Abstracts

Genetic and pharmacological manipulations indicate that BDNF plays an important role in epileptogenesis. Furthermore, epileptogenesis is associated with increased BDNF expression in the mossy fiber pathway of the hippocampus. This pathway contains both the presynaptic terminals of dentate granule cells (giant mossy fiber boutons), and the postsynaptic terminals of CA3 pyramidal cell proximal dendrites (thorny excrescences). To identify which, if any, of these process express BDNF, and to determine the likely targets of increases in BDNF protein, BDNF immunohistochemistry was examined in control and pilocarpine-treated transgenic mice expressing green fluorescent protein (GFP) in dentate granule cells and CA3 pyramidal cells. Colocalization of GFP in mossy fiber boutons or thorny excrescences with BDNF immunoreactivity permits establishing the cellular localization of BDNF.
Two to three month old GFP expressing mice from the M line (Feng et al., 2000) were treated either with scopalamine (1mg/kg) followed 15 minutes later by pilocarpine (340mg/kg) or sterile saline. All procedures conformed to NIH and institutional guidelines for the care and use of animals. Following 3 hours of status epilepticus (or control treatment) mice were treated with diazepam (10mg/kg) and allowed to recover for 48 hours before sacrifice. BDNF immunohistochemistry was conducted using a polyclonal rabbit anti-BDNF antibody (1ug/ml; Amgen) followed by Alexa Fluor 546 secondary antibodies (1:500; Molecular Probes). Neurons were imaged using a Leica confocal microscope equipped with a 63X objective.
In control animals, approximately 36% of GFP expressing mossy fiber boutons were also positive for BDNF (116 boutons from 5 animals). In contrast, no BDNF positive CA3 pyramidal cell thorny excrescences were found (37 from 4 animals). Interestingly, BDNF negative thorny excrescences were often observed adjacent to BDNF positive mossy fiber boutons. Forty-eight hours after pilocarpine-induced status epilepticus, approximately 80% of mossy fiber boutons were BDNF immunopositive. Insufficient numbers of thorny excrescences were available for analysis.
The present study establishes the localization of BDNF protein in dentate granule cell giant mossy fiber boutons in normal animals and demonstrates that the seizure-induced increase in BDNF is also, at least in part, in mossy fiber boutons. Since CA3 pyramidal cells are the postsynaptic targets of granule cell boutons in stratum lucidum, these findings indicate that seizures increase presynaptic BDNF at the granule cell/CA3 pyramidal cell synapse, and suggest that increased release of BDNF onto pyramidal cells may participate in epileptogenesis. Autocrine activation of TrkB receptors on mossy fiber boutons may occur as well.
[Supported by: SCD was supported by a postdoctoral fellowship from the American Epilepsy Society. This work was also supported by NIH grants NS07370 and NS32334 and NINDS grant NS17771.]