Immunohistochemical evidence of seizure-induced activation of trk receptors in the mossy fiber pathway of adult rat hippocampus

Abstract

Recent work suggests that limiting the activation of the trkB subtype of neurotrophin receptor inhibits epileptogenesis, but whether or where neurotrophin receptor activation occurs during epileptogenesis is unclear. Because the activation of trk receptors involves the phosphorylation of specific tyrosine residues, the availability of antibodies that selectively recognize the phosphorylated form of trk receptors permits a histochemical assessment of trk receptor activation. In this study the anatomy and time course of trk receptor activation during epileptogenesis were assessed with immunohistochemistry, using a phospho-specific trk antibody. In contrast to the low level of phosphotrk immunoreactivity constitutively expressed in the hippocampus of adult rats, a striking induction of phosphotrk immunoreactivity was evident in the distribution of the mossy fibers after partial kindling or kainate-induced seizures. The anatomic distribution, time course, and threshold for seizure-induced phosphotrk immunoreactivity correspond to the demonstrated pattern of regulation of BDNF expression by seizure activity. These results provide immunohistochemical evidence that trk receptors undergo activation during epileptogenesis and suggest that the mossy fiber pathway is particularly important in the pro-epileptogenic effects of the neurotrophins.

Fig. 1.

PY490 phosphotrk antibody recognizes phosphorylated trks. A₁, Western blot of PC12 cultures treated with vehicle or NGF and probed with pY490 phosphotrk antibody. A₂, Blot from A₁ stripped of antibody and reprobed with pan-trk antibody.B₁, Western blot of E18 cortical cultures treated with vehicle, BDNF, or NT-3 and probed with pY490 phosphotrk antibody. B₂, Blot from B₁ stripped of antibody and reprobed with pan-trk antibody.

Fig. 2.

Seizures increase phosphotrk immunoreactivity in hilus and CA3 stratum lucidum. A, Nissl-stained coronal section through hippocampus showing cell body layers (DG and CA1–CA3).B, Phosphotrk immunoreactivity in sham-stimulated animal. Note the presence of light immunoreactivity in neuropil but its absence in cell body layers. C, Phosphotrk immunoreactivity in an animal 24 hr after seven ventral hippocampal ESs. Note the marked increase in immunoreactivity in dentate hilus and stratum lucidum of CA3 (arrowheads); the remainder of hippocampal neuropil also appears slightly more immunoreactive, whereas the cell body layers still display an absence of immunoreactivity.

Fig. 3.

Peptide competition of phosphotrk immunoreactivity. Shown is phosphotrk immunoreactivity in coronal sections of hippocampus from a single animal killed 24 hr after seven ventral hippocampal ESs. A, No peptide.B, Preincubation of pY490 antibody with 300 nm phosphopeptide 490 immunogen. C, Preincubation with 300 nm unphosphopeptide 490.D, Preincubation with 30 μm phosphopeptide 674/5.

Fig. 4.

Time course of phosphotrk immunoreactivity in hippocampus and CA3 after seven ventral hippocampal electrographic seizures. Shown is phosphotrk immunoreactivity in representative coronal sections of hippocampus from sham-stimulated animals and animals killed 3, 12, and 24 hr and 1 week after seven ventral hippocampal electrographic seizures. The whole hippocampus is shown on the left, and the CA3 region is shown on theright. Note the temporal (24 hr only) and spatial (hilus and stratum lucidum of CA3) pattern of the increase in phosphotrk immunoreactivity.

Fig. 5.

Time course of phosphotrk immunoreactivity in CA3 after seven ventral hippocampal kindling stimulations. The data are expressed as a percentage of reduction in gray value in the given stratum as compared with stratum pyramidale (see Materials and Methods); thus, higher values reflect more intense immunoreactivity. Each symbol corresponds to one animal. Horizontal lines denote mean values. **p < 0.01 compared with all of the other time points by ANOVA with post hoc Bonferroni’s test.

Fig. 6.

Time course of phosphotrk immunoreactivity in dentate gyrus after seven ventral hippocampal kindling stimulations. The data are expressed as a percentage of reduction in gray value in the given stratum as compared with the granule cell layer (see Materials and Methods); thus, higher values reflect more intense immunoreactivity. Each symbol corresponds to one animal.Horizontal lines denote mean values. **p < 0.01 compared with all of the other time points by ANOVA with post hoc Bonferroni’s test.OML, Outer molecular layer; MML, middle molecular layer; IML, inner molecular layer.