Susana M. Silva

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.

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.

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 restores mRNA levels and the expression of neuropeptides in the suprachiasmatic nucleus of rats submitted to chronic ethanol treatment and withdrawal

Chronic ethanol treatment and withdrawal from alcohol decrease the synthesis and expression of neuropeptides in the hypothalamic suprachiasmatic nucleus. Given the existing evidence that neurotrophins modulate the synthesis and expression of neurotransmitters/neuromodulators in the mature brain, we have hypothesized that such alterations might result from the reduced biological activity or brain content of neurotrophic factors. To test this possibility, nerve growth factor (NGF) was delivered intraventricularly, over a 4-week period, to rats submitted to ethanol treatment for 6 months and to withdrawn rats. Vasopressin (AVP) and vasoactive intestinal polypeptide (VIP), and the respective mRNAs were detected by immunocytochemistry and in situ hybridization histochemistry, and their levels estimated using stereological methods and densitometry. In ethanol-treated and withdrawn rats, NGF produced increases in the number of AVP- and VIP-immunostained neurons to values identical to those of controls. Corresponding variations were detected in AVP and VIP mRNA levels, which indicates that NGF restored the expression of AVP and VIP by enhancing neuropeptide synthesis. These findings show that NGF can correct the changes induced by chronic ethanol treatment and withdrawal in the gene expression and protein content of the neuropeptides synthesized by suprachiasmatic neurons. They also reveal that NGF plays an important role in the maintenance of the neurochemical phenotype of the suprachiasmatic nucleus in the adult rat. Because suprachiasmatic neurons do not express trkA, NGF might have exerted its effects either through direct signalling of suprachiasmatic neurons via p75NTR activation or, indirectly, by enhancing the activity of the cholinergic and/or glutamatergic afferents to the suprachiasmatic nucleus, or both.

Sexually dimorphic response of the hypothalamo–pituitary–adrenal axis to chronic alcohol consumption and withdrawal

In males, long-term alcohol consumption provokes neurochemical changes in the medial parvocellular division of the PVN (PVNmp) that are partially reversed by withdrawal. Because gonadal steroids modulate the activity of the hypothalamo-pituitary-adrenal axis, we analyzed the possibility that the repercussions of chronic alcohol consumption and withdrawal on the anatomy and neurochemistry of the PVNmp might differ between the sexes. Male and female Wistar rats were examined after ingesting a 20% alcohol solution for 6 months or after 2 months of withdrawal from 6 months of alcohol consumption. The levels of gonadal steroids and the basal concentrations of corticosterone were also evaluated. Chronic alcohol consumption and withdrawal did not alter the global cytoarchitectonic features of the PVNmp in rats of both sexes. However, alcohol consumption was associated with a decrease in the number of vasopressin (VP) neurons only in females and of corticotropin releasing hormone (CRH) neurons in males and females. Further, the response to withdrawal was sexually dimorphic because in males there was a partial recovery of the number of CRH neurons whereas in females there was a further loss of VP and CRH neurons. Corticosterone levels were unchanged by alcohol consumption, but they were decreased by withdrawal in females. Alcohol consumption and withdrawal did not alter estrogen and progesterone concentrations in females, but decreased testosterone levels in males. These findings show that the response of CRH and VP neurons to excess alcohol is gender-specific, with females being more vulnerable during alcohol consumption and, most notably, after withdrawal.

Effects of chronic alcohol consumption and withdrawal on the response of the male and female hypothalamic–pituitary–adrenal axis to acute immune stress

The hypothalamic-pituitary-adrenal (HPA) axis plays a central role in the response to stress, and its activity is sexually dimorphic and modulated by sex steroids. Recent work indicates that HPA axis functioning is disturbed by chronic alcohol consumption and subsequent withdrawal in rats of both sexes, but particularly in females. To examine the influence of sex steroid hormones in HPA axis response to acute stress after ingestion of a 20% ethanol solution over 6months and subsequent withdrawal (2months), intact males, and estradiol- and oil-injected ovariectomized females received a single intraperitoneal injection of lipopolysaccharide (LPS). Six hours after LPS administration, corticosterone concentrations were increased in all male groups; however, in ethanol-treated rats they remained below those of control and withdrawn rats. mRNA levels of corticotrophin-releasing hormone (CRH) increased, and were identical in all groups after LPS stimulation, whereas those of vasopressin, although increased, remained below control levels. LPS stimulation elevated corticosterone concentrations in all oil-injected female groups, but did not alter those of estradiol-injected females. In oil- and estradiol-injected ethanol-treated females, CRH mRNA levels did not change in response to LPS stimulation, whereas those of vasopressin increased, but stayed below control levels. In withdrawn oil- and estradiol-injected females, CRH and vasopressin gene expression increased, but did not reach control levels. These data show that prolonged alcohol consumption produces long-lasting, possibly irreversible, changes in the neuroendocrine system that regulates the production of corticosteroids, and that these consequences are more profound in females, particularly when estrogen levels are low.

Timed hypocaloric food restriction alters the synthesis and expression of vasopressin and vasoactive intestinal peptide in the suprachiasmatic nucleus

In mammals, the main circadian pacemaker is located in the suprachiasmatic nucleus (SCN) and its most potent synchronizer is the daily variation of the intensity of light. However, other nonphotic cues, such as timed food restriction, can induce changes in the circadian rhythms, leading also to the appearance of a food-entrained oscillator. The present study was designed to establish if the alterations of the circadian rhythms induced by timed hypocaloric food restriction are accompanied by structural changes in the SCN. Two groups of adult rats, both maintained on 12-h light/12-h dark cycles, were used; in one group, animals had permanent free access to food, whereas in the other they were subjected to a restricted hypocaloric early morning feeding during 7 months. Using stereological techniques and in situ hybridization, we have examined the structure of the SCN and the synthesis and expression of vasopressin (AVP) and vasoactive intestinal peptide (VIP). The volume of the SCN and the total number of neurons did not vary between the two groups. However, the total number of AVP- and VIP-immunoreactive neurons and the AVP and VIP mRNA levels were significantly decreased in timed hypocaloric food-restricted animals. The results indicate that timed hypocaloric food restriction has led to changes of AVP and VIP content of the neurons. They furthermore suggest the existence of a coupling between the food-entrainable oscillator and the light-entrainable pacemaker.

Neuroanatomy: The added value of the Klingler method.

Undergraduate neuroanatomy students are usually not able to achieve a clear comprehension of the spatial relationships existing between the white matter fiber tracts in spite of numerous neuroanatomy textbooks, atlases and multimedia tools. The objective of this paper is to show the educational value of the application of the Klingler fiber dissection technique and the use of these dissections in the understanding of the three-dimensional intrinsic anatomy of the brain white matter for medical students. Four formalin-fixed brains were dissected using the Klingler methodology in order to reveal the inner anatomical organization of the brain white matter. The most important fiber systems were dissected and their relationships to the cerebral and cerebellar gray matter structures visualized. These dissections were used as a learning tool in teaching the brain white matter structural and topographical connectivity. The white matter fiber systems were presented to undergraduate medical students during a neuroanatomy course. They observed and manipulated the dissected specimens leading to a thorough understanding of the configuration and location of the white matter fiber tracts, and their relationships to the ventricular system and gray matter structures. Subsequently, students were asked to answer a survey concerning the importance of the utilization of this material in their understanding of the three-dimensional intrinsic anatomy of the brain white matter. The knowledge acquired with this technique, complemented by conventional formalin-fixed sections may improve the neuroanatomical knowledge and future retention of medical students.

Chronic ethanol intake induces partial microglial activation that is not reversed by long-term ethanol withdrawal in the rat hippocampal formation

HIGHLIGHTSAlcohol consumption lead to an increase in the total number of activated microglia.Chronic alcohol exposure induces microglial partial activation.Partial activation of microglia was not reversed by long‐term withdrawal.Chronic alcohol consumption produced no changes in the expression of TNF‐&agr; or COX‐2. ABSTRACT Neuroinflammation has been implicated in the pathogenesis of several disorders. Activation of microglia leads to the release of pro‐inflammatory mediators and microglial‐mediated neuroinflammation has been proposed as one of the alcohol‐induced neuropathological mechanisms. The present study aimed to examine the effect of chronic ethanol exposure and long‐term withdrawal on microglial activation and neuroinflammation in the hippocampal formation. Male rats were submitted to 6 months of ethanol treatment followed by a 2‐month withdrawal period. Stereological methods were applied to estimate the total number of microglia and activated microglia detected by CD11b immunohistochemistry in the hippocampal formation. The expression levels of the pro‐inflammatory cytokines TNF‐&agr;, COX‐2 and IL‐15 were measured by qRT‐PCR. Alcohol consumption was associated with an increase in the total number of activated microglia but morphological assessment indicated that microglia did not exhibit a full activation phenotype. These data were supported by functional evidence since chronic alcohol consumption produced no changes in the expression of TNF‐&agr; or COX‐2. The levels of IL‐15 a cytokine whose expression is increased upon activation of both astrocytes and microglia, was induced by chronic alcohol treatment. Importantly, the partial activation of microglia induced by ethanol was not reversed by long‐term withdrawal. This study suggests that chronic alcohol exposure induces a microglial phenotype consistent with partial activation without significant increase in classical cytokine markers of neuroinflammation in the hippocampal formation. Furthermore, long‐term cessation of alcohol intake is not sufficient to alter the microglial partial activation phenotype induced by ethanol.

Neurosurgical relevance of the dissection of the diencephalic white matter tracts using the Klingler technique

OBJECTIVEnThe Klingler fiber dissection technique is a relevant and reliable method for neurosurgery to identify with accuracy the fine structure of the brain anatomy highlighting white matter tracts. In order to demonstrate the significance of the application of this technique, we aimed to observe the course and relations of the mammillothalamic and habenulo-interpeduncular tracts as there are very few papers showing these important diencephalic tracts.nnnMATERIAL AND METHODSnTwelve formalin-fixed brains were dissected using the Klingler technique in order to expose the medial diencephalic surface. Diencephalic white matter tracts, particularly the mammillothalamic and habenulo-interpeduncular tracts, were dissected using wooden spatulas and metallic dissectors with different sizes and tips. Several measurements were performed in both dissected hemispheres relative to the mammillothalamic and habenulo-interpeduncular tracts.nnnRESULTSnThe course and length of these two tracts were visualized and the relations with other fiber systems and with the neighboring gray matter structures quantified and registered. The mammillothalamic tract approximately marks the anteroposterior coordinate of the anterior pole of the subthalamic nucleus in the anterior commissure - posterior commissure plane.nnnCONCLUSIONnThe present study helps to understand the three-dimensional architecture of the white matter systems of tracts when the Klingler technique is used. The numerical data obtained may be helpful to neurosurgeons while approaching brain paraventricular and ventricular lesions and deep brain stimulation. Finally, the anatomical knowledge can lower surgical complications and improve patient care particularly in the field of neurosurgery.