hippocampal astrocytes. Using the optical fractionator method, the total number of GFAP‐IR cells found in CA1 and CA3 areas was significantly higher in males compared to that in age‐matched females. This numerical pattern was reversed in TP‐females and FLU‐males in both hippocampal areas. In addition, more GFAP‐IR cells were found in dorsal hippocampus than in the ventral region in the CA1 area from all experimental groups, whereas this result was found in the CA3 area from males and TP‐females. Our results suggest an essential contribution of gonadal hormones to gender differences found in the astrocyte population of the rat hippocampus during development.

Involvement of the mammillary bodies in spatial working memory revealed by cytochrome oxidase activity

In view of the inconclusive findings relating the nuclei of the mammillary bodies (MB) with spatial memory, we evaluated the oxidative metabolic activity of the medial and lateral nuclei of the mammillary bodies (MB) after training young rats (30 days) of both sexes in the Morris water maze. Different groups were trained in spatial working (WM) or reference memory (RM) tasks, respectively. The corresponding naïve groups swam for the same amount of time as the trained groups but without the escape platform. Control groups were added that had not been manipulated in any way. No sex-related differences were detected in the working memory task although males exhibited better reference memory than females. Cytochrome oxidase (CO) activity, an endogenous metabolic marker for neuronal activity, was measured in all the groups. CO activity increased significantly in both MB nuclei of male and female rats only in the spatial working memory group. In addition, high CO activity in the lateral nucleus of the MB was linearly correlated with lower escape latencies in both sexes after training in the working memory task. No CO activity changes were found in the basolateral amygdala (BL) in any of the experimental groups. This nucleus was used as a control brain region because of its participation in emotional behavior. The results suggest a specific role of the MB nuclei in spatial working memory in both sexes.

Evidence for sexual difference in astrocytes of adult rat hippocampus

We quantified the number of glial fibrillary acidic protein immunoreactive (GFAP-IR) astrocytes in the CA1 and CA3 areas of the adult rat hippocampus. The dorsal and ventral regions of the hippocampus were taken into account to estimate the GFAP-IR cells using unbiased stereological techniques. Males had a higher number of GFAP-IR astrocytes in the CA3 area, whereas females had more in the CA1 area. No sex difference was found between dorsal and ventral regions, although most GFAP-IR astrocytes were located in the dorsal hippocampus.

Cortico-limbic-striatal contribution after response and reversal learning: a metabolic mapping study.

Learning of arbitrary stimulus-response associations is an adaptive behavior essential for species survival in an ever-changing environment. Particular subdivisions of the striatum have been shown to be critical for both motor-response learning and reversal learning. However, recent evidence suggests that different cortical and subcortical brain regions may be involved in response learning, a kind of learning more complex than previously thought. In fact, many brain regions subserving response learning seem to be also related to reversal learning, traditionally ascribed to the prefrontal cortex. The present study examined the role of different subdivisions of the rat prefrontal cortex, striatum, amygdala and the ventral tegmental area on both response and reversal learning evaluated in the water T-maze. Increased neuronal metabolic activity, as measured by cytochrome oxidase (CO) histochemistry, was found in most brain regions after training rats in a response learning task as compared to yoked controls. Reversal learning was associated with a return to baseline CO activity levels except for the orbitofrontal cortex and the ventral tegmental area. Analysis of functional connectivity among brain regions showed significant correlations in CO activity between particular cortical and striatal subdivisions in the reversal learning group. These findings suggest that the interaction of specific frontal and subcortical regions is required for reversal but not for response learning. However, our findings support the involvement of a cortico-limbic-striatal circuit in both types of learning.

Oxidative metabolism of limbic structures after acute administration of diazepam, alprazolam and zolpidem

The effects of acute administration of two benzodiazepines and a non-benzodiazepine hypnotic on behavior and brain metabolism were evaluated in rats. After testing the behavioral action of the benzodiazepines on the open field and the elevated plus-maze, the effects of the three drugs on neuronal metabolism of particular limbic regions were measured using cytochrome c oxidase (CO) histochemistry. Diazepam (5 mg/kg i.p.) and alprazolam (0.5 mg/kg i.p.) induced clear anxiolytic effects and a decrease in locomotion, whereas zolpidem (2 mg/kg i.p.) caused an intense hypnotic effect. The anxiolytic effects of alprazolam were distinguishable from diazepam due to the pharmacological and clinical profile of this triazolobenzodiazepine. CO activity decreased significantly in almost all the limbic regions evaluated after zolpidem administration. However, significant prominent decreases in CO activity were found after diazepam treatment in the medial mammillary nucleus, anteroventral thalamus, cingulate cortex, dentate gyrus and basolateral amygdala. Alprazolam caused similar decreases in CO activity, with the exception of the prelimbic and cingulate cortices, where significant increases were detected. In agreement with previous studies using other functional mapping techniques, our results indicate that particular benzodiazepines and non-benzodiazepine hypnotics induce selective changes in brain oxidative metabolism.

Induction of c-Fos expression in the mammillary bodies, anterior thalamus and dorsal hippocampus after fear conditioning.

The aim of the present study was to provide further evidence on the role of particular subdivisions of the mammillary bodies, anterior thalamus and dorsal hippocampus to contextual and auditory fear conditioning. We used c-Fos expression as a marker of neuronal activation to compare rats that received tone-footshock pairings in a distinctive context (conditioned group) to rats being exposed to both the context and the auditory CS without receiving footshocks (unconditioned group), and naïve rats that were only handled. Fos immunoreactivity was significantly increased only in the anterodorsal thalamic nucleus and the lateral mammillary nucleus of the conditioned group. However, the dorsal hippocampus showed the highest density of c-Fos positive nuclei in the naïve group as compared to the other groups. Together, our data support previous studies indicating a particular involvement of the mammillary bodies and anterior thalamus in fear conditioning.