Maria Dulce Madeira

Reorganization of the morphology of hippocampal neurites and synapses after stress-induced damage correlates with behavioral improvement.

We recently demonstrated that stress-induced cognitive deficits in rats do not correlate with hippocampal neuronal loss. Working on the premise that subtle structural changes may however be involved, we here evaluated the effects of chronic stress on hippocampal dendrite morphology, the volume of the mossy fiber system, and number and morphology of synapses between mossy fibers and CA3 dendritic excrescences. To better understand the mechanisms by which stress exerts its structural effects, we also studied these parameters in rats given exogenous corticosterone. Further, to search for signs of structural reorganization following the termination of the stress and corticosterone treatments, we analysed groups of rats returned to treatment-free conditions. All animals were assessed for spatial learning and memory performance in the Morris water maze. Consistent with previous findings, dendritic atrophy was observed in the CA3 hippocampal region of chronically stressed and corticosterone-treated rats; in addition, we observed atrophy in granule and CA1 pyramidal cells following these treatments. Additionally, profound changes in the morphology of the mossy fiber terminals and significant loss of synapses were detected in both conditions. These alterations were partially reversible following rehabilitation from stress or corticosterone treatments. The fine structural changes, which resulted from prolonged hypercortisolism, were accompanied by impairments in spatial learning and memory; the latter were undetectable following rehabilitation. We conclude that there is an intimate relationship between corticosteroid levels, hippocampal neuritic structure and hippocampal-dependent learning and memory.

Effects of corticosterone treatment and rehabilitation on the hippocampal formation of neonatal and adult rats. An unbiased stereological study

Elevations in the plasma levels of glucocorticoids are associated with cognitive impairments that have been ascribed to loss of neurons in the hippocampal formation. However, recent studies have strongly challenged this view. In order to clarify this issue, we have employed for the first time the optical fractionator and the Cavalieri principle, two unbiased stereological tools, to estimate respectively the total number of neurons and the volumes of the main subdivisions of the hippocampal formation of rats submitted to corticosterone treatment for different periods, either neonatally or in adulthood. A significant reduction in the number of neurons and in the volumes of the layers of the dentate gyrus and CA3 hippocampal field was found in rats exposed to glucocorticoids in the neonatal period; furthermore, animals treated with corticosterone from birth until 180 days of age had also a reduction in the volume of the stratum radiatum of the CA1 hippocampal field. Conversely, when the exposure occurred only during adulthood, no significant neuronal loss was observed, but there were significant reductions in the volume of layers in the dentate gyrus and CA3 hippocampal field. To search for signs of structural recovery, we incorporated a group of rats submitted to corticosterone treatment during the neonatal period in which the hormonal conditions were restored thenceforth. In this group we found a significant increase in the volume of the molecular layer of the dentate gyrus when compared with rats that were kept under corticosteroid treatment. In conclusion, these data provide a sound structural basis for the cognitive deficits observed during, and following, exposure to increased levels of glucocorticoids.

Maintenance of Hippocampal Cell Numbers in Young and Aged Rats Submitted to Chronic Unpredictable Stress. Comparison with the Effects of Corticosterone Treatment

Exposure of rats to sustained stress has been associated with behavioural impairments, the degree of impairment being greater with increasing age of the subject. Although the behavioural deficits have been frequently attributed to stress-induced neuronal loss in the hippocampus, the validity of that view may be disputed since it is based on data collected using conventional morphometric methods which are subject to bias. The question of whether stress per se does indeed induce hippocampal cell losses was therefore re-examined using unbiased stereological tools in the present work. Specifically, we used the optical fractionator and the Cavalieri principle, to respectively estimate the total number of neurons and volumes of the main divisions of the hippocampal formation of young and old rats which had been exposed for 1 month to an unpredictable stress paradigm. The efficacy of the treatment was confirmed by elevated serum corticosterone levels measured at various intervals during the experimental period. In order to evaluate whether any deleterious effects might have occurred merely due to the stress-induced elevations in corticosterone secretion, we conducted a parallel study on animals that were injected with corticosterone over a similar duration. Neither stress nor treatment with corticosterone was found to result in significant cell losses in any division of the hippocampal formation; likewise, neither treatment produced significant volumetric differences. Further, these results were not influenced by age of the experimental subjects. The present findings therefore call for a reappraisal of the hypothesis that hippocampal cell loss accounts for the behavioural impairments observed by others following prolonged stress and/or chronic elevation of serum corticosterone levels.

Sexual dimorphism in the mossy fiber synapses of the rat hippocampus

SummaryThe presence of sexual dimorphism in the hippocampal formation has long been recognized. Differences between male and female rats have been detected with respect to the number of dentate granule cells and branching patterns of dentate granule and hippocampal pyramidal cell dendrites. Groups of 6 male and 6 female Sprague-Dawley rats were studied at 180 days of age. Based on light microscopical Timm-staining and Golgi-impregnation and electron microscopy, and applying morphometric techniques, we now report that the total number of synapses between mossy fibers and the apical dendritic excrescences of CA3 pyramidal cells is the same in male and female rats, despite a higher numerical density in the latter. Moreover, the volume of the mossy fiber system was found to be smaller in females. Because the number of dentate granule cells is smaller in females than in males, the increased numerical density of synapses may be thought of as a compensatory mechanism to equalize the number of synaptic contacts between dentate granule and CA3 pyramidal cells in the two sexes. We demonstrate that an increase in the number of mossy fiber boutons in female rats is a determining factor for the sexual differences found.

Effects of chronic alcohol consumption and of dehydration on the supraoptic nucleus of adult male and female rats.

Ethanol ingestion affects the hypothalamo-neurohypophysial system resulting in increased diuresis, dehydration and hyperosmolality. We studied the supraoptic nucleus, of the hypothalamus, in ethanol-treated rats, to determine if ethanol alone and/or the associated disturbances of water metabolism lead to structural alterations in a nucleus known to play a central role in fluid homeostasis. Groups of male and female rats were ethanol-treated until 12 and 18 months of age and compared with age-matched pair-fed controls. Twelve and 18-month-old control groups and 12-month-old water control groups (rats submitted to chronic dehydration) were also included in this study in an attempt to differentiate between the effects of undernutrition and dehydration/hyperosmolality, and the specific neurotoxic effects of ethanol. We estimated the volume of the supraoptic nucleus and the numerical density of its neurons and calculated the total number of supraoptic neurons. The volume of both supraoptic neurons and neuropil were also estimated. In immunostained material the ratio of vasopressin to oxytocin neurons and the cross-sectional areas of the two neuronal types were evaluated. There was marked neuronal loss in alcohol-treated rats, but the volume of the supraoptic nucleus was increased. The increase in the volume of the supraoptic nucleus correlated with and was due to increases in the volume was particularly marked for vasopressin neurons. No significant differences were found between controls and pair-fed controls in any of the parameters investigated. In water control rats, the volume of the supraoptic nucleus and of the supraoptic neurons and neuropil was also greater than in pair-fed controls. However, the variations found were not as marked as in ethanol-treated rats and there was no cell loss. These findings reveal, for the first time, that chronic ethanol consumption affects the morphology of supraoptic neurons and neuropil and, consequently, the structure of the entire supraoptic nucleus. Moreover, this study supports the view that ethanol has direct neurotoxic effects on supraoptic neurons because the alterations that occur are not mimicked in animals in which water metabolism alone is disturbed.

Dendritic changes in the hippocampal formation of AIDS patients: a quantitative Golgi study

We have previously shown that in the hippocampal formation of patients with acquired immunodeficiency syndrome (AIDS) there is neuronal atrophy, without cell loss. Because reductions in neuronal size are suggestive of associated neuritic alterations, we decided to study the dendritic trees of the main neuronal populations in the hippocampal formation. Material was obtained in five male AIDS patients and five male controls. After Golgi impregnation, the dendritic arborizations of dentate granule and hilar basket cells, and of CA3 and CA1 pyramidal cells, were hand traced, and their segments classified, counted and measured. We found an impoverishment of the dendritic trees in all neuronal populations in the AIDS group, which was more striking in the hilus and CA3 field. Specifically, hilar neurons had fewer dendritic segments, and reduced branching density and dendritic extent; in CA3 pyramids there was a decrease in the number of terminal segments in the basal trees, and a reduction in the total number of segments, number of medium order terminals, dendritic branching density and dendritic extent in the apical trees. In CA1 pyramids, the terminals were shorter in the apical trees and the dendritic spine density decreased in the basal trees, whereas in granule cells only the dendritic spine density was reduced in AIDS patients. Subtle signs suggestive of dendritic reorganization were observed. These results point to a regional vulnerability of the hippocampal formation to HIV infection, and might contribute to explaining the occurrence of dementia, as a consequence of overall reduction in the hippocampal neuronal receptive surface.

The supraoptic nucleus of the adult rat hypothalamus displays marked sexual dimorphism which is dependent on body weight

The neurons of the supraoptic nucleus in the rat hypothalamus are reported not to possess receptors for gonadal steroids and sexual dimorphism has not previously been described in this nucleus. We have analysed this nucleus in groups of Sprague-Dawley rats (six males or six females per group), one, two, six, 12 and 18 months after birth. Body and brain weights were recorded, the volume of the nucleus was determined from the right hemisphere and all other quantitative parameters were determined from the left nucleus. In addition, different groups of four male and four female rats aged two and 18 months were analysed after immunocytochemical staining to distinguish between vasopressin and oxytocin neurons. The total number of neurons was constant in all groups studied, despite which the volume of the supraoptic nucleus increased progressively with age in both males and females. The cross-sectional areas and volumes of supraoptic neurons also increased with age. The volume density of the neuropil remained constant in all groups and there was a progressive decrease with age in the numerical density of neurons. Immunocytochemistry revealed that the age-dependent increases in the size of the neurons involved primarily the vasopressin neurons. The age-related changes were much greater in males than in females, resulting in significant differences between the sexes at two, six, 12 and 18 months with respect to the volume of the supraoptic nucleus, the cross-sectional areas of neuronal somata and nuclei, and the volume of supraoptic neurons. Thus the supraoptic nucleus and its vasopressin neurons are larger in adult males than in age-matched females. Since we have also shown that body weight is very closely correlated with changes in the size of supraoptic neurons, and adult male rats are heavier than females of the same age, we suggest that these size changes reflect adaptation of the vasopressin neurons of the supraoptic nucleus to increasing functional demands associated with the regulation of water balance in bodies of increasing size.

Structural alterations of the hippocampal formation of adrenalectomized rats: an unbiased stereological study

Previous studies have demonstrated that adrenalectomy rapidly induces cell death in hippocampal formation. However, these previous studies have involved only qualitative observations or biased estimates. Therefore, the selectivity of the effects of adrenalectomy and the magnitude of changes occurring, remain controversial. The present work employed unbiased stereological tools to examine the effects of adrenalectomy on the number of neurons in, and the volume of, the hippocampal formation. Male rats were adrenalectomized 15,30 or 120 days before sacrifice at 180 days of age. The total number of neurons in the somal layers and hilus of the hippocampal formation was estimated using the optical fractionator. The volume of the different layers of each subdivision in the hippocampal formation was determined according to the Cavalieri principle. A progressive reduction, reaching 43%, was found in the total number of granule cells. Adrenalectomized animals exhibited a reduction in the volume of all layers of the dentate gyrus. No other region of the hippocampal formation displayed significant cell loss or a reduction in volume. In addition, the main neuronal subpopulations of the dentate gyrus were also evaluated, and a reduction in the total number of GABA- and neuropeptide Y-immunoreactive neurons in the molecular and granule cell layers of adrenalectomized rats was found. No quantitative changes were observed in the hilus. To characterize the glial response to the neuronal degeneration, we estimated the total number of cells immunoreactive for glial fibrillary acidic protein in the dentate gyrus. Although no variation in the total number of glial cells was found, signs of astroglial activation were observed in the adrenalectomized group. The present data strengthen the evidence pointing to the critical role of corticosteroids in maintaining the structural integrity of the dentate gyrus.

The dendritic trees of neurons from the hippocampal formation of protein-deprived adult rats. A quantitative Golgi study

We have recently shown that lengthy periods of low-protein feeding of the adult rat lead to deficits in the number of hippocampal granule and pyramidal cells, and in the number of mossy fiber synapses. These findings prompted us to analyze the dendrites of these neurons to evaluate whether, under the same experimental conditions, degenerative and/or plastic changes also take place at the dendritic level. The hippocampal formations from five 8-month-old rats fed a low-protein diet (casein 8%) for 6 months from the age of 2 months and from five age-matched controls were Golgi-impregnated and the morphology of the dendritic trees quantitatively studied. We found that in malnourished animals there was a reduction in the number of dendritic branches in the dentate granule cells and in the apical dendritic arborizations of CA3 pyramidal neurons. In addition, in the dentate granule cells the spine density was markedly increased and the terminal dendritic segments were elongated in malnourished animals. No alterations were found in the apical dendrites of CA1 pyramidal cells. The results obtained show that long periods of malnutrition induce marked, although not uniform, changes in the dendritic domain of the hippocampal neurons, which reflect the presence of both degenerating and regrowing mechanisms. These alterations are likely to affect the connectivity pattern of the hippocampal formation and, hence, the activity of the neuronal circuitries in which this region of the brain is involved.

Stereological evaluation and Golgi study of the sexual dimorphisms in the volume, cell numbers, and cell size in the medial preoptic nucleus of the rat

The medial preoptic nucleus (MPN) and the sexually dimorphic nucleus of the preoptic area (SDN-POA) stand out as prominent sexually dimorphic cell groups of the rat brain. However, quantitative data on sex-related differences in these nuclei in the adult rat are confined to their volume. We have used stereological methods and Golgi-impregnated material to examine whether, in young adult rats, the sexual dimorphism in the volume of the MPN, including its divisions, and of the SDN-POA, reflect similar differences in the number and size of their neurons. We found that the total number of neurons in all MPN divisions is higher and the mean somatic volume larger in males than in females. In addition, the total dendritic length of MPN neurons is greater, but the dendritic spine density is smaller, in males than in females. Likewise, in the SDN-POA the total number and size of its neurons is greater in males than in females. The sex differences in all quantitative parameters evaluated accounted for the larger volume of the MPN and SDN-POA in males relative to females. In addition, the MPN neuropil also displays sex-related differences in its volume, and these differences closely match those detected for the volume of each MPN division. It deserves to be emphasised that the numerical density of neurons was the only parameter found to be significantly higher in females than in males in all MPN divisions and in the SDN-POA. Our results show that the MPN and the SDN-POA display sex differences in the volume, total number of neurons, and size of neuronal cell bodies and dendritic trees. Furthermore, they also indicate that the neuropil is critical for the establishment of sexual dimorphism in the size of the MPN.