Requiring collaboration: Hippocampal-prefrontal networks needed in spatial working memory and ageing. A multivariate analysis approach.

Ageing is characterized by a decline in the processes of retention and storage of spatial information. We have examined the behavioural performance of adult rats (3months old) and aged rats (18months old) in a spatial complex task (delayed match to sample). The spatial task was performed in the Morris water maze and consisted of three sessions per day over a period of three consecutive days. Each session consisted of two trials (one sample and retention) and inter-session intervals of 5min. Behavioural results showed that the spatial task was difficult for middle aged group. This worse execution could be associated with impairments of processing speed and spatial information retention. We examined the changes in the neuronal metabolic activity of different brain regions through cytochrome C oxidase histochemistry. Then, we performed MANOVA and Discriminant Function Analyses to determine the functional profile of the brain networks that are involved in the spatial learning of the adult and middle-aged groups. This multivariate analysis showed two principal functional networks that necessarily participate in this spatial learning. The first network was composed of the supramammillary nucleus, medial mammillary nucleus, CA3, and CA1. The second one included the anterior cingulate, prelimbic, and infralimbic areas of the prefrontal cortex, dentate gyrus, and amygdala complex (basolateral l and central subregions). There was a reduction in the hippocampal-supramammilar network in both learning groups, whilst there was an overactivation in the executive network, especially in the aged group. This response could be due to a higher requirement of the executive control in a complex spatial memory task in older animals.

Astrocytic plasticity as a possible mediator of the cognitive improvements after environmental enrichment in aged rats.

Currently, little is known about the effect of environmental enrichment (EE) on astrocytic plasticity, especially during aging. Given the newly discovered role of the astrocytes in regulating the synaptic transmission and thereby, the cognitive functions, we aimed to study the impact of EE on the performance in a spatial memory task and on the number and morphology of GFAP immunopositive cells in the dorsal hippocampus. After two months of EE (3 h/per day), the animals were tested in the Radial-Arm Water Maze (RAWM) for four days, with six daily trials. Next, we analyzed the changes in the GFAP immunopositive cells in CA1, CA3 and Dentate Gyrus (DG). Behavioral results showed that, even in advanced ages, EE improved the performance in a spatial memory task. Also, we found that aged rats submitted to EE had more GFAP immunopositive cells in the DG and more complex astrocytes, revealed by Sholl analysis, in all hippocampal subfields with respect to the other experimental conditions. Interestingly, the learning of a spatial memory task produced more morphological complexity and higher levels of GFAP immunopositive cells with regard to a standard control group, but not at the same level of the enriched groups. Thus, it is possible that the plastic changes found in the hippocampal astrocytes after EE are involved in a brain reserve to cope with age-related cognitive impairments.

Functional networks involved in spatial learning strategies in middle-aged rats.

Our aim was to assess the way that middle-aged rats solve spatial learning tasks that can be performed using different strategies. We assessed the brain networks involved in these spatial learning processes using Principal Component Analysis. Two tasks were performed in a complex context, a four-arm radial maze, in which each group must use either an allocentric or an egocentric strategy. Another task was performed in a simple T-maze in which rats must use an egocentric strategy. Brain metabolic activity was quantified to evaluate neural changes related to spatial learning in the described tasks. Our findings revealed that two functional networks are involved in spatial learning in aged rats. One of the networks, spatial processing, is composed of brain regions involved in the integration of sensory and motivational information. The other network, context-dependent processing, mainly involves the dorsal hippocampus and is related to the processing of contextual information from the environment. Both networks work together to solve spatial tasks in a complex spatial environment.

Coping with Stress During Aging: The Importance of a Resilient Brain.

BACKGROUND: Resilience is the ability to achieve a positive outcome when we are in the face of adversity. It supposes an active resistance to adversity by coping mechanisms in which genetic, molecular, neural and environmental factors are involved. Resilience has been usually studied in early ages and few is known about it during aging. METHODS: In this review, we will address the age-related changes in the brain mechanisms involved in regulating the stress response. Furthermore, using the EE paradigm, we analyse the resilient potential of this intervention and its neurobiological basis. In this case, we will focus on identifying the characteristics of a resilient brain (modifications in HPA structure and function, neurogenesis, specific neuron types, glia, neurotrophic factors, nitric oxide synthase or microRNAs, among others). RESULTS: The evidence suggests that a healthy lifestyle has a crucial role to promote a resilient brain during aging. Along with the behavioral changes described, a better regulation of HPA axis, enhanced levels of postmitotic type-3 cells or changes in GABAergic neurotransmission are some of the brain mechanisms involved in resilience. CONCLUSION: Future research should identify different biomarkers that increase the resistance to develop mood disorders and based on this knowledge, develop new potential therapeutic targets.

Environmental Enrichment Results in Both Brain Connectivity Efficiency and Selective Improvement in Different Behavioral Tasks.

Exposure to environmental enrichment (EE) has been a useful model for studying the effects of experience on brain plasticity, but to date, few is known about the impact of this condition on the brain functional networks that probably underlies the multiple behavioral improvements. Hence, we assessed the effect of an EE protocol in adult Wistar rats on the performance in several behavioral tasks testing different domains (Open field (OP): locomotor activity; Elevated-zero maze (EZM): anxiety-related behaviors; 5-choice serial reaction time task (5-CSRTT): attentional processes; 4-arm radial water maze (4-RAWM): spatial memory) in order to check its effectiveness in a wide range of functions. After this, we analyzed the functional brain connectivity underlying each experimental condition through cytochrome C oxidase (COx) histochemistry. Our EE protocol reduced both locomotor activity in the OP and anxiety-related behaviors in the EZM. On the other hand, enriched rats showed more accuracy in the 4-RAWM, whereas 5-CSRTT performance was not significantly ameliorated by EE condition. In relation to COx functional connectivity, we found that EE reduced the number of strong positive correlations both in basal and training conditions, suggesting a modulating effect on specific brain connections. Our results suggest that EE seems to have a selective effect on specific brain regions, such as prefrontal cortex and hippocampus, leading to a more efficient brain connectivity.

Environmental Enrichment as a Positive Behavioral Intervention Across the Lifespan.

BACKGROUND: In recent decades, the interest in behavioral interventions has been growing due to the higher prevalence of age-related cognitive impairments. Hence, behavioral interventions, such as cognitive stimulation and physical activity, and along with these, our lifestyle (education level, work position, frequency of cognitive and social activities) have shown important benefits during the cognitive impairment, dementia and even recovery after brain injury. This is due to the fact that this type of intervention and activities promote the formation of a cognitive and brain reserve that allows tolerating brain damage during a long period of time without the appearance of cognitive symptoms. With regard to this, animal models have proved very useful in providing information about the brain mechanisms involved in the development of these cognitive and brain reserves and how they interact with each other. METHODS: We summarize several studies showing the positive effects of Environmental Enrichment (EE), understood as a housing condition in which animals benefit from the sensory, physical, cognitive and social stimulation provided, on brain and cognitive functions usually impaired during aging. RESULTS: Most of studies have shown that EE is a successful protocol to improve cognitive functions and reduce anxiety-related behaviors across the lifespan, as well as in animal models of neurodegenerative diseases. CONCLUSION: Therefore, EE is a laboratory condition in which some aspects of an active lifestyle are reproduced.

Environmental enrichment as a therapeutic avenue for anxiety in aged Wistar rats: Effect on cat odor exposition and GABAergic interneurons.

The use of more ethological animal models to study the neurobiology of anxiety has increased in recent years. We assessed the effect of an environmental enrichment (EE) protocol (24h/day over a period of two months) on anxiety-related behaviors when aged Wistar rats (21months old) were confronted with cat odor stimuli. Owing to the relationship between GABAergic interneurons and the anxiety-related neuronal network, we examined changes in the expression of Parvalbumin (PV) and 67kDa form of glutamic acid decarboxylase (GAD-67) immunoreactive cells in different brain regions involved in stress response. Behavioral results revealed that enriched rats traveled further and made more grooming behaviors during the habituation session. In the cat odor session, they traveled longer distances and they showed more active interaction with the odor stimuli and less time in freezing behavior. Zone analysis revealed that the enriched group spent more time in the intermediate zone according to the proximity of the predator odor. Regarding the neurobiological data, the EE increased the expression of PV-positive cells in some medial prefrontal regions (cingulate (Cg) and prelimbic (PL) cortices), whereas the GAD-67 expression in the basolateral amygdala was reduced in the enriched group. Our results suggest that EE is able to reduce anxiety-like behaviors in aged animals even when ethologically relevant stimuli are used. Moreover, GABAergic interneurons could be involved in mediating this resilient behavior.

Housing condition-related changes involved in reversal learning and its c-Fos associated activity in the prefrontal cortex.

Our study examined how different housing conditions modulated the acquisition of a spatial reference memory task and also, a reversal task in the 4-radial arm water maze (4-RAWM). The animals were randomly assigned to standard or enriched cages, and, as a type of complementary stimulation along with the environmental enrichment (EE), a group of rats also ran 15 min/day in a Rotarod. Elevated-zero maze results allowed us to discard that our exercise training increased anxiety-related behaviors. 4-RAWM results revealed that the non-enriched group had a worse performance during the acquisition and also, during the first trial of each session with respect to the enriched groups. Regarding the reversal task, this group made more perseverative errors in the previous platform position. Interestingly, we hardly found differences between the two enriched groups (with and without exercise). We also analyzed how the reversal learning, depending on the previous housing condition, modulated the expression of c-Fos-positive nuclei in different subdivisions of the medial prefrontal cortex (cingulate (Cg), prelimbic (PL) and infralimbic (IL) cortices) and in the orbitofrontal (OF) cortex. The enriched groups had higher c-Fos expression in the Cg and OF cortices and lower in the IL cortex respect to the non-enriched animals. In the PL cortex, we did not find significant differences between the groups that performed the reversal task. Therefore, our short EE protocol improved the performance in a spatial memory and a reversal task, whereas the exercise training, combined with the EE, did not produce a greater benefit. This better performance seemed to be related with the specific pattern of c-Fos expression in brain regions involved in cognitive flexibility.

Age-related changes of the nucleolar organizer regions without neuron loss in medial mamillary bodies (hypothalamus) in old rats.

Age-related morphological and functional changes were studied in the medial mamillary nuclei of the hypothalamus (MMN). The number of nerve cells and the volume of MMN were estimated in both groups of Wistar rats, adult and old (3 and 22 months, respectively) using stereologic methods such as the optical fractionator and Cavalieri's method respectively. The number of neurons and glial cells remain inalterable with ageing but there was an age-dependent reduction in MMN volume. In the second experiment, functional changes in the MMN neurons were observed although there was no loss in neuron number. Several functional parameters of the Ag-NORs were quantified by a computer analysis system: area and number of Ag-NORs per neuron; number of neurons with one or several Ag-NORs and also surface of neuronal nucleus. The present study shows how ageing causes volumetric and functional changes in the MMN whereas the number of neurons and glial cells remain unchanged in the mamillary region. All these results confirm the effects of age on proteic synthesis activity in neurons of the MMN, showing a decreased neuronal activity in this region.

Unbiased estimation of the total number of nervous cells and volume of medial mammillary nucleus in humans.

In this study, we demonstrate that aging does not provoke any changes in neuronal number or in the glial cells of the medial mammillary nucleus (MMN) in humans. Three age groups were used: young (age 17-35), adult (age 50-57), and aged (age 70-88). Furthermore, no age-dependent volumetric changes were observed in the MMN. All the estimations were performed with stereological methods: an optical fractionator and Cavalier's principle. The total number of neurons cells was estimated using an optical fractionator and amounted to 32x10(3) in the young group, 24x103 in the adult group, and 29x103 in the aged group. The number of glial cells was 164x10(3), 187x103, 185x103, respectively. Thus, all three age groups had a neuron/glial ratio of about 1:5, 1:8, and 1:6, respectively. The MMN volume was estimated using the Cavalier's principle. The total volume was 6.98 mm3 in the young group, 6.66 mm3 in the adult group, and 6.80 mm3 in the aged group. We have demonstrated that neither the total number of neurons and glial cells nor the volume of MMN are affected by age.

Functional sex differences in the accessory olfactory bulb of the rat.

The aim of this study is to determine whether sex-related differences exist in the biosynthetic activity of the mitral cells within the mitral layer of the AOB. Possible functional changes over the estrus cycle and the potential effects of castration and androgenization are assessed. Biosynthetic activity was measured using silver staining of the argyrophilic proteins associated with the nucleolar organizer regions (Ag-NOR). Assisted by stereological methods, the following parameters were studied: mean number, percentage and mean area of Ag-NOR in estrus and diestrus females, intact males, castrated and androgenizated rats. We detected sex differences in a histochemical marker related to synthetic activity, an estrus cycle effect and changes resulting from the perinatal treatments. We conclude that this structurally dimorphic region is also functionally dimorphic.

Effects of mammillary body lesions on spatial reference and working memory tasks.

This work examines the effects of electrolytic mammillary body (MB) lesions on the performance of rats in different spatial memory tasks in the Morris water maze. The first experiment assessed the effect of MB lesion on performance in a spatial reference memory task (place learning with multiple trials). The second experiment examined the effect of a lesion in this nucleus on performance in a spatial working memory task (single-trial place learning). The results show that lesion of the MB impairs the animals performance in spatial working memory tasks but does not impair acquisition in spatial reference memory tasks (place learning, transfer task, reversal task) or in a visual-cued task. However, the deficit in the spatial working memory task does not appear to vary with the delay between acquisition and retention trials (30 s and 5 min). Our results demonstrate a clear role of the mammillary bodies in the processing of spatial information in a working memory task. Lesion of the MB impairs performance in a working memory task but does not affect reference memory processes.

Improvement of spatial memory by (R)-alpha-methylhistamine, a histamine H(3)-receptor agonist, on the Morris water-maze in rat.

This work aims to clarify the role of histamine in learning and memory. This is done by studying the effect of administration of the histamine precursor, L-histidine (His), and the agonist of the H(3) receptor (R)-alpha-methylhistamine (RAMH), on acquisition and retention of spatial reference memory in rats. Treatment with RAMH (10 mg/kg i.p.) facilitates recovery of spatial memory. In contrast, administration of His (500 mg/kg i.p.) does not affect the performance of this task. Moreover, pharmacological modulation of the cerebral histaminergic system does not impair the animal's behavioral flexibility, i.e. their ability to adapt to a new learning task in the same stimular context. Improved retention of spatial memory after a reduction in cerebral histamine confirms the modulating role of this neurotransmitter in memory processes.

Effects of histamine precursor and (R)-alpha-methylhistamine on the avoidance response in rats.

The aim of this work is to clarify the role of histamine in learning and memory processes. In order to do this, the effect of administration of the histamine precursor, L-histidine (HIS) and of the agonist of the H(3) receptor, (R)-alpha-methylhistamine (RAMH), on active avoidance response in rats is studied. Treatment with RAMH (10 mg/kg i.p.) increased the number of avoidance responses produced during acquisition and retention of the learning. In contrast, administration of L-his (500 mg/kg i.p.) impairs performance in the shuttle-box. These results are consistent with a role for histamine in cognitive processes and suggest that a increase in cerebral histamine levels impair the acquisition of avoidance response, whereas reduced levels facilitate this acquisition.

Behaviour laterality in male rats: influence of practice and stress.

During recent decades, the existence of cerebral and behavioural asymmetries has been manifested in many different animal species. In this work, the asymmetric spatial preference recorded in the water T-maze has been analysed. Fifty-five male rats were tested in this maze for 8 consecutive days. Spatial preference, the number of times each arm was chosen, and the percentages of animals that showed right, left, or no spatial preference were calculated. The results show the evolution of this asymmetric behaviour. Most of the animals tested preferred the right arm (populational laterality). Recently, the influence of factors such as stress and practice in the process of acquiring this behaviour has been investigated. We used 40 male rats divided into two groups with two different stress levels: water temperature at 21 degrees C and 14 degrees C. Our results, based on these two criteria, hypothesize that both variables, stress and practice, are involved in the acquisition of spatial preference behaviour.

c-Fos expression in supramammillary and medial mammillary nuclei following spatial reference and working memory tasks.

To investigate brain substrates of spatial memory, neuronal expression of c-Fos protein was studied. Two groups of rats were trained in two spatial memory tasks in the Morris water maze, where the rats have to apply a reference memory rule or a working memory rule. In addition to the experimental groups, two control groups were used to study c-fos activation not specific to the memory processes studied. After immunohistochemical procedures, the number of c-Fos positive neuronal nuclei was quantified in the mammillary body (MB) region (medial mammillary nucleus [MMn] and supramammillary nucleus [SuM]). The results have shown that some MMn neurons expressed c-Fos nuclear immunoreactivity related to spatial working memory but not to spatial reference memory. The increased number of c-Fos immunoreactive neuronal nuclei in the SuM was related to spatial training but not to either working or reference memory demands of the tasks.

Ethanol- and diazepam-induced cytochrome oxidase activity in mammillary bodies.

This study aims to analyze the effect of the administration of diazepam and alcohol on cytochrome c oxidase activity (COX) in the mammillary bodies (MB) with a quantitative densitometry method. The histochemical reaction of the COX is used as a reflection of energy consumption. Our results show an increase in the COX activity after treatment with diazepam in the different nuclei of MB: medial medial nucleus (MMNm), lateral medial nucleus (MMN1), and lateral nucleus (LMN) of the MB, the MMNm and LMN being significantly more active compared to the MMN1. Furthermore, the consequences of administering these drugs become manifest in spatial learning (water T maze). The performance in a spatial discrimination task did not prove to be impaired.

Effects of chronic alcohol consumption on spatial reference and working memory tasks.

The aim of this work was to determine the spatial memory impairments induced by chronic alcohol consumption in rats. The alcoholization process began on the 21st postnatal day and alcohol concentrations were gradually increased to reach a concentration of 20% that was maintained for 4 mon. Behavioral tests were performed in the Morris Water Maze (MWM). The first study assessed the effects of chronic alcohol intake on two reference memory tasks (a place learning with multiple trials and a new place learning carried out in the same experimental context). Alcohol-treated animals presented no overall impairment in their ability to process spatial information. Deficits were restricted to reduced behavioral flexibility in spatial strategies. The second study assessed working memory in two tasks in which information about platform location was only valid for one trial. In the first working memory task, the animals had to perform one trial per day and in the second task they were submitted to four trials per day. At the end of the second experiment, all animals were trained in a visual-cued task. In the second experiment, the most important deficits in alcohol-treated animals occur in spatial working memory tasks, and this impairment was independent of the intertrial interval used. In the second spatial working memory task, performance of the alcohol-treated animals in the earlier trials affected their performance in subsequent trials, suggesting that a process of proactive interference had taken place. The visual-cued task demonstrated that these behavioral impairments were produced without visuoperceptive impairments.

Non-effects of mammillary body lesions on spontaneous alternation: pre and postoperative study.

This study was designed to investigate the potential effects of medial mammillary nucleus (MMn) lesions on spontaneous alternation behavior in rats. Behavioral measurements were made in a water T maze in which the side arms were not differentially reinforced. Spontaneous alternation was tested before and after surgery alone. In both experiments, volumetric estimates of the MMn lesion were made by stereologic calculations. The results obtained do not support the direct participation of the MMn in retrograde or anterograde memory processes related to spontaneous spatial alternation.

Portacaval shunt control animals: physiological consequences derived from the sham operation.

The portacaval shunt in the rat is a frequently used experimental model of portosystemic encephalopathy. Among other consequences of this surgical preparation is an important decrease in hepatic and testicular volume. Different sham-operation methods including a laparotomy were used as controls in each case. Given that the liver volume varies greatly in comparison to body weight in the sham-operated animals, this paper aims to evaluate the possible consequences of the sham operation. It concludes that control animals without manipulation, in addition to the respective controls of portacaval shunt, should be used in every case.