A. Cadete-Leite

Hippocampal mossy fiber-CA3 synapses after chronic alcohol consumption and withdrawal

A quantitative study of the CA3 pyramidal cells and of the mossy fiber-CA3 synapses (MF-CA3) of the rat hippocampal formation was performed in rats alcohol-fed for 6, 12 and 18 months and respective age-matched controls. Additional groups alcohol-fed for 6 and 12 months and withdrawn for 6 months were also studied. The numerical densities of the CA3 pyramids and of the synapses were calculated applying the disector method to adjacent sections of the CA3 pyramidal cell layer and the stratum lucidum respectively. The results showed a progressive loss of pyramidal cells in alcohol-treated and withdrawal groups and a significant decrease of MF-CA3 synapses after 18 months of alcohol feeding. Taking into account that both hippocampal granule and CA3 pyramidal cells are reduced, the maintenance of the relative number of MF-CA3 synapses in 6- and 12-month alcohol-fed rats suggests the formation of new contacts. The increased proportion of the MF plasmalemma occupied by synapses can also be interpreted as an additional compensation process. These data show that MF-CA3 synapses display plastic and degenerative changes after chronic alcohol consumption and withdrawal which presumably will lead to functional modifications of the hippocampal circuitry.

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.

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.

Effects of chronic alcohol intake and withdrawal on the prefrontal neurons and synapses

Previous studies demonstrated that alcohol induces marked deterioration of the cerebral cortex. However, quantitative evaluations of neurons and synapses of the prefrontal cortex from chronic alcohol-fed and withdrawn animals are nonexistent, in spite of the functional implications that can underlie structural changes in this cortical area. To achieve this evaluation, we used groups of rats alcohol-fed for 6, 12 and 18 months, their respective controls as well as groups of rats alcohol-fed for 6 and 12 months and then switched to water for 6 months--withdrawal groups. The thickness of the prelimbic cortical layers I-III was reduced in the alcohol-fed and withdrawal groups. A significant reduction in the density of cells was found, which was more marked after withdrawal. Conversely, the density of synapses increased after alcohol exposure and withdrawal; such numerical shifts lead to a significant increase in the synapse-to-neuron ratio. These results indicate that the cortical circuitry of the prelimbic cortex is vulnerable to prolonged periods of alcohol exposure and withdrawal and allow to advance the view that the alterations can induce functional implications.

A quantitative study of frontal cortex dendritic microtubules in patients with Alzheimer's disease

The dendritic microtubules (MT) of the frontal cortex layers II and III were studied in 9 patients with Alzheimers disease (AD) and the results compared with 9 case controls. Dendrites with abnormally oriented MTs and others depleted of these structures were seen. A significant reduction in the number of MTs per unit area was found in AD. It is suggested that microtubular changes in AD can interfere with neuroplasmic transport and thus, be implicated with the dendritic degeneration present in this disease.

Nerve growth factor prevents cell death and induces hypertrophy of basal forebrain cholinergic neurons in rats withdrawn from prolonged ethanol intake

We have previously reported that the hippocampal cholinergic fiber network is severely damaged in animals withdrawn from ethanol, and that a remarkable recovery in fiber density occurs following hippocampal grafting, a finding that we suggested to be underpinned by the graft production of neurotrophic factors, which are known to be decreased after ethanol exposure. It is widely accepted that nerve growth factor (NGF) signals the neurons of the brain cholinergic system, including those of the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) nuclei, from which the septohippocampal projection arises. Because neurons in these nuclei are vulnerable to ethanol consumption and withdrawal we thought of interest to investigate, in withdrawn rats previously submitted to a prolonged period of ethanol intake, the effects of intraventricular delivery of NGF upon the MS/VDB cholinergic neurons. Stereological methods were applied to estimate neuron numbers and neuronal volumes in choline acetyltransferase (ChAT)-immunostained and Nissl-stained material. We have found that in ethanol-fed rats there was a significant reduction in the total number of Nissl-stained and cholinergic neurons in the MS/VDB, and that the suppression of ethanol intake further decreased neuron numbers. In addition, the somatic size of ChAT-IR neurons was reduced by ethanol intake, and withdrawal further aggravated neuronal atrophy. NGF treatment prevented the withdrawal-associated loss, and induced hypertrophy, of cholinergic neurons. These findings show that exogenous NGF protects the phenotype and prevents the withdrawal-induced degeneration of cholinergic neurons in the MS/VDB. These effects might be due to the trophic action of NGF upon the basal forebrain cholinergic neurons, including the hippocampal fiber network that conveys this neurotrophin retrogradely to the MS/VDB, and/or upon their targets, that is, the hippocampal formation neurons.

Long-term low-protein diet reduces the number of hippocampal mossy fiber synapses

Previously we have reported a loss of the dentate granule cells and hippocampal CA3 pyramidal cells in adult animals after lengthy periods of low-protein diet. In this study we examined the effects of this cell loss upon the synaptic connections between the granule cell axons (the mossy fibers) and CA3 pyramidal cell dendrites. Three groups of five rats each were given a low-casein (8%) diet for 6, 12, and 18 months, respectively, and the results of the ultrastructural morphometric analysis compared with similarly processed control rats kept on a control diet. The numerical density of synapses was decreased in undernourished rats and the fraction of the mossy fiber terminal membrane occupied by synaptic specializations was reduced. It can be inferred that the synaptic connectivity pattern between mossy fiber terminals and CA3 dendrites is altered due to a reduction in the number of contacts. Besides, as the synapses of low-protein-treated animals do not display any increase in the length of their active zones, evidence is not provided for the existence of morphological synaptic plasticity, contrary to what has been recently described in other experimental circumstances.

Effects of chronic alcohol consumption on the cholinergic innervation of the rat hippocampal formation as revealed by choline acetyltransferase immunocytochemistry.

The specific aim of this study was to evaluate whether the cholingeric innervation of the hippocampal formation is affected by chronic alcohol consumption in the rat. Choline acetyltransferase-immunoreactive fibres and neurons were analysed in both alcohol-fed and control rats using a monoclonal antibody against choline acetyltransferase and quantitative methods. We found a global reduction in the cholinergic plexus, which was more pronounced in the hippocampus proper than in the dentate gyrus. The areal density of choline acetyltransferase immunoreactive neurons was also reduced. Differences from controls in neuronal number were particularly striking in the stratum lacunosum moleculare of the regio superior, which is precisely the zone of the hippocampal formation where choline acetyltransferase immunoreactive neurons are more abundant in controls. In conclusion, our results show that prolonged ethanol consumption leads to a substantial reduction in the cholinergic innervation of the hippocampal formation, as there was a loss of cholinergic fibres and also an apparent loss of hippocampal cholingeric neurons. These findings may help to explain the cognitive dysfunctions observed after chronic alcohol consumption.

Basal forebrain neurons modulate the synthesis and expression of neuropeptides in the rat suprachiasmatic nucleus.

We tested the hypothesis that efferents from the nucleus basalis magnocellularis (NBM) play a direct role in the regulation of neuropeptide synthesis and expression by neurons of the rat suprachiasmatic nucleus (SCN). Adult male rats in which the NBM was destroyed with quinolinic acid, either unilaterally or bilaterally, were compared with rats injected with physiological saline and with control rats. The estimators used to assess the effects of cholinergic deafferentation on the neuroanatomy and neurochemistry of the SCN were the total number of SCN neurons, the total number and somatic size of SCN neurons producing vasopressin (VP) and vasoactive intestinal polypeptide (VIP), and the respective mRNA levels. Bilateral destruction of the NBM did not produce cell death in the SCN, but caused a marked reduction in the number and somatic size of SCN neurons expressing VP and VIP, and in the mRNA levels of these peptides. The decrease in the number of VP- and VIP-producing neurons provoked by unilateral lesions was less striking than that resulting from bilateral lesions. It was, however, statistically significant in the ipsilateral hemisphere, but not in the contralateral hemisphere. The results show that the reduction of cholinergic inputs to the SCN impairs the synthesis, and thereby decreases the expression of neuropeptides by SCN neurons, and that the extent of the decline correlates with the amount of cholinergic afferents destroyed. This supports the notion that acetylcholine plays an important, and direct role in the regulation of the metabolic activity of SCN neurons.

Nerve growth factor improves spatial learning and restores hippocampal cholinergic fibers in rats withdrawn from chronic treatment with ethanol

Abstract. The cholinergic septohippocampal pathway has long been known to be important for learning and memory. Prolonged intake of ethanol causes enduring memory deficits, which are paralleled by partial depletion of hippocampal cholinergic afferents. We hypothesized that exogenous supply of nerve growth factor (NGF), known to serve as a trophic substance for septal cholinergic neurons, can revert the ethanol-induced changes in the septohippocampal cholinergic system. Adult rats were given a 20% ethanol solution as their only source of fluid for 6 months. During the first 4 weeks after the animals were withdrawn from ethanol, they were intraventricularly infused with either NGF or vehicle alone via implanted osmotic minipumps. The vehicle-infused withdrawn animals showed impaired performance on a spatial reference memory version of the Morris water maze task, both during the task acquisition and on the retention test. In contrast, NGF-treated withdrawn rats were able to learn the task as well as controls, and significantly outperformed the vehicle-infused withdrawn rats. The histological analysis revealed that, in the latter group, the length density of fibers immunoreactive to choline acetyltransferase was reduced relative to control values by approximately 25%, as measured in the dentate gyrus and regio superior of the hippocampal formation. However, in NGF-treated withdrawn rats, the length density of these fibers was identical to that of control rats. These data provide support to the notion that NGF is capable of ameliorating memory deficits and restoring septohippocampal cholinergic projections following chronic treatment with ethanol.