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pilepsy Research (2012) 102, 126—130

j ourna l ho me pag e: www.elsev ier .com/ locate /ep i lepsyres

HORT COMMUNICATION

onditional deletion of TrkC does not modify limbicpileptogenesis

. Soren Leonarda, Ram S. Puranama,b, Jeffrey Helgagera, Gumei Liua,ames O. McNamaraa,b,c,∗

Department of Neurobiology, Duke University Medical Center, Durham, NC, United StatesDepartment of Medicine (Neurology), Duke University Medical Center, Durham, NC, United StatesDepartment of Pharmacology and Molecular Cancer Biology, Duke University Medical Center, Durham, NC, United States

eceived 26 August 2011; received in revised form 16 July 2012; accepted 30 July 2012vailable online 12 September 2012

KEYWORDSTropomyosin-related

Summary The neurotrophin receptor, tropomyosin-related kinase B (TrkB), is required forepileptogenesis in the kindling model. The role of a closely related neurotrophin receptor, TrkC,in limbic epileptogenesis is unknown. We examined limbic epileptogenesis in the kindling model

kinase C;

TrkC;Kindling model;Epileptogenesis;Cre recombinase

in TrkC conditional null mice, using a strategy that previously established a critical role of TrkB.Despite elimination of TrkC mRNA, no differences in development of kindling were detectedbetween TrkC conditional null and wild type control mice. These findings reinforce the centralrole of TrkB as the principal neurotrophin receptor involved in limbic epileptogenesis.© 2012 Elsevier B.V. All rights reserved.

Br22nsoN

ntroduction

lthough simultaneous overexpression of exogenous BDNFnd FGF2 can limit epileptogenesis in the pilocarpine modelParadiso et al., 2009), converging lines of evidence demon-trate that excessive activation of the receptor tyrosineinase, TrkB, by endogenous ligands is critical for induc-ion of limbic epileptogenesis (reviewed by McNamara et al.,

006; Brooks-Kayal et al., 2009). Specifically, the geneticerturbation of brain-derived neurotrophic factor (BDNF)Kokaia et al., 1995; Croll et al., 1999; He et al., 2004;

∗ Corresponding author at: Department of Neurobiology, Duke Uni-ersity Medical Center, Durham, NC 27710, United States.el.: +1 919 684 4241; fax: +1 919 684 8219.

E-mail address: jmc@neuro.duke.edu (J.O. McNamara).

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920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.eplepsyres.2012.07.019

arton and Shannon, 2005) or its receptor, tropomyosin-elated kinase B (TrkB) (Lähteinen et al., 2002; He et al.,004, 2010; Kotloski and McNamara, 2010; Heinrich et al.,011), has convincingly demonstrated that this particulareurotrophin pathway is required for limbic epileptogene-is. In contrast to TrkB, whether TrkC is activated by seizuresr is required for epileptogenesis has not been investigated.evertheless, this remains a relevant question because thelimination of just one allele of neurotrophin-3 (NT-3), anndogenous agonist of TrkC, has been shown to delay epi-eptogenesis in the kindling model (Elmér et al., 1997).

We examined the role of TrkC in limbic epileptogenesissing a genetic strategy identical to that which established

critical role for TrkB in the kindling model (He et al.,004). Because deletion of TrkC from the germline is lethalhortly after birth (Klein et al., 1994), we used a condi-ional approach in which mice with floxed alleles of TrkC

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Figure 1 In situ hybridization and qRT PCR of TrkC mRNA inthe hippocampus of WT and TrkC−/− mutant mice. (A—C) Thegranule cell layer (GCL) of the dentate gyrus adjacent to thehilus (H) and molecular layer (ML) exhibited a clear decreasein TrkC mRNA in the TrkC−/− mutant mice (A, right panel) rela-tive to WT controls (A, left panel). The CA3 pyramidal layer (P)adjacent to the stratum oriens (SO) and stratum lucidum (SL)exhibited a clear decrease in TrkC mRNA in the TrkC−/− mutantmice (B, right panel) relative to WT controls (B, left panel).The TrkC mRNA levels in the CA1 pyramidal layer (P) adjacentto the SO and stratum radiatum (SR) exhibited a modest reduc-tion in the TrkC−/− mutant mice (C, right panel) compared toWT controls (C, left panel). Scale bar = 50 �m. (D) qRT PCR anal-

TrkC is not required for epileptogenesis

were mated with mice expressing Cre recombinase driven bya synapsin-1 promoter (Syn-Cre) (Zhu et al., 2001), result-ing in mice in which both TrkC alleles were eliminated froma subset of central nervous system neurons. We assessedepileptogenesis by quantifying development of kindling andused both RT-PCR and in situ hybridization to verify deletionof TrkC mRNA.

Methods

All animal experiments were carried out in accordance withcurrent IACUC guidelines under animal protocol A298-09-03.Detail of the animals used, genotyping, qRT-PCR, in situhybridization, electrode implantation and kindling proce-dure are described in the Supplemental Methods section.

Electrode implantation and kindling procedure

Surgical and kindling procedures followed that of Heet al., 2010 (see Supplemental Methods). In brief, theelectrographic seizure threshold (EST) was determined byadministering a 1 s train at 50 �A with additional stimu-lations increasing by 25 �A (at 1 min intervals) until anelectrographic seizure was detected. Stimulations were sub-sequently administered twice per day at an intensity of theEST until the animals exhibited 3 consecutive seizures ofclass 4 or greater. Seizures were classified according to amodified Racine (1972) scale. Kindling data are presentedas the mean ± SEM for each group.

Results

Neuron-specific TrkC conditional knockout mice

To selectively eliminate TrkC expression from CNS neurons,mice in which exon 14 of the TrkC gene was flanked by loxPsites (Chen et al., 2005) were crossed to Syn-Cre transgenicmice. Reduction of TrkC mRNA was evidenced by qRT PCRstudy of hippocampal homogenates which revealed levels inTrkC−/− mutant mice approximating 57.2 ± 0.2% of WT mice(p < 0.05, Fig. 1D, right and middle). Importantly, the floxedTrkC mice in the absence of Syn-Cre exhibited no decrease inTrkC mRNA levels (Fig. 1D, right) relative to non-floxed wild-type controls (Fig. 1D, left). In situ hybridization revealedstriking reductions of TrkC mRNA in the dentate granule andCA3 pyramidal cells with lesser reductions in CA1 pyramidalcells of TrkC−/− mutant compared to WT mice (Fig. 1A—C),a pattern identical to that found with TrkB mRNA usingthe same Cre driver line (He et al., 2004). Together thesefindings demonstrate the efficacy of Cre recombinase inreducing TrkC mRNA expression.

Development and persistence of kindling isequivalent in WT and TrkC−/− mice

The development of kindling as measured by electrophysio-

logical and behavioral responses to stimulation of amygdalaproceeded similarly in WT and TrkC−/− mice. No differenceswere found between WT (n = 7) and TrkC−/− (n = 7) micewith respect to the following measures: first, the current

ysis demonstrating TrkC−/− (n = 7; Cre+, floxed/floxed) mutantmice exhibit reduced hippocampal mRNA content (†, p < 0.05)relative to WT (n = 3; Cre−, floxed/floxed) and non-floxed WT*(n = 4) controls.

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Figure 2 Kindling development is equivalent in WT (n = 7;open squares) and TrkC−/− (n = 7; closed squares) mutant mice.(A and B) Kindling development is presented as electrographicseizure duration (A) and behavioral seizure class (B). Stimula-tion number (x axis) refers to the number of stimulations thatevoked an electrographic seizure with duration of least 5 s. (C)Number of stimulations to reach different seizure classes (y axis)in WT and TrkC−/− mice. Left to right: First class 1, class 2 andclass 4/5 behaviors; Fully kindled refers to the third consecu-tw

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ive class 4/5 behavioral seizures. The maintenance stimulationas to assess persistence of the kindled state.

equired to evoke the initial electrographic seizure wasimilar in WT and TrkC−/− mice (279 ± 42 �A and 271 ± 50 �Aor WT and TrkC−/−, respectively). Second, the duration ofhe initial electrographic seizure and the progressive length-ning of electrographic seizure duration were similar inT and TrkC−/− mice (Fig. 2A). Third, the development of

ehavioral seizure intensity progressed similarly in mice ofoth genotypes (Fig. 2B). No significant differences were

ound in the number of stimulations required to evoke therst clonic motor seizure (class 4 or greater) or the thirdonsecutive clonic or tonic motor seizure lasting at least0 s (Fig. 2C).

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A. Soren Leonard et al.

To determine whether a null mutation of TrkC influencedhe persistence of the hyperexcitability following the com-letion of kindling, WT (n = 7) and TrkC−/− mutants (n = 7)ere stimulated following a stimulation-free period of 2eeks after the third consecutive class 4 or 5 seizure hadeen evoked. No significant differences were detected inST assessed after a 2 week stimulation-free period (aver-ge ESTs of 338 ± 132 �A and 343 ± 144 �A of WT and TrkC−/−

utants). Moreover, the number of additional stimulationsequired to evoke a class 4 or 5 seizure did not differetween the WT and TrkC−/− mice (1.9 ± 0.4 and 1.2 ± 0.2timulations, respectively; Fig. 2C, far right column). Inddition, there was no significant difference in the dura-ion of electrographic seizure (23.1 ± 1.2 and 26.0 ± 1.7 s)r seizure class evoked in the WT and TrkC−/− mutants.hus, the persistence of the hyperexcitable state estab-

ished by kindling was unaffected in the TrkC−/− mutantice.

iscussion

he objective of this study was to test the hypothesis that aonditional deletion of TrkC inhibits epileptogenesis in theindling model. Two principal findings emerged: (1) crossingyn-Cre transgenic mice to floxed-TrkC mutant mice reducedrkC mRNA content as assessed by two independent methodsnd (2) this reduction of TrkC content did not affect epi-eptogenesis as revealed by the development of kindling orersistence of hyperexcitability. We conclude that the par-ial reduction of TrkC expression in this conditional mutantouse does not modify limbic epileptogenesis in the kindlingodel.To compare the effects of TrkC with TrkB on epileptogen-

sis in the kindling model, the identical genetic strategysing the same Cre driver line was used. Despite reduc-ions of TrkC in a pattern similar to that observed for TrkBn earlier studies (He et al., 2004), no differences wereetected in the development of kindling in TrkC−/− com-ared to WT control mice. The contrast is striking becausehe development of kindling was eliminated altogether inhe conditional TrkB−/− mice (He et al., 2004). Even moreodest reductions of TrkB content in the conditional TrkB+/−

eterozygous mice resulted in a 50% increase in the num-er of stimulations required to induce the development ofindling (He et al., 2004).

The absence of detectable inhibition of the developmentf kindling in the conditional TrkC−/− null mutant mice isarticularly surprising in light of previous studies of NT-3eterozygotes in the kindling model. That is, NT-3, a neu-otrophin with high affinity and efficacy for TrkC (Lamballet al., 1991), is thought to function as the principal neu-otrophin agonist of TrkC in vivo. Mutant mice carrying justne allele of NT-3 exhibit a 50% increase in the numberf stimulations required to induce kindling (Elmér et al.,997). While the present results were surprising in light ofhe studies of NT-3+/− mice, our findings are consistent witharlier studies of Binder et al. (1999) that examined the

ffects on kindling development of intraventricular (ICV)nfusion of recombinant proteins in which the ligand recog-ition domain of TrkA or B or C was fused in frame withhe Fc portion of human IgG1, proteins that bind to and

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TrkC is not required for epileptogenesis

scavenge the endogenous neurotrophins. Whereas ICV infu-sion of TrkB-Fc markedly inhibited the development ofkindling, infusion of either TrkA- or TrkC-Fc had no effect(Binder et al., 1999). The failure of ICV infused TrkC-Fc to inhibit kindling development is consistent with thesimilarity of kindling development in WT and TrkC−/−

conditional mutant mice in the present study. The pos-sibility that endogenous NT-3 promotes the developmentof kindling by activation of TrkB seems unlikely becauseICV TrkC-Fc would be expected to scavenge NT-3 andpartially inhibit development of kindling, yet it did not(Binder et al., 1999).

The present findings underscore several unansweredquestions arising with respect to NT-3 in epileptogene-sis. What is the cellular and molecular mechanism bywhich the development of kindling is inhibited in NT-3+/−

mice? What is the functional consequence of the seizure-induced reduction of NT-3 mRNA described in multipleanimal models (Gall, 1993; Kokaia et al., 1996; Elméret al., 1997; Ferencz et al., 1997)? Does TrkC undergo acti-vation following seizures, a possibility suggested by thesmall increases of pTrk immunoreactivity detected in West-ern blot analyses of TrkB mutant mice (He et al., 2010)?Addressing this last question is hampered by the lack ofantibodies that selectively detect TrkC in western blot orby immunoprecipitation. The answers to these questionsnotwithstanding the present results demonstrate that TrkCexerts neither a detectable pro- nor anti-epileptogeneicaction in the kindling model. These findings underscorethe specificity of TrkB among the neurotrophin tyro-sine kinase receptors regulating epileptogenesis in animalmodels.

Conflict of interest

The authors have no conflicts of interest.

Acknowledgments

We thank Dr. David Ginty for the TrkC−/− mice, and Ms. Clau-dia Clarke for technical support. This work was supported bya grant from the National Institutes of Health (5R01NS56217)to J.O.M. and by a Ruth L. Kirschstein National ResearchService Award (F32 NS051064-01) to A.S.L.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.eplepsyres.2012.07.019.

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