Functional interaction between the dorsal hippocampus and the striatum in visual discrimination learning.

The hippocampus and the striatum have traditionally been considered as part of different and independent memory systems. However, there is evidence that supports a functional interaction between the hippocampus and the dorsal striatum at least in particular learning tasks. Here, we evaluated the functional contribution of both brain regions in a visual discrimination learning task using cytochrome c oxidase (CO) quantitative histochemistry. Compared with other brain metabolic mapping techniques, CO activity reflects steady-state neuronal energy demand. Rats were trained for 6 days in a water T-maze to find a hidden escape platform associated with an intramaze visual cue. A control group of animals swam for an equivalent amount of time compared as the trained group but without any escape platform available. After finishing the behavioral task, CO activity was measured in subdivisions of the dorsal hippocampus and the dorsal striatum in both groups. Results show significantly higher CO activity in the CA1 area and the dentate gyrus of the dorsal hippocampus in the trained rats compared with the control group. In addition, a significant negative functional cross-correlation between area CA1 of the dorsal hippocampus and the anterodorsal striatum was found. Our results support current theories on competitive interaction of different memory systems during visual discrimination learning.

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.

Evaluation of two experimental models of hepatic encephalopathy in rats.

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Functional interactions between dentate gyrus, striatum and anterior thalamic nuclei on spatial memory retrieval.

The standard model of memory system consolidation supports the temporal reorganization of brain circuits underlying long-term memory storage, including interactions between the dorsal hippocampus and extra-hippocampal structures. In addition, several brain regions have been suggested to be involved in the retrieval of spatial memory. In particular, several authors reported a possible role of the ventral portion of the hippocampus together with the thalamus or the striatum in the persistence of this type of memory. Accordingly, the present study aimed to evaluate the contribution of different cortical and subcortical brain regions, and neural networks involved in spatial memory retrieval. For this purpose, we used cytochrome c oxidase quantitative histochemistry as a reliable method to measure brain oxidative metabolism. Animals were trained in a hidden platform task and tested for memory retention immediately after the last training session; one week after completing the task, they were also tested in a memory retrieval probe. Results showed that retrieval of the previously learned task was associated with increased levels of oxidative metabolism in the prefrontal cortex, the dorsal and ventral striatum, the anterodorsal thalamic nucleus and the dentate gyrus of the dorsal and ventral hippocampus. The analysis of functional interactions between brain regions suggest that the dorsal and ventral dentate gyrus could be involved in spatial memory retrieval. In addition, the results highlight the key role of the extended hippocampal system, thalamus and striatum in this process. Our study agrees with previous ones reporting interactions between the dorsal hippocampus and the prefrontal cortex during spatial memory retrieval. Furthermore, novel activation patterns of brain networks involving the aforementioned regions were found. These functional brain networks could underlie spatial memory retrieval evaluated in the Morris water maze task.

NOR activity in hippocampal areas during the postnatal development and ageing.

The silver staining of the nucleolar organizer regions (Ag-NORs) was used in order to estimate the biosynthetic activity of three hippocampal areas (dentate gyrus, CA1 and CA3) during postnatal development and ageing. 32 Wistar rats were used and 4 groups were formed according to the age of the animals (14, 21, 90 days and 23 months). Several Ag-NOR parameters such as mean Ag-NOR area and the ratio between Ag-NOR and nuclear areas per neuronal cell were quantified using an image analysis system. High values of these parameters are associated with a high rate of rRNA transcription. In this way, the neural biosynthetic activity in all regions studied decreased as the older ages are reached. Differences between areas are shown with the dentate gyrus and CA1 areas decreasing faster. The different activity among these areas is discussed, taking into account the particular affect on these areas of some injuries and the ageing process. Our results support the hypothesis of NOR loss as a main cause of ageing as reported by other authors.

Glial and neuronal cell numbers and cytochrome oxidase activity in CA1 and CA3 during postnatal development and aging of the rat.

Stereological methods (neuron and glial cell numbers) and histochemical methods (cytochrome c oxidase) were used to study postnatal development and aging of the CA1 and CA3 hippocampal areas in male rats. No changes were observed in 10 microns sections in the neuronal population of areas CA1 and CA3 in any of the groups (14 days, 21 days, adult-90 days and elderly-22 months). Statistical differences were found in the number of glial cells in both the CA1 and CA3 areas. An increase was observed in cytochrome oxidase (CO) activity in the CA1 area in the 14 day old rats compared to the other groups while in area CA3 this parameter increased in the 14 and 21 day old groups and the group of adult rats. No significant changes in CO activity were found in the elderly rats in both areas. These results are discussed in the light of those recorded in other areas of the limbic system.

Brain differences in newborn rats predisposed to helpless and depressive behavior.

Inborn brain differences in metabolic capacity were mapped in congenitally helpless rats, a genetically selected strain predisposed to show helpless and depressive behavior. There are a number of brain regions showing abnormal metabolism in adult congenitally helpless rats. Some of these alterations may be innate while others may be due to environmental factors, such as maternal care and postnatal stress. To identify which brain structures show innate differences, brains of newborn rats from congenitally helpless and non-helpless strains were compared using cytochrome oxidase histochemistry, an endogenous marker of regional metabolic capacity. A smaller subset of regions affected in adults showed significantly less metabolic activity in the newborn brains, including paraventricular hypothalamus, habenula, hippocampus, subiculum, lateral septal nucleus, anterior cingulate cortex, infralimbic cortex, and medial orbitofrontal cortex. A covariance analysis further revealed a striking reduction of functional connectivity in the congenitally helpless brain, including a complete decoupling of limbic forebrain regions from midbrain/diencephalic regions. This pattern of brain metabolism suggests that helplessness vulnerability is linked to altered functioning of limbic networks that are key to controlling the hypothalamic-pituitary-adrenal axis. This implies that vulnerable animals have innate deficits in brain systems that would normally allow them to cope with stress, predisposing them in this manner to more readily develop helpless and depressive behaviors.

Postnatal development of argyrophilic nucleolar organizer regions in the mammillary body of undernourished rats.

The effects of undernutrition during pregnancy and/or lactation periods on cortical structures have already been reported. However, its effect on non-cortical areas like the hypothalamic, that participates in the regulation of the autonomic nervous system or food intake, has not been extensively investigated. We studied the postnatal development of the medial mammillary nucleus (hypothalamus) in the rat offspring that had a dietary restriction in utero and during lactation. The argyrophilic nucleolar regions (Ag-NORs), that represent the transcriptional activity of the cell, were quantified in their neurons. No statistically significant differences were found in the results between the control and undernourished groups in the ages studied (7, 14, 21 and 30 days). However, a delay in the neuronal activity of the latter group was observed. A critical period in the development of this nucleus, at about 21 days of age, was also seen in both groups. After this age, the activity levels remained steady.

Changes of cytochrome oxidase activity in rat suprachiasmatic nucleus.

This paper evaluates the changes of cytochrome oxidase (CO) activity that take place in the suprachiasmatic nucleus (SCN) during the light-dark cycle. CO is a mitochondrial energy-generating enzyme used as a marker of neural oxidative metabolism. We measured CO activity using quantitative histochemistry calibrated with brain tissue standards and a computerized analysis image system. The results indicate that the CO enzyme activity changes on the basis of a circadian pattern, with the higher levels during the light phase (P < 0.0001). These changes are detected over a period of hours, in accordance with other studies on the possible short-term regulation of CO activity in the nervous system. It is, therefore, possible to apply this methodology to the study of the SCN and other brain areas which show functional rhythmicity.

Quantitative histochemical assessment of oxidative metabolism in the subfornical organ after partial aortic ligature in rats.

Circumventricular organs are considered to be involved in hydromineral homeostatic responses. In this study we used quantitative histochemistry of cytochrome oxidase to evaluate the oxidative metabolic activity of the subfornical organ of rats with a partial aortic occlusion. These rats showed a significant increase in water intake from the second day after the ligature, while natriophilia was already significant on the first day. Greater levels of cytochrome oxidase activity were found in subfornical organs of partial aortic ligated rats when compared with control, providing further evidence for the involvement of this circumventricular structure in fluid homeostasis at least in this hyperdipsic, hypernatriophilic, hyperreninemic and hypertensive experimental model.

The development of oxidative metabolism in diencephalic structures of the rat: a quantitative study.

A new method for quantitative determination of cytochrome oxidase (C.O.) activity was applied to diencephalic structures of the limbic system that are closely connected anatomically, that is, the mammillary bodies (MB) and the anterior nucleus of the thalamus (AT). This method makes it possible to easily evaluate the oxidative metabolic capacity of brain regions, an index of their functionality. By using this technique, we studied the postnatal development of both structures in Wistar rats of 14, 21, 30, and 120 days of age. Furthermore, animals of 730 days were included in order to evaluate the effects of aging on C.O. activity of these structures. The results showed a significant increase in the C.O. activity of the subdivisions of the AT, its levels remaining constant until the adult age, with a significant decrease in its activity in aged animals. In the MB, only the increase in C.O. activity of the medial mammillary nucleus (pars medialis) was significant until the adult age. A decrease of C.O. values with aging was significant only in the lateral mammillary nucleus. These data suggest that there is a wide heterogeneity in the maturation and aging of brain oxidative metabolism in diencephalic structures.

Sex-related differences in spatial learning during the early postnatal development of the rat.

Some authors have reported that male rats younger than 21 days old are unable to perform spatial learning correctly because they have still not developed the ability to use extra-maze cues. In experiment 1, we analyzed spatial learning in 14-, 21-, 30- and 42-day-old rats using the Morris water maze (MWM). According to our results, a good performance was observed in 30-day-old male rats whereas this was not observed in female rats until they were 42 days old. In experiment 2 we studied the role of sex hormones in this kind of learning using the MWM and 30-day-old rats (castrated male rats and female rats treated with testosterone propionate (TP) after birth). The latter group, the male control group and the castrated males all solved the task correctly. The objective of experiment 3 was to determine possible differences between the sexes in the use of taxon strategies in the T water maze. To summarize, sexual dimorphism was only observed in spatial learning during development.

Cytochrome oxidase activity in splanchnic organs of portal hypertensive rats.

OBJECTIVE: Portal hypertension is characterized by hyperdynamic splanchnic circulation associated with the development of portosystemic portal collateral circulation. Since blood flow regulation mechanisms in the splanchnic organs can be metabolic, its metabolic capacity has been studied using the mitochondrial enzyme cytochrome C oxidase as histochemical marker. METHOD: Cytochrome oxidase was quantified with a histochemical technique in the liver, pancreas and small bowel of Wistar rats in the control group (n = 8) and in rats with portal hypertension by triple stenosing ligation of the portal vein (n = 9) at 28 days of evolution. RESULTS: All rats with portal hypertension develop portosystemic collateral circulation. In these animals, cytochrome oxidase activity increases (p < 0.01) in the liver (left lateral lobe, periportal zone: 91.81 +/- 5.18 vs. 86.03 +/- 2.82) exocrine pancreas (125.6 +/- 7.25 vs 117.57 +/- 6.43; p < 0.05) as well as in the mucosa (crypts) and duodenum serosa, jejunum and ileum while it decreases in the pericentral zone of the hepatic acinus and intestinal villi. CONCLUSION: Cytochrome oxidase is considered an endogenous marker of local tissular metabolic capacity, so that its increased activity in the small bowel mucosa, crypts, exocrine pancreas and visceral peritoneum may be a metabolic factor that induces splanchnic hyperdynamic circulation in short-term portal hypertensive rats.

Different brain networks underlying the acquisition and expression of contextual fear conditioning: a metabolic mapping study.

The specific brain regions and circuits involved in the acquisition and expression of contextual fear conditioning are still a matter of debate. To address this issue, regional changes in brain metabolic capacity were mapped during the acquisition and expression of contextual fear conditioning using cytochrome oxidase (CO) quantitative histochemistry. In comparison with a group briefly exposed to a conditioning chamber, rats that received a series of randomly presented footshocks in the same conditioning chamber (fear acquisition group) showed increased CO activity in anxiety-related brain regions like the ventral periaqueductal gray, the ventral hippocampus, the lateral habenula, the mammillary bodies, and the laterodorsal thalamic nucleus. Another group received randomly presented footshocks, and it was re-exposed to the same conditioning chamber one week later (fear expression group). The conditioned group had significantly higher CO activity as compared with the matched control group in the following brain regions: the ventral periaqueductal gray, the central and lateral nuclei of the amygdala, and the bed nucleus of the stria terminalis. In addition, analysis of functional brain networks using interregional CO activity correlations revealed different patterns of functional connectivity between fear acquisition and fear expression groups. In particular, a network comprising the ventral hippocampus and amygdala nuclei was found in the fear acquisition group, whereas a closed reciprocal dorsal hippocampal network was detected in the fear expression group. These results suggest that contextual fear acquisition and expression differ as regards to the brain networks involved, although they share common brain regions involved in fear, anxiety, and defensive behavior.

A role for dorsal and ventral hippocampus in response learning.

The hippocampus and the striatum have been traditionally considered as part of different and independent memory systems despite growing evidence supporting that both brain regions may even compete for behavioral control in particular learning tasks. In this regard, it has been reported that the hippocampus could be necessary for the use of idiothetic cues in several types of spatial learning tasks. Accordingly, the ventral striatum receives strong anatomical projections from the hippocampus, suggesting a participation of both regions in goal-directed behavior. Our work examined the role of the dorsal and ventral hippocampus on a response learning task. Cytochrome c oxidase (C.O.) quantitative histochemistry was used as an index of brain oxidative metabolism. In addition, determination of C.O. subunit I levels in the hippocampus by western blot analysis was performed to assess the contribution of this subunit to overall C.O. activity. Increased brain oxidative metabolism was found in most of the studied hippocampal subregions when experimental group was compared with a swim control group. However, no differences were found in the amount of C.O. subunit I expressed in the hippocampus by western blot analysis. Our results support that both the dorsal and ventral hippocampus are associated with the use of response strategies during response learning.

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.

Functional sexual differences in rat mammillary bodies: a quantitative Ag-NOR study.

The mammillary bodies of the posterior hypothalamus are one of the CNS structures in which structural sexual dimorphism has already been described. This study quantifies the argyrophilic nucleolar organizer regions (NORs) of the neurons from this region in the male and female rats, the latter during two major phases of the estrous cycle (estrus-diestrus). The number and relative area of these NORs stained with a silver nitrate technique are considered as an index of the global protein synthesis of the neurons in the different nuclei from the mammillary bodies. Our results show the existence of statistically significant differences between sexes and estrous cycle phases. These findings suggest a significant influence of gender and the hormonal state on the neural activity of the MB.

Undernutrition and Postnatal Development of Brain Oxidative Metabolism in Limbic Structures: A Quantitative Study.

The effects of food restriction during gestation, lactation and post-weaning were studied in rat brain structures (14,21 and 30 days). Oxidative metabolism was quantified in neurons from the anterior thalamus and mammillary bodies using a quantitative histochemical method for cytochrome c oxidase (CO). In all the rat brains studied, a significant increase in activity occurred in the control group from 14 to 21 days after birth which then remained constant up to 30 days. A similar pattern was observed in the undernourished group, although in the anterodorsal and anteromedial thalamic nuclei the rise in CO only occurred between day 14 and 30 and there were no significant age-related changes in the lateral mammillary nucleus. Undernutrition produced a significant drop in CO activity after 21 days in all the nuclei except the lateral mammillary nucleus. In the latter nucleus and also in the pars medialis of the medial mammillary nucleus this parameter decreased at 30 days. Our results suggest that undernutrition and nutritional rehabilitation have different effects on the diencephalic regions studied, which depends on age and region.

Evaluation of two experimental models of hepatic encephalopathy in rats

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.