Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study.

The effects of hypothyroidism upon the structure of the central nervous system of adult rats are poorly understood in spite of evidence that the mature brain is vulnerable to this condition. Existing developmental studies show that the morphological changes induced by thyroid hormone deficiency are related to alterations in neurogenesis. We studied the granular layer of the dentate gyrus under different experimental conditions of hypothyroidism, because in rodents the neurogenesis of the granule cells continues during adulthood. The following groups of rats were analysed: 1) control; 2) hypothyroid from day 0 until day 180 (hypothyroid group); 3) hypothyroid until day 30 and henceforth maintained euthyroid (recovery group); and 4) hypothyroid since day 30 (adult hypothyroid group). Groups of 6 male rats and 6 female rats were analysed separately. The volume of the dentate gyrus granular layer and the numerical density of its neurons were evaluated, so we were able to estimate the total number of granule cells. Because in the experimental groups the volume of the granular layer and the numerical density of its neurons were reduced, the total number of granule cells was decreased. In the hypothyroid and recovery groups the alterations were identical and more striking than in the adult hypothyroid groups. The total number of granule cells displayed sexual differences in all groups studied except in the hypothyroid groups. The present results support the view that thyroid hormone deficiency interferes with the process of cell acquisition by reducing neuronal proliferation and that it also leads to increased cell death. These events underlie the irreversible morphological changes observed in the brain of hypothyroid rats, either during development or at maturity. The referred structural alterations are probably related to the functional deficits observed in this condition.

Selective vulnerability of the hippocampal pyramidal neurons to hypothyroidism in male and female rats.

Thyroid hormone deficiency has long been considered to affect profoundly such cognitive functions as learning and memory, which are known to depend on the structural integrity of the hippocampal formation. Since we previously found that the number of granule cells of the dentate gyrus is reduced in hypothyroid animals, we decided to extend our observations to the pyramidal cells of the hippocampus in order to gain further insight into the effects of hypothyroidism upon the other neuronal links of the hippocampal trisynaptic circuitry, inasmuch as CA1 neurons are known to be particularly vulnerable to aggressive agents. Groups of 6 male and 6 female rats aged 30 and 180 days were analysed separately after being treated as follows: (1) hypothyroid from day 0 until day 30 (30-day-old hypothyroid group); (2) respective 30-day-old control; (3) hypothyroid from day 0 until day 180 (180-day-old hypothyroid group); (4) hypothyroid until day 30 and thenceforth maintained euthyroid (recovery group); (5) hypothyroid since day 30 (adult hypothyroid group); and (6) respective 180-day-old control. The volume of the pyramidal cell layer of the CA1 and CA3 regions and the numerical density of the respective neurons were evaluated, thereby allowing us to estimate the total number of pyramidal cells in each hippocampal region. The areal density and the mean nuclear volume of CA1 and CA3 pyramidal cells were also estimated. In the CA3 region, we found that hypothyroidism, whatever its duration and time of onset, induces a reduction in the volume of the pyramidal cell layer and a parallel increase in the numerical density of its neurons, without interfering with the total number of pyramidal cells. Conversely, in the CA1 region, thyroid hormone deficiency started either neonatally or during maturity was found to lead to a decrease in the total number of pyramidal cells. Reductions ranging between 14.2 and 22.5% were found in 30 and 180-day-old hypothyroid groups. The reestablishment of a euthyroid state did not ameliorate the referred neuronal loss. The present results support the view that hypothyroidism induces small alterations in the structural organization of the hippocampal CA3 region, contrary to what happens in CA1 in which neuronal death occurs. Furthermore, the data presented herein demonstrate that the total number of CA1 pyramidal cells displays sexual dimorphism that is not affected by thyroid hormone manipulations.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The supraoptic nucleus in hypothyroid and undernourished rats: an experimental morphometric study.

The supraoptic nuclei of both male and female 30-day-old rats rendered hypothyroid by daily subcutaneous injection of propylthiouracil were studied and the results were compared with age- and sex-matched rats fed ad libitum (control rats) and with undernourished rats. Morphometric methods were used to evaluate the volume of the supraoptic nucleus and the areal and numerical densities of its neurons. These parameters allowed us to estimate the total number of neurons of this nucleus. In addition, the mean cross-sectioned area and mean nuclear diameter of the same neurons were also evaluated. The volume of the supraoptic nucleus was reduced both in hypothyroid and undernourished animals when compared with normal controls. The areal and numerical densities of neurons from the former groups were increased and the volume density of the neuropil was reduced. As a consequence, the total number of neurons was found to be identical in all groups studied. Furthermore, the mean nuclear diameters and cross-sectioned areas of the supraoptic neurons were similar in all groups. The volumetric differences and the increased packing found were always more marked for hypothyroid than for undernourished rats. Differences were not detected between male and female groups. The present results support the view that the increased cell packing in hypothyroid animals depends upon a reduction in the neuropil of the nucleus, as has been described under similar conditions in other central nervous system areas displaying identical patterns of neurogenesis. In addition, it was found that the effects of undernourishment cannot be discriminated from those dependent on neonatal hypothyroidism.

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.

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.

Intracerebral grafts promote recovery of the cholinergic innervation of the hippocampal formation in rats withdrawn from chronic alcohol intake. An immunocytochemical study.

We have previously found that alcohol withdrawal aggravates the neuronal cell loss induced by chronic alcohol consumption in the rat hippocampal formation. We have also shown that intracerebral grafts of immature hippocampal tissue could reverse the progressive degeneration that occurs during this withdrawal. Furthermore, we have shown that chronic alcohol consumption reduces the areal density of choline acetyltransferase-immunoreactive neurons and the density of choline acetyltransferase-immunoreactive fibres in the hippocampal formation. Thus, we thought it would be of interest to investigate the effects of alcohol withdrawal in the hippocampal cholinergic innervation and to determine whether the intracerebral grafting of immature hippocampal tissue would have beneficial effects upon the cholinergic system in this condition. Choline acetyltransferase-immunoreactive fibres and perikarya were analysed in 14-month-old control, alcohol-fed, withdrawal and withdrawal-grafted groups of rats. The areal density of choline acetyltransferase-immunoreactive neurons was reduced in all experimental groups when compared to controls. The density of choline acetyltransferase-immunoreactive fibres was lower in the alcohol-fed and withdrawal groups than in the control and withdrawal-grafted groups. We conclude that the grafted tissue probably produced neurotrophic factors which allowed a recovery of the hippocampal cholinergic fibre network. This recovery might be of importance to reverse the cognitive dysfunction described after chronic alcohol consumption and withdrawal.

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.

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 Alzheimer's 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.

Granule cell loss and dendritic regrowth in the hippocampal dentate gyrus of the rat after chronic alcohol consumption.

The effects of chronic alcohol consumption (CAC) on the relative number of dentate gyrus granule cells and their dendritic trees, were studied in animals fed alcohol for 6, 12 and 18 months and in their respective controls. The granule cell density was estimated with the unbiased disector method. Following 6 months of alcohol consumption, the thickness of the dentate gyrus granular layer and the relative number of dentate granule cells were significantly decreased when compared with controls. The granule cell dendritic arborizations showed an increase of their dendritic extent in alcohol-treated rats. No significant differences were found in the density of dendritic spines between alcohol-fed and control animals. These results indicate the existence of hippocampal granule cell dendritic regrowth in alcohol-fed rats, probably occurring as a compensatory response to the granule cell deficit which follows the alcohol-induced granule cell degeneration. These degenerative and regenerative changes might have functional implications for the organization of the synaptic hippocampal circuitry after long periods of alcohol consumption.

Alcohol withdrawal does not impede hippocampal granule cell progressive loss in chronic alcohol-fed rats.

Using the unbiased disector method we have shown that chronic alcohol consumption induces a significant decrease of dentate gyrus granule cell density in alcohol-fed rats for 18 months. A still more dramatic reduction was observed in a group of age-matched rats alcohol-fed for 12 months and switched to water for 6 months (recovery group). These results indicate that a progressive neuronal loss of the hippocampal granule cells in not impeded after cessation of alcohol intake. It is thus suggested that once the mechanisms underlying the alcohol-induced neuronal degeneration are triggered, they continue to act even after withdrawal.

Giant multivesicular bodies in the rat hippocampal pyramidal cells after chronic alcohol consumption.

Multivesicular bodies (MVBs) with diameters up to 4.5 microns were observed in the hippocampal pyramidal cells of rats submitted to chronic alcohol consumption. A significant increase in the volumetric density (Vv) of these organelles was found in CA1 pyramidal cells. Transitional forms of MVBs towards lysosomes were seen. A failure in MVB's enzymatic hydrolytic mechanisms, due to the prolonged alcohol aggression, could underlie its formation.

Structural changes in the hippocampal formation after long-term alcohol consumption and withdrawal in the rat.

The effects of long-term alcohol consumption and withdrawal upon the structure of the rat hippocampal formation were studied by applying morphometric methods to material processed for light and electron microscopy. The somatostatinergic neurons of the hilus were also studied. Groups of 6 rats were treated as follows: (a) given alcohol for 6, 12 and 18 months; (b) paired controls; and (c) rats switched to a normal diet in the 6 months after 6 and 12 months of alcohol intake. A progressive loss of hippocampal neurons after chronic alcohol consumption was found. The loss was aggravated during withdrawal from alcohol, with the exception of the hilar cells. The dendrites of granule cells from the alcohol-treated rats displayed signs of regrowing, but they did not do so in rats withdrawn from alcohol. The synapses between mossy fibre terminals and CA3 dendrites appear to be rather resistant to alcohol insult, and evidence of morphological plasticity was found in withdrawn rats. If an homology can be established between humans and rodents then the changes observed in alcohol-fed rats can be regarded as underpinning some of the functional and behavioural alterations depicted under these circumstances. The peculiar changes found in some nerve cell populations after withdrawal of alcohol could be related to the deficient or incomplete functional recovery often seen after abstinence from alcohol.

Piracetam impedes hippocampal neuronal loss during withdrawal after chronic alcohol intake.

In previous studies we have demonstrated that prolonged ethanol consumption induced hippocampal neuronal loss. In addition, we have shown that withdrawal after chronic alcohol intake augmented such degenerative activity leading to increased neuronal death in all subregions of the hippocampal formation but in the CA3 field. In an attempt to reverse this situation, we tested, during the withdrawal period, the effects of piracetam (2-oxo-1-pyrrolidine acetamide), a cyclic derivative of gamma-aminobutyric acid, as there is previous evidence that it might act as a neuronoprotective agent. The total number of dentate granule, hilar, and CA3 and CA1 pyramidal cells of the hippocampal formation were estimated using unbiased stereological methods. We found out that in animals treated with piracetam the numbers of dentate granule, hilar, and CA1 pyramidal cells were significantly higher than in pure withdrawn animals, and did not differ from those of alcohol-treated rats that did not undergo withdrawal. These data suggest that piracetam treatment impedes, during withdrawal, the pursuing of neuronal degeneration.

GM1 and piracetam do not revert the alcohol-induced depletion of cholinergic fibers in the hippocampal formation of the rat.

Chronic alcohol consumption causes a depletion of the cholinergic fiber network in the rat hippocampal formation, which is not ameliorated by alcohol withdrawal. Following withdrawal from alcohol, there is a further loss of intrinsic hippocampal cholinergic neurons. In this study, we investigated whether treatment with putative neuroprotective agents during the entire withdrawal period would have beneficial effects upon the hippocampal cholinergic innervation. Adult male rats were alcohol-fed for 6 months and subsequently withdrawn from alcohol for 6 months. Some animals were treated with either ganglioside GM1 (35 mg/kg body weight s.c.), vehicle (saline s.c.), or piracetam (800 mg/kg body weight p.o.) for the entire withdrawal period. Choline acetyltransferase (ChAT) immunoreactive (IR) fibers and neurons were analyzed quantitatively in all four animal groups. There were no significant differences in the density of the ChAT-IR hippocampal fiber network when the pure withdrawal and withdrawal + vehicle groups were compared to the withdrawal + GM1 or withdrawal + piracetam groups. In contrast, the number of ChAT-IR interneurons in the hippocampal formation was higher in the withdrawal + GM1 or withdrawal + piracetam groups than in the pure withdrawal and withdrawal + vehicle groups. These results indicate that, in the doses used, neither neuroprotective agent had an effect upon the extrinsic cholinergic innervation, but they had a beneficial effect upon the hippocampal intrinsic cholinergic system.

Effects of GM1 ganglioside upon neuronal degeneration during withdrawal from alcohol.

In previous studies we demonstrated that chronic alcohol consumption induced hippocampal cell and synapse loss in offset with an increase in the length of granule cell dendrites. In addition we observed that withdrawal after long periods of alcohol intake worsened the degenerative processes and that dendritic alterations were no longer apparent. In an attempt to reverse these structural changes we tested the action of GM1 ganglioside during the withdrawal period as there is evidence that GM1 may enhance neuronal recovery after different kinds of brain lesions. Cell and synaptic quantifications were performed and the branching pattern of the granule cell dendritic arborizations was analysed. The number of dentate granule and CA3 pyramidal cells from GM1-treated animals was found not to be significantly different from that of the alcohol-treated and withdrawal groups. No quantitative changes were found in the number of mossy fiber-CA3 pyramidal cell synapses when the aforementioned groups were compared. Whether the lack of effectiveness of GM1 can be related to the model employed or not is thoroughly discussed.

Piracetam promotes mossy fiber synaptic reorganization in rats withdrawn from alcohol.

Prolonged alcohol intake affects the morphology of the hippocampal formation of the rat and the resulting alterations do not reverse after withdrawal. Actually, an increase of the degenerative activity might occur in this condition. This unexpected observation prompted us to test the efficacy of neuronoprotective drugs during withdrawal. Because in a previous study we found that piracetam, a cyclic derivative of GABA, once added during withdrawal impedes hippocampal cell loss, we decided to evaluate the effect of this compound at the synaptic level. Using unbiased stereological techniques, we estimated the total number of contacts between mossy fibers and CA3 pyramids, as well as the volume and the surface area of the respective pre- and postsynaptic compartments. We found that in piracetam-treated withdrawn rats the number of synapses was higher than that observed in nonpiracetam-treated and alcohol-fed animals. The mechanisms leading to the synaptic reorganization took place at the mossy fiber level. The postsynaptic compartment does not seem to participate in the reorganization. It is suggested that the role of piracetam in this process might depend on the protective effect that this compound has upon glutamatergic receptors.

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.

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.