Center of Experimental Morphology

Departamento de Anatomia - Faculdade de Medicina da Universidade do Porto

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Plasticity of Neuronal Circuits in the Limbic Cortex


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

OBJECTIVES

We have previously found that pilocarpine-induced status epilepticus (SE) leads to a decrease in the density of neurons which express neuropeptide Y (NPY) in layers II-IV and VI of the rat retrosplenial granular b (Rgb) cortex and that brief generalized seizures elicited by electroshock (ECS) cause an increase in the density of NPY neurons in Rgb layer V. These data suggest that moderate brain damage induced by brief seizures can be compensated, partly at least, by plastic changes in local circuit NPY-producing neurons and that more severe damage involving loss of NPY neurons can lead to the formation of permanent epileptogenic foci in the brain.

However, it remained unknown whether the seizure-induced plasticity of NPY neurons is specific to brain areas characterized by cell loss and implicated in epileptogenesis. Therefore, one of our goals was to extend the analysis of the effects of seizures on NPY neurons to other brain regions, namely the dentate gyrus, a brain region also known to be implicated in epileptogenesis, and the primary somatosensory barrel field (S1bf) cortex, which appears not to be critically involved in this disorder.

It was also not clear whether the seizure-induced plasticity of inhibitory neurons is specific to those that produce NPY or it is more widespread. Thus, another objective of these experiments was to test the effects of ECS seizures and SE on the number of the dentate hilus cells immunoreactive (IR) to another neuropeptide coexpressed by GABAergic interneurons, somatostatin (SS), whose synthesis is also transiently enhanced by seizures. However, the seizure-related changes in SS-IR cells, at least in the dentate hilus, appear to be less pronounced when related to NPY.

There is growing body of evidence that alterations in the cholinergic modulation of neuronal excitability may initiate seizure events in epileptic cortex and thus contribute to epileptogenesis. However, very little is known about what actually happens with cholinergic neurons and their fibers following epileptogenesis. We hypothesized that, if Rgb cortex is involved in seizure activity, as suggested by our prior findings, and if limbic seizures are indeed modulated by cortical cholinergic afferents, then the specific markers of cholinergic activity in this cortical area must be altered in epileptic rats. To test this hypothesis, we measured the densities of fiber varicosities immunoreactive to the vesicular acetylcholine transporter (VAChT) protein in different layers of Rgb of rats treated with pilocarpine which developed spontaneous motor seizures, of rats treated with ECS and of sham-treated control rats.

MAIN ACHIEVEMENTS

In the first experiment, we estimated the total number of NPY-containing neurons in the dentate gyrus hilus and their areal density in various layers of the S1bf cortex following either the induction of SE with pilocarpine or repeated administration of brief ECS seizures. ECS seizures were administered using a modified stimulation protocol such that the animals were given six seizures, the first five of which were spaced by 24-h intervals, whereas the last two were only 2 h apart. The main findings of this experiment are as follows: 1) repeated brief seizures elicited by ECS in rats caused an increase in the total number of NPY-IR cells in the dentate hilus, but not in the somatosensory (S1bf) cortex; 2) pilocarpine-induced SE led to a marked loss of NPY-IR in the dentate hilus; SE was also associated with a loss of NPY-IR cells in the S1bf cortex (layer VI). These results strongly support the main hypothesis of this experiment in showing that repeated brief seizures produce chronic (lasting at least 10 weeks) increase in the expression of NPY in brain regions involved in the pathogenesis of TLE, i.e. dentate hilus and Rgb layer V, but not in the S1bf cortex whose role in this process is not established.

In another experiment we examined the effects of the seizures on the total number hilar cells immunoreactive to SS. It was found that 1) ECS seizures caused no changes in the total number of SS-IR cells in the dentate hilus; 2) pilocarpine-induced SE led to a loss of hilar SS-IR cells, similar in extent to that of NPY-IR cells. Thus, the effect of brief seizures on interneurons seems to be specific to NPY because the total number of hilar cells immunoreactive to SS, a neuropeptide known to be coexpressed by the majority of hilar GABAergic cells including those which produce NPY, was unchanged following the administration of ECS seizures. The later observation is consistent with the results of prior studies which showed that the number of hilar SSergic cells remains constant in rats after administration of as many as 110 ECS seizures as well as in fully kindled animals. Our finding that pilocarpine-induced SE causes a significant reduction in the number of interneurons immunoreactive to both NPY and SS in epilepsy-related brain regions strongly supports the hypotheses of causal relationships between dysfunction of GABAergic inhibition and the process of epileptogenesis.

In testing the cholinergic hypothesis of epileptogenesis, we found that 1) pilocarpine-induced SE leads to a significant increase in the densities of VAChT-IR varicosities in all layers of Rgb cortex with the exception of layer V, 2) ECS administration does not produce significant changes in the densities of cholinergic varicosities in the Rgb cortex, at least, as measured two months after the termination of the treatment. In fact, there was a slight increase in the densities of VAChT-IR varicosities in all layers of Rgb in the ECS group, but this effect was not statistically significant. These data support the hypothesis of cholinergic dysfunction in the initiation and/or propagation of seizures, as well as in seizure-related neurodegeneration.

PUBLICATIONS IN PEER REVIEW JOURNALS

Cardoso A, Freitas-da-Costa P, Carvalho LS, Lukoyanov NV. (2010) Seizure-induced changes in neuropeptide Y-containing cortical neurons: Potential role for seizure threshold and epileptogenesis. Epilepsy and Behavior 19, 559-567.

Carvalho IP, Pais VG, Almeida SS, et al. (2010) Learning clinical communication skills: Outcomes of a program for professional practitioners. Patient Educ Couns., doi:10.1016/j.pec.2010.05.010

INTERNATIONAL PUBLICATIONS

Lukoyanov N. V., Freitas-Da-Costa P., Carvalho L. S. & Cardoso A. Divergent effects of brief seizures and status epilepticus on the expression of neuropeptide Y in cortical neurons. Amsterdam. Julho de 2010. FENS Abstr., vol.5, p. 50.

Cardoso A., Lukoyanova E. A., Madeira M. D. & Lukoyanov N. V. Comparison of behavioral deficits and neuronal loss in the rat hippocampal formation following brief seizures and status epilepticus. Amsterdam. Julho de 2010. FENS Abstr., vol.5, p. 305.

Carvalho IP, Almeida SS, Correia LM, et al. Empathy and the process of learning communication skills in clinical settings. Programme and Abstracts of the International Conference on Communication in Healthcare. 2010; 10D.5.

Carvalho IP, Pais VG, Silva FR, et al. Communication skills in clinical settings with simulated and real patients: a comparative study. Programme and Abstracts of the International Conference on Communication in Healthcare. 2010; P2.5.24.

NATIONAL PUBLICATIONS

José Luís Marques Ferreira (2010) The cholinergic transmission in the retrosplenial cortex of the epileptic rat, Master Thesis. Porto Medical School (Supervisor: N. Lukoyanov).

Carvalho IP, Ribeiro-Silva R, Pais VG, et al. (2010) O ensino da comunicação na relação médico-doente: Uma proposta em prática. Acta Med Port. 23(3): 527-32.

Dores AR, Carvalho IP, Castro-Caldas A. (2010) O desenvolvimento de um programa de reabilitação cognitiva com recurso a tecnologias informáticas. In Actas do VII Simpósio Nacional de Investigação em Psicologia. Edts Nogueira C, Silva I, Lima L, Almeida AT, Cabecinhas R, Gomes R, Machado C, Maia A, Sampaio A e Taveira MC. Universidade do Minho, Braga, pp 645-659. http://www.actassnip2010.com

Contactos
Centro de Morfologia Experimental
Faculdade de Medicina da Universidade do Porto
Al. Professor Hernâni Monteiro
4200-319 Porto
tel. (+351) 22 551 36 16
fax (+351) 22 551 36 17
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