Physical activity as a promoter of stress resilience: An analysis of behavioral effects and brain connectivity with cytochrome c-oxidase activity in adult male Wistar rats.

Physical activity (PA) is very beneficial for physical and mental health. This study aims to examine the resilience-inducting effect of PA in adult male Wistar rats exposed to unpredictable chronic mild stress (UCMS). Furthermore, we analyzed the influence of PA on behavioral tasks and functional brain connectivity with cytochrome c oxidase technique. The cytochrome c oxidase (CCO) is a mitochondrial enzyme involved in oxidative phosphorylation and ATP generation. For this analysis, we included five groups: Basal (n = 10, to determine the basal level of brain activity), Behav (n = 15, subjected exclusively to behavioral tests), PA (n = 10, exposed to physical activity), UCMS (n = 15, subjected to a stress protocol) and PA + UCMS (n = 15, exposed to PA prior to stress). The UCMS protocol consisted of randomly presenting several different stressors over four consecutive weeks. We evaluated several behaviors of the Behav, UCMS, and PA + UCMS groups. This assessment includes the hedonic responses using the sucrose consumption task, unconditioned anxiety with the zero maze, and coping strategies assessed with the cat odor test. The UCMS group showed an anhedonia profile and increased anxiety compared with the other groups. Although in the exposure to cat odor test, the PA + UCMS remained for the same time in the cat odor compartment as the other groups, it did not approach the odor, showing that it detected the risk. This response is more adaptive than the responses of the UCMS and Behav groups. An exploratory analysis of the cerebral connections showed an increase in CCO activity in the UCMS group compared to the other groups. This overactivity was reduced in dorsal Cornu Ammonis 3(dCA3) by prior PA. In this region, PA + UCMS showed similar activity as the groups not subjected to chronic stress. Therefore, PA can prevent the harmful effects of chronic stress on dCA3.

An evaluation of hedonic responses in taste-potentiated odor aversion using the analysis of licking microstructure and orofacial reactivity.

Two experiments examined the hedonic responses conditioned to odor cues in the phenomenon of taste-potentiated odor aversion. Experiment 1 analyzed the microstructure of licking behavior during voluntary consumption. A tasteless odor (amyl acetate) was delivered to rats either diluted in water or mixed with saccharin before being injected with LiCl. At test, subjects which had received the odor-taste compound during conditioning showed both lower odor consumption and lick cluster size, a result indicating an increased negative evaluation of the odor. Experiment 2 examined the orofacial reactions elicited by the odor as index of its hedonic impact. During conditioning, the rats were intraorally infused with either the odor alone or the odor-saccharin compound before being injected with LiCl. At test, they were infused with the odor and their orofacial responses video recorded. More aversive orofacial responses were elicited by the odor cue in rats that had compound conditioning, again a result indicating a strengthened negative hedonic reactivity compared to animals experiencing odor aversion conditioning alone. Taken together, these results indicate that taste-mediated potentiation of odor aversion conditioning impacts on the acquisition of conditioned hedonic reactions as well as consumption.

Impact of environmental enrichment on the GABAergic neurons and glucocorticoid receptors in the hippocampus and nucleus accumbens of Wistar rats: Pro-resilient effects.

The unpredictable chronic mild stress (UCMS) model has been used to induce depressive-like symptoms in animal models. Our work aims to evaluate the impact of environmental enrichment on male Wistar rats in an animal model for depression. For this purpose, we aim to assess changes in GR and GABAergic (PV+) density in cerebral regions related to cognitive-affective processes associated with depressive disorder, such as the dorsal- ventral hippocampus and accumbens nuclei. Three groups of rats were used: UCMs (unpredictable chronic mild stress), EE+ UCMs (enrichment + stress) and CONT (behavioral tests only). Hedonic responses elicited by sucrose solution were examined by licking behavior analysis; the anxiety level was evaluated using the elevated zero maze and the forced swimming (passive coping) tests. The environmental enrichment reduced the effects of chronic stress, promoting greater resilience. Thus, the UCMs group showed an anhedonia response, more anxiety and immobility behavior than either the control or the EE+ UCMs groups. Regarding immunochemistry results, there was a reduction in GABAergic activity coupled with increased activation of GR in UCMs in the dorsal hippocampus, but there were no differences between groups in the ventral hippocampus. These results suggest environmental enrichment could enhance greater resilience, reducing the vulnerability of the subjects to develop disorders such as depression and anxiety.

Odor-taste pairings lead to the acquisition of negative hedonic qualities by the odor in aversion learning.

Three experiments examined the affective responses conditioned to an odorous stimulus in the taste-mediated odor aversion learning paradigm. Experiment 1 analyzed the microstructure of licking behavior during voluntary consumption. Before conditioning, water-deprived rats had access to a bottle containing either a tasteless odor (0.01% amyl acetate) diluted in water or mixed with 0.05% saccharin. Next, the rats were injected with either LiCl or saline immediately after drinking saccharin. At test, they received the odor and taste solutions on separate days. Lick cluster size was used as a direct measure of the hedonic response to the odor cue. Rats receiving odor-taste pairings prior to the saccharin devaluation showed both lower consumption and lick cluster size, reflecting a reduced hedonic evaluation of the odor. Experiments 2a and 2b used the orofacial reactivity method. After pretraining in the drinking boxes with the odor alone or mixed with saccharin, the rats were intraorally infused with saccharin before injection with LiCl or saline. At test, they were infused in separate sessions with the odor and taste and their orofacial reactions video recorded. There were increased aversive orofacial responses to the odor in rats that had prior odor-taste experience, a result indicating a negative hedonic evaluation of the odor. These results provide evidence of conditioned changes in affective value of odor cues through taste-mediated learning and are consistent with the idea that odor-taste pairings lead to the acquisition of taste qualities by the odor.

Reorganization of Brain Networks as a Substrate of Resilience: An Analysis of Cytochrome c Oxidase Activity in Rats.

The unpredictable chronic mild stress (UCMS) model has been used to induce depressive-like symptoms in animal models, showing adequate predictive validity. Our work aims to evaluate the effects of environmental enrichment (EE) on resilience in this experimental model of depression. We also aim to assess changes in brain connectivity using cytochrome c oxidase histochemistry in cerebral regions related to cognitive-affective processes associated with depressive disorder: dorsal hippocampus, prefrontal cortex, amygdala, accumbens, and habenula nuclei. Five groups of rats were used: UCMS, EE, EE + UCMS (enrichment + stress), BG (basal level of brain activity), and CONT (behavioral tests only). We assessed the hedonic responses elicited by sucrose solution using a consumption test; the anxiety level was evaluated using the elevated zero maze test, and the unconditioned fear responses were assessed by the cat odor test. The behavioral results showed that the UCMS protocol induces elevated anhedonia and anxiety. But these responses are attenuated previous exposure to EE. Regarding brain activity, the UCMS group showed greater activity in the habenula compared to the EE + UCMS group. EE induced a functional reorganization of brain activity. The EE + UCMS and UCMS groups showed different patterns of connections between brain regions. Our results showed that EE favors greater resilience and could reduce vulnerability to disorders such as depression and anxiety, modifying metabolic brain activity.

Characterizing Hedonic Responses to Flavors Paired with Internal Pain and Nausea through the Taste Reactivity Test in Rats.

Feeding behavior is a complex experience that involves not only sensory (i.e., visual, odor, taste, or texture) but also affective or emotional aspects (i.e., pleasure, palatability, or hedonic value) of foods. As such, behavioral tests that assess the hedonic impact of foods are necessary to fully understand the factors involved in ingestive behavior. In this protocol, we use the taste reactivity (TR) test to characterize the hedonic responses of rats to flavors paired with either lithium chloride-induced nausea or internal pain produced by hypertonic NaCl, two treatments that reduce voluntary consumption. This application of the TR test demonstrates how emetic and non-emetic (somatic pain in particular) treatments produce dissociable patterns of hedonic reactions to fluids: only emetic treatments result in the production of aversive orofacial responses, reflecting conditioned nausea, whereas somatic pain produces immobility, reflecting conditioned fear. Other methods, such as the microstructural analysis of licking behavior, do not reliably distinguish conditioned nausea and fear, a key advantage of the more selective TR procedure. This protocol also contains guidance for adaptation to other species and designs.

Long-term consequences of alcohol use in early adolescent mice: Focus on neuroadaptations in GR, CRF and BDNF.

Our aim was to assess the cognitive and emotional state, as well as related-changes in the glucocorticoid receptor (GR), the corticotropin-releasing factor (CRF) and the brain-derived neurotrophic factor (BDNF) expression of adolescent C57BL/6J male mice after a 5-week two-bottle choice protocol (postnatal day [pd]21 to pd52). Additionally, we wanted to analyse whether the behavioural and neurobiological effects observed in late adolescence (pd62) lasted until adulthood (pd84). Behavioural testing revealed that alcohol during early adolescence increased anxiety-like and compulsive-related behaviours, which was maintained in adulthood. Concerning cognition, working memory was only altered in late adolescent mice, whereas object location test performance was impaired in both ages. In contrast, novel object recognition remained unaltered. Immunohistochemical analysis showed that alcohol during adolescence diminished BDNF+ cells in the cingulate cortex, the hippocampal CA1 layer and the central amygdala. Regarding hypothalamic-pituitary-adrenal axis (HPA) functioning, alcohol abuse increased the GR and CRF expression in the hypothalamic paraventricular nucleus and the central amygdala. Besides this, GR density was also higher in the prelimbic cortex and the basolateral amygdala, regardless of the animals' age. Our findings suggest that adolescent alcohol exposure led to long-term behavioural alterations, along with changes in BDNF, GR and CRF expression in limbic brain areas involved in stress response, emotional regulation and cognition.

Influence of Pre-reproductive Maternal Enrichment on Coping Response to Stress and Expression of c-Fos and Glucocorticoid Receptors in Adolescent Offspring.

Environmental enrichment (EE) is an experimental setting broadly used for investigating the effects of complex social, cognitive, and sensorimotor stimulations on brain structure and function. Recent studies point out that parental EE experience, even occurring in the pre-reproductive phase, affects neural development and behavioral trajectories of the offspring. In the present study we investigated the influences of pre-reproductive EE of female rats on maternal behavior and adolescent male offspring's coping response to an inescapable stressful situation after chronic social isolation. For this purpose female Wistar rats were housed from weaning to breeding age in enriched or standard environments. Subsequently, all females were mated and housed in standard conditions until offspring weaning. On the first post partum day (ppd 1), mother-pup interactions in undisturbed conditions were recorded. Further, after weaning the male pups were reared for 2 weeks under social isolation or in standard conditions, and then submitted or not to a single-session Forced Swim Test (FST). Offspring's neuronal activation and plastic changes were identified by immunohistochemistry for c-Fos and glucocorticoid receptors (GRs), and assessed by using stereological analysis. The biochemical correlates were measured in the hippocampus, amygdala and cingulate cortex, structures involved in hypothalamic-pituitary-adrenocortical axis regulation. Enriched dams exhibited increased Crouching levels in comparison to standard reared dams. In the offspring of both kinds of dams, social isolation reduced body weight, decreased Immobility, and increased Swimming during FST. Moreover, isolated offspring of enriched dams exhibited higher levels of Climbing in comparison to controls. Interestingly, in the amygdala of both isolated and control offspring of enriched dams we found a lower number of c-Fos immunopositive cells in response to FST and a higher number of GRs in comparison to the offspring of standard dams. These results highlight the profound influence of a stressful condition, such as the social isolation, on the brain of adolescent rats, and underline intergenerational effects of maternal experiences in regulating the offspring response to stress.

c-Fos activity in the insular cortex, nucleus accumbens and basolateral amygdala following the intraperitoneal injection of saccharin and lithium chloride.

This study examined c-Fos expression in selected brain areas consequent to intraperitoneal (IP) administration of saccharin and lithium chloride. Rats were tested for aversion to the saccharin as measured by flavor consumption and orofacial reactions in the taste reactivity (TR) test. It was found that intraperitoneal conditioning resulted in the reduction in voluntary consumption but not in the production of aversive orofacial responses to the saccharin. The immunohistochemistry quantification revealed increased c-Fos activity in the insular cortex, the shell and core regions of the nucleus accumbens, and the basolateral nucleus of the amygdala. These results show that a conditioned taste aversion can be induced without direct oropharyngeal gustatory stimulation at the time of conditioning. In addition, this study provide evidence of increased neural activity in response to intraperitoneal saccharin injections.

Functional brain networks underlying latent inhibition of conditioned disgust in rats.

The present experiment examined the neuronal networks involved in the latent inhibition of conditioned disgust by measuring brain oxidative metabolism. Rats were given nonreinforced intraoral (IO) exposure to saccharin (exposed groups) or water (non-exposed groups) followed by a conditioning trial in which the animals received an infusion of saccharin paired (or unpaired) with LiCl. On testing, taste reactivity responses displayed by the rats during the infusion of the saccharin were examined. Behavioral data showed that preexposure to saccharin attenuated the development of LiCl-induced conditioned disgust reactions, indicating that the effects of taste aversion on hedonic taste reactivity had been reduced. With respect to cumulative oxidative metabolic activity across the whole study period, the parabrachial nucleus was the only single region examined which showed differential activity between groups which received saccharin-LiCl pairings with and without prior non-reinforced saccharin exposure, suggesting a key role in the effects of latent inhibition of taste aversion learning. In addition, many functional connections between brain regions were revealed through correlational analysis of metabolic activity, in particular an accumbens-amygdala interaction that may be involved in both positive and negative hedonic responses.

Reversal learning impairment and alterations in the prefrontal cortex and the hippocampus in a model of portosystemic hepatic encephalopathy.

Patients with liver dysfunction often suffer from hepatic encephalopathy (HE), a neurological complication that affects attention and memory. Various experimental animal models have been used to study HE, the most frequently used being the portocaval shunt (PCS). In order to determine brain substrates of cognitive impairment in this model, we assessed reversal learning and c-Fos expression in a rat model of portosystemic derivation. PCS and sham-operated rats (SHAM) were tested for reversal learning. Brains were processed for c-Fos immunocytochemistry. The total number of c-Fos positive nuclei was quantified in the prefrontal cortex and hippocampus. The spatial reference memory task showed no differences between groups in escape latencies. The no-platform probe test showed that both the PCS and the SHAM learned the location of platform. However, the PCS group perseverated in the old target during reversal. The PCS group presented less c-Fos- positive cells in prelimbic cortex, CA1 and dentate gyrus of the dorsal hippocampus than SHAM. Overall, these results suggest that this specific model of portosystemic hepatic encephalopathy produces reversal learning impairment that could be linked to dysfunction in neuronal activity in the prefrontal cortex and hippocampus.

Assessment of the global intelligence and selective cognitive capacities in preterm 8-year-old children.

The aim of this study was to assess various cognitive abilities such as attention, IQ, reasoning, and memory related to academic achievement in 8- and 9-year-old preterm children. A total of 141 children were assessed. The preterm group (=37 weeks) comprised 63 children and was compared to 78 full-term children. Attention was evaluated using the d2 Selective Attention test, and the IQ by the L-M form of the Stanford-Binet Intelligence Scale, establishing a profile of abilities: perception, memory, comprehension, reasoning, and verbal fluency. Significant differences in IQ were found between the preterm and full-term children. Of the cognitive abilities assessed, the only significant differences were found in verbal fluency, with preterm boys showing lower verbal fluency scores than full-term children. In conclusion, all preterm groups have attention ability similar to that of full-term children. However, preterm children obtain lower scores in intelligence measures. In addition, preterm boys have verbal fluency difficulties. Taking into account the increase in preterm births, suitable intervention programs must be planned to attend the difficulties found.

Spatial short-term memory in rats: effects of learning trials on metabolic activity of limbic structures.

The oxidative metabolism was assessed in the hippocampus and related regions in rats that were trained in a spatial short-term memory task in the water maze following distinct training schedules. The cytochrome oxidase (COx) histochemistry was evaluated in groups of rats that received a daily session made up of either two or three learning trials. An untreated group was added to determine baseline levels of COx. We found that the 3-Trials group exhibited a better performance concerning the differences in latency between trials 1 and 2. Trained groups showed higher COx activity than the untreated group in the medial prefrontal cortex, dentate gyrus, CA1, and the mammillary region. However, a decrease in COx activity was found in the dentate gyrus, CA1, and supramammillary region of the 3-Trials group. In addition, COx activity levels found in this group were similar to those of the untreated group in some thalamic nuclei. Most of the regions that presented significant correlations between COx activity and behavioral scores were found in the 3-Trials group. These findings suggest an influence of task difficulty in the oxidative metabolism of brain regions involved in spatial learning.

Portal hypertension in 18-month-old rats: memory deficits and brain metabolic activity.

Portal hypertension is a major complication of cirrhosis that frequently leads to a neuropsychiatric disorder that affects cognition. We compared the performance of 18-month-old prehepatic portal hypertensive rats (PH) and 18-month-old normal rats (CO) in spatial short-term and reference memory tasks in the Morris water maze and in active avoidance task. The PH group showed worse spatial short-term memory than the CO group. Also, the PH group tended to perform worse than the CO group in the reference memory task, but it presented a correct acquisition of the active avoidance task. We assessed the brain metabolic activity of the animals by means of cytochrome c-oxidase (COx) histochemistry. We found that the PH group developed prefrontal dysfunction characterized by increased COx activity in this region compared to the CO group. Similar results were found in the medial mammillary nucleus and dentate gyrus, whereas the CA1 area, bed nucleus of the stria terminalis, and supramammillary nucleus showed lower COx activity in the PH group as compared to the CO group. We conclude that the 18-month-old portal hypertensive rats present spatial memory impairment without alteration of implicit learning. This deficit could be related to the alteration of the metabolic activity of the brain regions involved in the processing of spatial memories.

Effects of medial prefrontal cortex lesions on anxiety-like behaviour in restrained and non-restrained rats.

The medial prefrontal cortex has been associated with fear, anxiety and stress regulation, and has recently been suggested to play a crucial role in the development of behavioural changes in response to stress. In this study, we evaluated medial prefrontal cortex (mPFC) involvement in both anxiety-like behaviour and increased anxiety-like responses induced by uncontrollable restraint. Rats with mPFC electrolytic lesions (n=7) and sham-lesioned (n=8) were tested in the elevated T-maze (ETM). Restrained rats with mPFC lesions (n=8) and sham-lesioned rats (n=6) were tested in the elevated T-maze 24h after restraint. Both two-trial passive avoidance and one-trial escape behaviours were assessed. The results revealed that mPFC lesions impair passive avoidance, but not escape behaviour. In addition, decreased anxiety-like behaviour in both passive avoidance and escape behaviours were observed in restrained rats with mPFC lesions. Our results suggest that mPFC is important in mediating both anxiety-like behaviour expression and long-term anxiogenic-like effects induced by acute restraint.

Temporary inactivation of the supramammillary area impairs spatial working memory and spatial reference memory retrieval.

The aim of our study was to examine the supramammillary (SuM) area involvement in spatial memory. Sprague-Dawley rats with chronically implanted cannula in the supramammillary area were trained in two spatial memory tasks with different memory demands: reference and working memory. In the spatial reference memory task, the rats received microinjections in the SuM area of tetrodotoxin (TTX) (0.5 ng diluted in 0.5 microL of saline) or saline (0.5 microL). The microinjections were administered 30 min before the spatial training (day 4) (to assess the effect on acquisition) and on the following two days (days 5 and 6) the training was conducted without microinjections (to study the effect on consolidation). On the last training day (day 7), in order to assess the retrieval of spatial information, the rats received the microinjections 30 min before the spatial training. The spatial working memory used was a delayed-matching-to-position (DMTP) task. Spatial training was performed for seven days. During the first three days of the spatial training, the rats achieved a good spatial knowledge and learnt the working memory rule necessary to solve the spatial task. On days 4 and 6, the rats received microinjections to study involvement of the SuM area in working memory. The results showed that temporary inactivation of SuM area impairs both the rat's ability to solve a spatial working memory task with DMTP demands and the recovery of spatial information in a spatial reference memory task. We suggest that SuM area is involved in the rearrangement of spatial information during spatial working memory tasks with DMTP memory demands.

Hippocampal and caudate metabolic activity associated with different navigational strategies.

Hippocampal and striatal systems are widely related to spatial tasks. Depending on the strategies used, different memory systems can be activated. In this study, the authors used the cytochrome c-oxidase technique as a functional marker of the hippocampal and dorsal striatum activity related to training in several water maze tasks. Current results show a differential participation of the hippocampal and striatal systems in navigation. When spatial information is relevant, participation of the hippocampal system is more important, and when the task is similar to a response learning one, the striatal system is more active. According to computational models, CA3 seems to be more active when the associative demand is higher, whereas CA1 and dentate gyrus activity are higher when spatial information processing is required.

Astroglial distribution and sexual differences in neural metabolism in mammillary bodies.

The sexual differences in cerebral nuclei are produced by the organizational and the activational function of gonadal hormones. The different performances by male and female rats in memory tasks requiring use of the mammillary bodies (MBs), could be due to structural and functional sexual dimorphic differences. Our work quantifies the number of glial fibrillary acidic protein immunoreactive (GFAP-IR) astrocytes, and neuronal metabolic activity measured by the cytochrome oxidase (CO) histochemistry in the MBs in rats of both sexes. We find that there is no difference in astroglial number in the medial mammillary nucleus (MMN) and in the lateral mammillary nucleus (LMN) of males, females in estrus and diestrus adult rats. However, we do find statistically significant differences between the sexes in the neuronal oxidative metabolism influenced by the estrous cycle. We, therefore, conclude that there are functional and not structural sex differences in the MBs.

Supramammillary and adjacent nuclei lesions impair spatial working memory and induce anxiolitic-like behavior.

The present study assesses the involvement of the supramammillary and adjacent nuclei in spatial memory and anxiety-like behaviors. Rats with electrolytic lesions in the supramammillary nucleus were pre- and post-operatively trained in two spatial memory tasks and two anxiety tasks. Spatial memory tasks were performed in an open field with seven different goal positions containing the reward. Anxiety-like behaviors were tested in the elevated T-maze. In the spatial reference memory task, neither lesioned nor sham-lesioned groups were impaired. In the working memory task, lesioned animals were permanently impaired in their ability to solve the delayed-matching-to-position task. This working memory deficit is not related to increased proactive interference. It could be related to impairment of the rats ability to reorganize spatial stimuli. Consequently, rats were not able to achieve an optimal performance level to solve spatial tasks with continuous changes in the place location. In the elevated T-maze, lesioned rats reduced passive avoidance response but no changes in the escape response were observed. These results suggest a clear involvement of the supramammillary nucleus in working memory and behavioral inhibition but not in either spatial reference memory or in escape responses.