Marcelino Cuesta

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 Cavaliers 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 Cavaliers 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 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.

Age-dependent effects of environmental enrichment on brain networks and spatial memory in Wistar rats

We assessed the effect of 3h of environmental enrichment (EE) exposure per day started at different ages (3 and 18months old) on the performance in a spatial memory task and on brain regions involved in the spatial learning (SPL) process using the principal component analysis (PCA). The animals were tested in the four-arm radial water maze (4-RAWM) for 4days, with six daily trials. We used cytochrome c oxidase (COx) histochemistry to determine the brain oxidative metabolic changes related to age, SPL and EE. Behavioural results showed that the enriched groups, regardless of their age, achieved better performance in the spatial task. Interestingly, in the case of the distance travelled in the 4-RAWM, the effect of the EE was dependent on the age, so the young enriched group travelled a shorter distance compared to the aged enriched group. Respect to COx histochemistry results, we found that different brain mechanisms are triggered in aged rats to solve the spatial task, compared to young rats. PCA revealed the same brain functional network in both age groups, but the contribution of the brain regions involved in this network was slightly different depending on the age of the rats. Thus, in the aged group, brain regions involved in anxiety-like behaviour, such as the amygdala or the bed nucleus of the stria terminalis had more relevance; whereas in the young enriched group the frontal and the hippocampal subregions had more contribution.

Effects of Modeling the Heterogeneity on Inferences Drawn from Multilevel Designs

This article uses Monte Carlo techniques to examine the effect of heterogeneity of variance in multilevel analyses in terms of relative bias, coverage probability, and root mean square error (RMSE). For all simulated data sets, the parameters were estimated using the restricted maximum-likelihood (REML) method both assuming homogeneity and incorporating heterogeneity into multilevel models. We find that (a) the estimates for the fixed parameters are unbiased, but the associated standard errors are frequently biased when heterogeneity is ignored; by contrast, the standard errors of the fixed effects are almost always accurate when heterogeneity is considered; (b) the estimates for the random parameters are slightly overestimated; (c) both the homogeneous and heterogeneous models produce standard errors of the variance component estimates that are underestimated; however, taking heterogeneity into account, the REML-estimations give correct estimates of the standard errors at the lowest level and lead to less underestimated standard errors at the highest level; and (d) from the RMSE point of view, REML accounting for heterogeneity outperforms REML assuming homogeneity; a considerable improvement has been particularly detected for the fixed parameters. Based on this, we conclude that the solution presented can be uniformly adopted. We illustrate the process using a real dataset.

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.

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 Cavalieris 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.

Metabolic brain activity underlying behavioral performance and spatial strategy choice in sedentary and exercised Wistar rats.

We have studied the performance of a spatial reference memory task, the navigation strategy and the changes in the cytochrome c oxidase activity (COx) in different brain regions in exercised (forced exercise, 10 consecutive days, 15min/day) and non-exercised adult Wistar rats. The spatial learning task was carried out in the radial-arm water maze (RAWM) for four days with six daily trials, and on the fifth day, a probe session was run, in which we rotated the position of the distal cues 90° in a clockwise direction. During the four days of training, the exercised group showed shorter latency and distance traveled to find the platform, as well as fewer memory errors and reduced use of non-appropriate navigation strategies according to the protocol of the task (egocentric). Interestingly, the rotation of the cues did not affect the performance of the exercised group, in contrast to the non-exercised group, which spent more time in the center of the maze and traveled longer distance to find the platform. Finally, higher COx activity in the cingulate and the retrosplenial cortices, as well as in the dorsal CA1 and CA3 was found in the exercised group. All in all, it seems that the exercise favored the configuration of an efficient and accurate cognitive map of the environment, which was supported by our finding that the rotation of the cues, without altering their overall configuration, did not affect performance. The brain regions with higher COx activity in the exercised group seem to be involved in this function.

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

&NA; 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 (3 months old) and aged rats (18 months 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 5 min. 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. HighlightsDiscriminant analysis is a useful multivariate tool in brain networks assessment.New spatial working memory task reflects age‐related difficulties in performance.Poor anticonsolidation response in ageing compensated with the executive network.

Undernutrition and postnatal development of transcriptional activity in the rat medial mamillary nucleus (hypothalamus)

This paper aims to examine the effects of food restriction during gestation and lactation regarding the neuronal rRNA synthesis activity. In order to evaluate this activity, the distribution of the neuronal population was quantified on the basis of the argyrophilic nucleolar organizer region (Ag-NOR) numbers stained with silver nitrate. The CNS region chosen was the medial mamillary nucleus belonging to the mamillary bodies (posterior hypothalamus) of the rat, because of the unclear affectation of non-cortical regions by malnutrition and due to its implication in emotional behavior. The control group was made up of rats of 7, 14, 21 and 30 days of age and the undernourished group of animals of the same ages whose mothers received a restrictive diet during gestation and lactation. The results indicate that the distribution of neurons with one Ag-NOR increases with age while the neuron population with more than one clearly decreases with age. Our results show an overall decrease of the neuronal biosynthetic needs with age. No significant effect of undernutrition was found regarding the Ag-NOR distribution in any of the ages studied. In summary, our paper remarks the accuracy of a novel technique used to study the effects of malnutrition, as an indirect index of protein biosynthesis, which is simple, affordable and rapid as compared to the conventional autoradiographic studies of radioactive amino-acid incorporation for the same purpose.