Research ReportDoublecortin-positive newly born granule cells of hippocampus have abnormal apical dendritic morphology in the pilocarpine model of temporal lobe epilepsy
Introduction
The role of hippocampal neurogenesis in physiological and pathological conditions has been intensely investigated in recent years (for a review, see Ming and Song, 2005). The hippocampus participates in learning and memory but also in the most common human epileptic syndrome, the temporal lobe epilepsy (TLE; Sutula et al., 1989). Plastic alterations such as aberrant zinc-enriched synaptic terminals are one of the hippocampal sclerosis hallmarks in TLE patients. These aberrant terminals are present in the inner molecular layer and hilus of dentate gyrus (DG) resulting from the sprouting of granule cells axons, named mossy fibers (Tauck and Nadler, 1985, Sutula et al., 1998).
Along with the plastic change of mossy fibers sprouting, an increase in hippocampal neurogenesis rate was reported in epilepsy experimental models using immunohistochemical staining for bromodeoxyuridine (BrdU; Parent et al., 1997, Bengzon et al., 1997). In recent studies, the use of an endogenous neurogenesis marker such as doublecortin (DCX), a microtubule-associated phosphoprotein expressed by migrating and differentiating neurons, allowed the study of new granular neuron dendritic arborization (Rao and Shetty, 2004, Couillard-Després et al., 2005). Alterations in the apical dendritic tree of adult-generated granular neurons were observed in aging, employing DCX immunostaining (Rao et al., 2005), and with voluntary exercise, employing Golgi-Cox staining (Redila and Christie, 2006). Adult-generated granular neurons exhibit transient basal dendrites, an immature characteristic of these neurons; basal dendrites are more frequent in epileptic hippocampus (Ribak et al., 2000, Spigelman et al., 1998). Together, these results show that plastic changes occur in the hippocampus and newly born granule cells are susceptible to morphological changes in physiological conditions.
The behavioral and histopathological characteristics of TLE can be modeled in experimental animals with systemic injection of pilocarpine, a cholinergic muscarinic agonist (Turski et al., 1989, Cavalheiro et al., 1991, Mello et al., 1992, Cavazos et al., 2004). Pilocarpine induces status epilepticus (SE) and, after a latent period, spontaneous recurrent limbic seizures in the animals (Cavalheiro et al., 1991, Mello et al., 1992).
Since the newly generated neurons differentiate into functional granule cells integrated in the DG (van Praag et al., 2002), we wanted to investigate plastic changes in DCX-positive (DCX+) granular neurons morphology in the TLE pilocarpine model with three-dimensional digital reconstruction techniques. Dendrites length, nodes and spatial distribution were quantified and analyzed in control and epileptic animals 30 days after treatment with pilocarpine.
Section snippets
Pilocarpine treatment and spontaneous recurrent seizures
Of the twenty seven animals treated with pilocarpine, seventeen (63%) developed SE. Ten of these animals died in the acute phase of treatment. The video monitoring of the remaining seven animals recorded a mean of 8.4 limbic seizures with a minimum of 2 and a maximum of 15 seizures observed in each animal for the entire observation period. The control animals were seizure-free. The most frequent seizure class observed was 5 with the animal rearing and falling (Racine, 1972); a mean value of 3.3
Discussion
Here, we demonstrate that pilocarpine-induced seizures altered the newly born granule cell dendritic morphology. The epileptic animals had DCX+ apical dendrites with more bifurcations inside the granular cell layer and more endings in the inner molecular layer. The new granule cell dendrites were concentrated at mossy fiber sprouting sites like the hilus and the inner molecular layer. We confirm with doublecortin immunostaining previous results obtained with BrdU immunostaining that
Animals and housing
A total of 34 Wistar male rats were used in this study, weighing 250–300 g and aging 6–7 weeks in the beginning of experiments. The animals were maintained in a 12-h light–dark cycle (06:00–18:00) with food and water ad libitum. The experimental procedure was approved by the Commission on Animal Experimentation Ethics of the University of São Paulo Medical School at Ribeirão Preto (protocol #243/2005).
Seizure induction
Twenty seven animals were treated with methyl-scopolamine (1 mg/kg, i.p.; all drugs provided
Acknowledgments
This work was financially supported by CAPES, FAPESP, PRONEX, and CNPq. The abstract form of this work was awarded the Aristides Leão Prize in basic research by the Brazilian League Against Epilepsy (supported by Novartis). We thank Maira Licia Foresti and Maria Luiza C. Dal-Cól for helping with the animals care. G.M.A. holds a CAPES PhD Fellowship (Brazil).
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