Chronic temporal lobe epilepsy is associated with severely declined dentate neurogenesis in the adult hippocampus
Introduction
Production of new neurons from proliferating stem/progenitor cells in the subgranular zone (SGZ) of the dentate gyrus (DG) is maintained all through life in multiple species including humans (Cameron and McKay, 1998, Eriksson et al., 1998, Gould and Gross, 2002, Kaplan and Hinds, 1977, Kornack and Rakic, 1999, Kuhn et al., 1996, van Praag et al., 2002). Interestingly, the enormity of dentate neurogenesis exhibits a close relationship with the hippocampal functions of learning and memory (Feng et al., 2001, Gross, 2000, Monje et al., 2003, Shors et al., 2001). Further, alterations in the microenvironment of the stem/progenitor cells in the SGZ can block or increase dentate neurogenesis. For example, cranial irradiation can damage the neurogenic microenvironment in the SGZ and thereby block neurogenesis and lead to deficits in learning and memory (Monje and Palmer, 2003, Monje et al., 2002). In contrast, several other types of brain/hippocampal injury such as ischemia, stroke, and hypoxia enhance dentate neurogenesis (Choi et al., 2003, Felling and Levison, 2003).
Additionally, status epilepticus increases cell proliferation and neurogenesis in the SGZ of the DG (Bengzon et al., 1997, Ekdahl et al., 2001, Madsen et al., 2000, Nakagawa et al., 2000, Parent et al., 1997, Scott et al., 2000). Hippocampal injury inflicted by excitotoxins such as kainic acid also enhances the production of new neurons in the adult DG (Gray and Sundstrom, 1998). It has been demonstrated that status epilepticus or hippocampal injury induces an initial, transitory proliferative surge in the SGZ with the number of new neurons increasing several folds during the first few weeks after injury (Nakagawa et al., 2000, Parent et al., 1997). This is likely due to the release of mitogenic factors from dying neurons, deafferented granule cells and reactive glia, as studies suggest that several neurotrophic factors are upregulated in the hippocampus following seizures or excitotoxic injury (Lowenstein et al., 1993, Shetty et al., 2003). Dentate neurogenesis however reaches the baseline level after 2–3 weeks of the insult with normalization of the rate of proliferation of stem/progenitor cells (Nakagawa et al., 2000, Parent et al., 1997), which likely parallels the normalization in the levels of neurotrophic factors. Nevertheless, it is unknown whether the progression of the initial precipitating injury into chronic epilepsy alters neurogenesis in the adult DG. Furthermore, the effects of spontaneous recurrent motor seizures (SRMS) and multiple epileptogenic changes in the chronically injured hippocampus, such as the aberrant mossy fiber sprouting (Okazaki et al., 1995, Shetty and Turner, 1997, Shetty and Turner, 1999b, Shetty et al., 2003), the loss of calbindin (Shetty and Turner, 1995), and reductions in the number of GABA-ergic interneurons (Esclapez et al., 1997, Franck et al., 1988, Mathern et al., 1995, Shetty and Turner, 2000, Shetty and Turner, 2001, Sloviter, 1987), on dentate neurogenesis are unknown. Addressing these issues are imperative, as decreased hippocampal-dependent learning and memory functions have been observed in both human chronic temporal lobe epilepsy (TLE) and animal models of chronic epilepsy (Alessio et al., 2004, Helmstaedter et al., 2003, Mikati et al., 2001, Roozendaal et al., 2001), which could be due to decreased dentate neurogenesis in chronic epileptic conditions.
We hypothesize that epileptogenic changes and SRMS that ensue after hippocampal injury or status epilepticus considerably decrease dentate neurogenesis. To address this issue, we quantified cells that are positive for doublecortin (DCX, a marker of new neurons) in the DG of adult Fischer 344 rats at 16 days and 5 months after an intracerebroventricular kainic acid (ICV KA) administration or after three graded intraperitoneal KA (IP KA) injections, models of TLE. The absolute number of DCX-positive neurons was also quantified from the DG of age-matched intact animals for comparison. An ICV KA administration in rat induces selective loss of a greater fraction of granule cell targets (i.e., hippocampal CA3 pyramidal neurons and dentate hilar neurons) ipsilateral to the KA administration, which eventually leads to multiple epileptogenic changes and hippocampal hyperexcitability (Cornish and Wheal, 1989, Perez et al., 1996, Shetty and Turner, 1996, Shetty and Turner, 1997, Shetty and Turner, 2000). On the other hand, graded IP KA injections in rat induces acute seizures for over 4 h and leads to degeneration of fractions of dentate hilar neurons and CA3 and CA1 pyramidal neurons bilaterally in the hippocampus. Additionally, virtually all rats exhibiting acute seizures after IP KA injections develop multiple epileptogenic changes in the hippocampus as well as chronic epilepsy, characterized by SRMS (Dudek et al., 2002, Hellier et al., 1998, Hellier et al., 1999). Thus, the two TLE models chosen in this study exhibit both epileptogenic changes and chronic epilepsy after the initial precipitating injury. However, in the IP KA model, the frequency of SRMS is greater but the extent of CA3 pyramidal neuron loss (targets of granule cells) is less than the ICV KA model. The choice of DCX as a marker of new neurons in the DG is based on our recent finding that neurons visualized with DCX immunostaining in the adult rat DG are new neurons that are predominantly born during the 12 days prior to euthanasia (Rao and Shetty, 2004).
Section snippets
Animals and intracerebroventricular kainic acid lesions
Adult (4 months old) male Fischer 344 rats were obtained from Harlan Sprague–Dawley (Indianapolis, IN). Animals were individually housed in an environmentally controlled room (approximately 23°C) with a 12:12-h light/dark cycle, and were given food and water ad libitum. All experiments were performed as per the animal protocol approved by the Institutional Animal Care and Use Committee of the Duke University Medical Center and the animal studies subcommittee of the Durham Veterans Affairs
Behavioral activity of animals after ICV KA administration
The behavioral activity of animals receiving ICV KA was monitored for 4 h after the KA administration. None of the KA-treated rats showed behavioral changes indicative of seizure activities that are commonly seen after systemic injections of KA in cognizant rats (Hellier et al., 1999). These observations are consistent with the earlier findings that ICV KA administration as performed in this study (i.e., at the low dose and under anesthesia), does not produce acute motor seizures but
Discussion
By analyzing neurogenesis in two different kainate models of rat TLE at both early and delayed time-points after status epilepticus and/or hippocampal injury, we provide novel evidence that chronic TLE is associated with severely diminished addition of new neurons to the adult DG. The overall reductions in the addition of new neurons to the chronically epileptic hippocampus ranged from 64% to 81%, in comparison to the age-matched intact hippocampus. Interestingly, the overall decrease in
Conclusions
Analysis of neurogenesis at both early and extended periods after ICV or IP KA administration in this study demonstrates, for the first time, that chronic TLE is associated with dramatically declined production of new neurons in the adult DG. Because a fraction of newly born neurons become inhibitory basket cells in the DG (Liu et al., 2003), a dramatically decreased neurogenesis following chronic injury may contribute to the increased seizure susceptibility of the DG in the chronically
Acknowledgments
We thank Ms. Geetha A. Shetty for her excellent contributions to ELISA experiments in this study, and the two anonymous reviewers for useful suggestions and comments on the manuscript. This research was supported by NIH grants from the National Institute of Neurological Disorders and Stroke (NINDS Grant RO1 NS043507 to A. K. S.) and the National Institute for Aging (NIA Grant RO1 AG20924 to A.K.S.), and a VA Merit Review Award (A. K. S.) from the Department of Veterans Affairs.
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