M. Dulce Madeira

Behavioral and neuroanatomical consequences of chronic ethanol intake and withdrawal.

We have examined if long-term (13 months) alcohol consumption and the same treatment followed by a 6-week withdrawal period cause different neuropathological changes in rats. Spatial reference and working memory of alcohol-consuming and withdrawn rats were evaluated by comparison of their performance with age-matched controls in the Morris water maze. In the reference memory task we did not observe significant cognitive deficits in rats continuously exposed to ethanol, whereas withdrawn animals showed an obvious impairment of their overall performance. The reference memory deficit in withdrawn rats was evident in the spatial probe trial; these animals required significantly longer swimming distances to approach the former position of the platform when compared with controls and alcohol-consuming animals. In contrast, working memory was not significantly altered in either experimental group. Stereological methods were applied to compare the neurodegenerative changes produced by alcohol intake and withdrawal in the hippocampal formation. In the alcohol-consuming animals there was a significant cell loss in CA1 (18%) and CA3 (19%) hippocampal regions. Moreover, in withdrawn rats there was a further decay in the total number of pyramidal neurons, which amounted to 15% relative to nonwithdrawn animals. In the granular layer of the dentate gyrus there was a trend in the same direction, but it did not reach significance. Thus, our findings indicate that withdrawn rats are cognitively impaired relative to animals submitted to continuous alcohol consumption and to age-matched controls, which fits the morphological data showing that withdrawal aggravates ethanol-induced degenerative processes in the hippocampal formation.

Influence of sex and estrus cycle on the sexual dimorphisms of the hypothalamic ventromedial nucleus: Stereological evaluation and golgi study

Neurons in the ventromedial nucleus of the hypothalamus (VMN) display structural and biochemical sex differences in response to estrogen. Despite this fact, reports on sex differences in the morphology of the VMN are restricted to its volume and synaptic patterning. The aim of this study was to characterize the neuroanatomical sexual dimorphisms in the VMN and to investigate whether endogenous changes in ovarian steroid secretion influence such dimorphisms. The VMN of adult male rats and intact, aged‐matched female rats killed on proestrus and diestrus day 1 was examined by using stereological methods applied to conventionally stained sections and Golgi‐impregnated material. The VMN contained 55,000 neurons in rats of both sexes, but its volume was, on average, 1.25 times larger in males than in females. The volume was greater in proestrus than in diestrus rats due to parallel changes in the neuronal somatic size. Unlike the dorsomedial division, neurons in the ventrolateral division had longer dendritic trees in proestrus than in diestrus females and males. The spine density was consistently higher in females than in males in both VMN divisions. In addition, in the ventrolateral part the magnitude of the sex differences varied across the estrus cycle, and reached the greatest value when females were in proestrus. The volume of the neuropil was significantly larger in males than in females, and was not affected by the estrus phase. Our results reveal that the magnitude of the neuroanatomical sex differences in the VMN vary across the estrus cycle due to the trophic influence of estrogen upon its neurons. They also show that the fundamental sex difference in the structure of the VMN is accounted for by the neuropil components. J. Comp. Neurol. 432:329–345, 2001.

Prolonged alcohol intake leads to irreversible loss of vasopressin and oxytocin neurons in the paraventricular nucleus of the hypothalamus.

Previous data revealed that numerous neurons in the supraoptic nucleus degenerate after prolonged ethanol exposure, and that the surviving neurons increase their activity in order to prevent dramatic changes in water metabolism. Conversely, excess alcohol does not induce cell death in the suprachiasmatic nucleus, but leads to depression of neuropeptide synthesis that is further aggravated by withdrawal. The aim of the present study is to characterize the effects of prolonged ethanol exposure on the magnocellular neurons of the paraventricular nucleus (PVN) in order to establish whether or not magnocellular neurons display a common pattern of reaction to excess alcohol, irrespective of the hypothalamic cell group they belong. Using conventional histological techniques, immunohistochemistry and in situ hybridization, the structural organization and the synthesis and expression of vasopressin (VP) and oxytocin (OXT) in the magnocellular component of the PVN were studied under normal conditions and following chronic ethanol treatment (6 or 10 months) and withdrawal (4 months after 6 months of alcohol intake). After ethanol treatment, there was a marked decrease in the number of VP- and OXT-immunoreactive magnocellular neurons that was attributable to cell death. The surviving neurons were hypertrophied and the VP and OXT mRNA levels in the PVN unchanged. Withdrawal did not alter the number of VP- and OXT-producing neurons or the gene expression of these peptides. These results substantiate the view that after prolonged ethanol exposure numerous neurons of the hypothalamic magnocellular system degenerate, but the mRNA levels of VP and OXT are not decreased due to compensatory changes undergone by the surviving neurons.

Arcuate nucleus of the hypothalamus: Effects of age and sex

The arcuate nucleus of the hypothalamus (ARN) is involved in a variety of functions known to be sexually dimorphic and altered by aging. Although the effects of sex and age on the synaptic organization and neurochemistry of the ARN have been extensively analyzed, data regarding sex‐related differences and age‐induced effects on the total number of neurons and volume of the ARN in adult and aged male and female rats are controversial. To address this issue, we have quantitatively analyzed the ARN of male and female Wistar rats aged 6 and 24 months. The optical fractionator, the optical rotator, and the Principle of Cavalieri were used as the estimators of the total number of neurons, mean nuclear volume of ARN neurons, and volume of the ARN, respectively. In addition, a Golgi study was carried out to analyze the dendritic trees of its neurons. We found that in young adult rats, the volume of the ARN is 0.9 mm3in males and 0.7 mm3in females, whereas the total number of neurons is 100 × 103 in males and 86 × 103 in females. ARN neurons of males and females have identical mean nuclear volumes, which we estimated to be 300 μm3. No significant effects of age were found in these parameters, both in males and in females. In adult rats, no sex‐related differences were detected in the number of dendritic segments and in the total dendritic length, but the dendritic branching density and the spine density were greater in females than in males. In aged rats there was a significant reduction in the number of dendritic segments, in the total dendritic length, and in the branching and spine densities that, although evident in both sexes, was more marked in females. Our results show that the total number of neurons and the volume of the ARN are sexually dimorphic in adult and aged rats and that neither of these parameters is altered by aging. Conversely, aging induces regressive changes in the dendritic arborizations of ARN neurons of males and females and abolishes the sexual dimorphic pattern of their organization. J. Comp. Neurol. 401:65–88, 1998.

Effects of alcohol on the synthesis and expression of hypothalamic peptides

Studies aimed at analyzing the deleterious effects of excess alcohol in the brain have revealed structural alterations that are often associated with functional and behavioral disturbances. Among the neuronal damage related to prolonged alcohol exposure, alterations in the synthesizing capabilities and levels of expression of neuroactive peptides have been increasingly reported. Actually, such changes frequently represent the sole repercussion of acute and short-term exposure to ethanol. This review gathers the existing data on the effects of ethanol exposure on the synthesis and expression of hypothalamic peptides. Amid those that can act both as neurotransmitters and neurohormones, we allude to vasopressin, corticotropin-releasing hormone, thyrotropin-releasing hormone and pro-opiomelanocortin and related peptides produced by paraventricular, supraoptic and arcuate neurons. With respect to peptides that act exclusively as neurotransmitters, we address the effects of alcohol on vasoactive intestinal polypeptide, gastrin-releasing peptide, somatostatin and vasopressin synthesized by suprachiasmatic neurons. Hypothalamic neurons that produce peptides that act as neurotransmitters are supposed to be modulated primarily by influences exerted by neuronal afferents, whereas those producing peptides that additionally act as neurohormones are also regulated by peripheral stimuli (e.g., plasma levels of circulating hormones, osmotic challenges). These peculiar features endue the hypothalamus with characteristics that are particularly propitious to enlighten the still cryptic mechanisms underlying the ethanol effects on protein synthesis.

Synaptic reorganization in the hippocampal formation of alcohol-fed rats may compensate for functional deficits related to neuronal loss

We have examined the behavioral and neuroanatomical effects of long-term alcohol intake in rats ingesting a 20% solution of ethanol for 30 weeks. Previous studies have shown that this treatment provokes neuronal degeneration in the hippocampal formation, which occurs in parallel with remodeling processes. Spatial reference and working memory of alcohol-fed rats were evaluated during last 4 weeks of treatment by comparison of their performance with age-matched controls on the Morris water maze. Alcohol consumption did not affect the performance of rats in the reference memory task as indicated by the measures derived from the acquisition trials and from the probe-trial, which were highly similar for alcohol-fed and control animals. Also, performance in the working memory task was not significantly altered in alcohol-treated animals. No treatment-related changes in swim speed or impairments of sensorimotor abilities, tested in the visible platform task, were detected. Stereological methods were applied to evaluate the damage inflicted by alcohol intake in the structure of the hippocampal formation. In the alcohol-treated animals, there was a noticeable cell loss in the granular layer of the dentate gyrus (10%), and in CA3 (18%) and CA1 (19%) hippocampal subdivisions. In spite of the neuronal loss, the total number of synapses between mossy fibers and CA3 pyramids was unaffected by alcohol treatment suggesting that new synaptic contacts were formed between the surviving neurons. We show that, regardless the marked hippocampal cell loss in rats exposed to chronic alcohol intake, the reorganization that takes place at the synaptic level may alleviate the expected functional deficits.

Effects of age and sex on the water maze performance and hippocampal cholinergic fibers in rats

We have examined if age-related deterioration of spatial memory and cholinergic innervation of the dentate gyrus is gender-specific. Aging progressively affected the performance of male and female rats in place discrimination version of the water maze task. On repeated acquisition task, only old males, but not old females, were significantly impaired relative to young and adult animals of both sexes. In parallel, we found that the age-associated reduction of the density of cholinergic fibers in the dentate gyrus was significantly more profound in old males than in age-matched females. These results suggest that, although male and female rats have an identical pattern of reference memory decline, impairment of the working memory and deterioration of the hippocampal cholinergic system are slower to develop in females than in males.

Prolonged alcohol intake leads to reversible depression of corticotropin-releasing hormone and vasopressin immunoreactivity and mRNA levels in the parvocellular neurons of the paraventricular nucleus

The ability of alcohol to activate the hypothalamic-pituitary-adrenal (HPA) axis is well documented in investigations based in acute and short-term experimental paradigms. Herein, we have addressed the possibility that the prolonged exposure to ethanol concentrations that are initially effective in stimulating corticosteroid secretion might induce alterations in the response of the HPA axis that cannot be evinced by shorter exposures. Using conventional histological techniques, immunohistochemistry and in situ hybridization, we have examined the medial parvocellular division of the paraventricular nucleus (PVNmp), and the synthesis and expression of corticotropin-releasing hormone (CRH) and vasopressin (VP) by its constituent neurons, in rats submitted to 6 months of ethanol treatment and to withdrawal (2 months after 6 months of alcohol intake). Ethanol treatment and withdrawal did not produce neuronal loss in the PVNmp. However, the total number of CRH- and VP-immunoreactive neurons and the CRH mRNA levels were significantly decreased by ethanol treatment. In withdrawn rats, the number of CRH- and VP-immunostained neurons and the gene expression of CRH were increased relative to ethanol-treated rats and did not differ from those of controls. No significant variations were detected in VP mRNA levels as a result of ethanol treatment or withdrawal. These results show that prolonged alcohol intake blunts the expression of CRH and VP in the parvocellular neurons of the PVN, and that this effect is, partially at least, reversible by withdrawal. They also suggest that the development of tolerance to the effects of ethanol involve changes that take place at the hypothalamic level.

Loss of synapses in the entorhinal-dentate gyrus pathway following repeated induction of electroshock seizures in the rat.

The goal of this study was to answer the question of whether repeated administration of electroconvulsive shock (ECS) seizures causes structural changes in the entorhinal‐dentate projection system, whose neurons are known to be particularly vulnerable to seizure activity. Adult rats were administered six ECS seizures, the first five of which were spaced by 24‐hr intervals, whereas the last two were only 2 hr apart. Stereological approaches were employed to compare the total neuronal and synaptic numbers in sham‐ and ECS‐treated rats. Golgi‐stained material was used to analyze dendritic arborizations of the dentate gyrus granule cells. Treatment with ECS produced loss of neurons in the entorhinal layer III and in the hilus of the dentate gyrus. The number of neurons in the entorhinal layer II, which provides the major source of dentate afferents, and in the granular layer of the dentate gyrus, known to receive entorhinal projections, remained unchanged. Despite this, the number of synapses established between the entorhinal layer II neurons and their targets, dentate granule cells, was reduced in ECS‐treated rats. In addition, administration of ECS seizures produced atrophic changes in the dendritic arbors of dentate granule cells. The total volumes of entorhinal layers II, III, and V–VI were also found to be reduced in ECS‐treated rats. By showing that treatment with ECS leads to partial disconnection of the entorhinal cortex and dentate gyrus, these findings shed new light on cellular processes that may underlie structural and functional brain changes induced by brief, generalized seizures.

Estrogen modulates the sexually dimorphic synaptic connectivity of the ventromedial nucleus.

Neurons in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) display a remarkable estrogen‐dependent functional and structural plasticity, which is likely to be mediated, in part at least, by neuronal afferents. The present study was designed to determine whether the number of synapses per neuron and the size of individual synapses in the VMNvl vary across the estrus cycle and, also, whether they differ between the sexes. To accomplish this, the VMNvl of adult female rats at proestrus or diestrus day 1 and of age‐matched male rats was analyzed using electron microscopy. We found that a single VMNvl neuron receives around 7,000 synapses during diestrus and ∼10,000 during proestrus. This estrus cycle‐related variation is accounted for by increases in the number of all types of synapses. In males, the number of synapses received by each VMNvl neuron is similar to that of diestrus rats (∼7,500). However, in males the number of axodendritic and axospinous synapses is smaller than in proestrus rats, whereas the number of axosomatic synapses is higher than in diestrus rats. In addition, we found that the size of the postsynaptic densities of axospinous and axosomatic synapses is consistently larger in males than in females. Our results show that the synaptic organization of the VMNvl is sexually dimorphic, with females having more dendritic synapses and males more somatic synapses. They also show that the synaptic plasticity induced by estrogen in the VMNvl is characterized by changes in the number, but not the size, of the synapses. J. Comp. Neurol. 484:68–79, 2005.