Center of Experimental Morphology

Departamento de Anatomia - Faculdade de Medicina da Universidade do Porto

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Stress and Alcohol in the Brain

Research Area: Health Sciences
Status: In progress  
Project leaders:  
In cooperation with
  • Faculdade de Medicina - Universidade do Porto


Prolonged alcohol consumption modifies the activity of the hypothalamo-pituitary-adrenal (HPA) axis by down-regulating the neurons located in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVNmp) that synthesize the ACTH secretagogues corticotrophin releasing hormone (CRH) and vasopressin (VP). Because after prolonged alcohol consumption plasma corticosterone is within normal levels, those changes have been interpreted as adaptation of the HPA axis to excess alcohol, instead of ethanol-induced neurotoxicity.

Actually, the observation that ethanol withdrawal does not reverse the neurochemical changes induced by prolonged alcohol consumption strongly suggests such a possibility. Thus, one of the objectives was to evaluate whether the neurochemical changes displayed by PVNmp neurons after prolonged alcohol consumption and withdrawal reflect neuronal adaptation or, or the contrary, ethanol neurotoxicity. To achieve this goal, we examined the response of the HPA axis of rats submitted to these experimental conditions to a different stressor, an acute systemic stress induced by the intraperitoneal injection of the bacterial endotoxin lipopolysaccharide (LPS).

Gonadal sex steroids modulate the activity of the HPA axis, both in basal conditions and during stress. In addition, previous data obtained by our group revealed that excess alcohol differentially affects the HPA axis of males and females because ethanol withdrawal further aggravates the changes induced by prolonged alcohol consumption in females, but not in males. Thus, another objective was to investigate if the effects of acute stress on the synthesizing activity of CRH and VP neurons in the PVNmp of rats submitted to prolonged alcohol consumption and withdrawal are sex-specific and, in females, influenced by the circulating levels of estrogens. To achieve this goal, the studies were done in male rats and in female rats that after ovariectomy (2 weeks before the end of the experiments) were treated with either estradiol benzoate (EB) or vehicle. Six hours before sacrifice, all rats received an intraperitoneal injection of LPS. Behavioral studies were performed 1 and 5 hours after the injection. Blood samples were collected for corticosterone assay immediately after decapitation, and brains sections were hybridized in order to detect and measure CRH and VP mRNA levels.

Since the PVNmp is involved in the regulation of sexually dimorphic functions, such as the production of glucocorticoids, we though of interest to examine if the PVNmp, the hypothalamic cell group that regulates the activity of the HPA axis, would be, by itself sexually dimorphic. To achieve this goal, we have quantitatively studied this PVN subdivision in gonadally-intact adult males and females, in castrated adult males and females, and in ovariectomized females later treated with either EB or vehicle.The volume of the PVNmp, and the total number and mean somatic volume of its constituent neurons was estimated in all groups, using stereological methods. In addition, we have also evaluated the size and complexity of the dendritic arborizations of PVNmp neurons, and the density of their dendritic spines in Golgi-impregnated neurons.

The activity of the adrenal cortex is also regulated by autonomic nerves, whose cell bodies are under the influence of the dorsal parvicellular (PVNdp) subdivision of the PVN, one of the autonomic-related descending regulatory divisions of the PVN. We have also studied this cell group in the search for sex differences in its volume, total number and mean volume of its neurons, and in the number of its VP- and CRH-positive neurons.


The plasma levels of corticosterone obtained from control, ethanol-treated and withdrawn groups in basal conditions and after LPS administration are now under re-analysis. The results obtained using a chemoluminescence ELISA assay were unexpected and that prompted us to re-analyse sera by means of RIA techniques.

The data obtained from the sections processed using in situ hybridization techniques for detection of the mRNA levels of CRH and VP showed that:
  1. Under basal conditions, females treated with estradiol benzoate (EB) have higher CRH and VP mRNA levels than oil-treated females and males. This indicates that the sex differences in basal corticosterone levels are determined not only by the downregulating effects of testosterone on the synthesizing activity of PVNmp neurons, as demonstrated in earlier studies, but also by the activating effects of estrogens.
  2. The variations in the number of CRH and VP neurons provoked by prolonged alcohol consumption and withdrawal, and described by our group in earlier studies, are a consequence of the effects of alcohol on the synthesizing activity of these neuronal subpopulations.
  3. LPS injection activates the HPA axis of control males by provoking an increase of about 180% in the mRNA levels of VP, and a slighter increase (23%) in the mRNA levels of CRH. In females, the activation of the HPA is exclusively dependent on VP-producing neurons, whatever the circulating levels of estrogens. However, the effects of LPS were more evident in oil-treated (VP mRNA levels increased by 160%) than in EB-treated (VP mRNA levels increased by 146%) rats.
  4. In ethanol-treated male and female rats, the activation provoked by LPS was also particularly evident in VP-producing neurons and the increments in the mRNA levels of this neuropeptide were similar to, but less marked than, those noticed in control rats. Again, only in males there was a significant increase in CRH mRNA levels.
  5. In withdrawn rats, VP and CRH mRNA levels were also increased after LPS administration, both in males and females. However, only in females, the increase in CRH mRNA levels reached a statistical significant level.
  6. Despite the activation of PVNmp neurons provoked by LPS in ethanol-treated and withdrawn rats, the mRNA levels of VP in all groups and of CRH in female groups did not reach the levels provoked by LPS administration in control rats.
The results of the studies designed to identify and characterize the sex-related differences in the structural organization of the PVNmp showed that this cell group has a larger volume in males than in females, and that this sex dimorphism is not attributable to differences in the number of neurons, but to differences in neuronal size. The total dendritic length and the mean length of the terminal segments were significantly higher in intact females than in intact males. No significant differences were found between gonadectomized groups. In addition, the spine density was consistently higher in intact females than in males, as well as in the EB-treated group relative to the vehicle-treated group.

We also found that the PVNdp is larger in males than in females, despite the absence of sex differences in the total number of its neurons. Intact females have larger neurons than intact males, a sex-related difference that was annulled by gonadectomy. The total number of CRH-immunoreactive neurons was not influenced by sex or gonadectomy. Conversely, intact females had significantly more VP neurons than intact males, a sex difference that was dependent on the circulating levels of sex steroid hormones, as they were not apparent in gonadectomized rats.


Barbosa M, Veríssimo R. Cross-cultural psychometric assessment of Leeds's Pain Scale for the Assessment of Neuropathic Symptoms and Signs (LANSS) in the Portuguese population (abstract 219) European Journal of Pain Supplements April 2010; 4(1): 64.


Ana Luísa Aires da Silva (2010) Influence of sex steroids and gonadectomy in the anatomy and neurochemical organization of the dorsal parvicellular division of the hypothalamic paraventricular nucleus. Master Thesis. Porto Medical School (Supervisor: M. Dulce Madeira).

Ricardo Jorge Nogueira Rodrigues da Rocha (2010) Effect of chronic alcohol consumption and withdrawal on the hippocampal formation. Master Thesis. Porto Medical School (Supervisor: M. Dulce Madeira).


Scientic commitee of 28th European Conference on Psychosomatic Research — Innsbruck, June 30-July 3, 2010.

Organization of the workshop “The neural underpinnings of affect regulation” in the framework of the 28th European Conference on Psychosomatic Research, Innsbruck, June-July 2010.


Coelho SM, Silva SM. Influence of sex steroids on the morphology of parvocellular neurons of the paraventricular nucleus of hypothalamus. V YES Meeting. Porto, Setembro de 2010.

Centro de Morfologia Experimental
Faculdade de Medicina da Universidade do Porto
Al. Professor Hernâni Monteiro
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