Elsevier

Neurobiology of Disease

Volume 75, March 2015, Pages 142-150
Neurobiology of Disease

PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

https://doi.org/10.1016/j.nbd.2014.12.029Get rights and content

Abstract

Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy.

Introduction

Dentate granule cells are a major component of the classic hippocampal trisynaptic circuit, receiving information from the entorhinal cortex and passing it through long, unmylenated mossy fiber axons onto the apical dendrites of the CA3 pyramidal cells. In a normal brain, the dentate granule cells are hypothesized to block the throughput of excess excitation into the hippocampus, acting as a gate or filter (Hsu, 2007). It is hypothesized that a breakdown of this filtering occurs during temporal lobe epilepsy (TLE), resulting in excessive signaling to CA3. Changes in granule cell structure and connectivity coincide with the onset of spontaneous seizures and might facilitate this breakdown (Ben-Ari and Dudek, 2010, Cameron et al., 2011, Dudek and Sutula, 2007, Hester and Danzer, 2013, Hester and Danzer, 2014, Murphy et al., 2012, Parent and Kron, 2012, Santos et al., 2011, Scharfman and Pierce, 2012, Singh et al., 2013).

Mossy fiber axons have three types of presynaptic terminals: giant mossy fiber boutons, filopodial extensions of these boutons and en passant terminals. Mossy fiber boutons synapse with elaborate clusters of spines – thorny excrescences – located on the basal and apical dendrites of the CA3 pyramidal cells. Each mossy fiber axon gives rise to approximately 15 giant boutons, and individual CA3 pyramidal cells can receive input from up to 50 granule cells (Amaral et al., 1990). Filopodial extensions and en passant terminals, on the other hand, form synapses with the GABAergic interneurons (Acsády et al., 1998, Frotscher, 1989, Seress et al., 2001). The filopodial and en passant terminals are responsible for another 40 to 50 synapses per mossy fiber axon, allowing for feed-forward inhibition to regulate CA3 network excitability (Acsády et al., 1998). Structural plasticity of the mossy fiber axons and boutons has been noted in animal models of TLE. In fact, epileptogenesis has been associated with increased bouton density, increased number of release sites, increased active zone length and changes in the distribution of thorny excrescences of the CA3 pyramidal cells (Danzer et al., 2010, Goussakov et al., 2000, McAuliffe et al., 2011, Upreti et al., 2012). Enhanced connectivity between granule cells and CA3 pyramidal cells, therefore, may promote epileptogenesis in traditional models of TLE.

Recently, our lab described a novel transgenic mouse model of TLE, in which the mammalian target of rapamycin (mTOR) pathway inhibitor phosphatase and tensin homologue (PTEN) could be selectively deleted from adult born granule cells (Pun et al., 2012). These mice developed spontaneous seizures beginning 4–6 weeks following gene deletion. Enhanced mTOR signaling among granule cells is a common feature of a variety of TLE models (Brewster et al., 2013, Lasarge and Danzer, 2014, Wong, 2013), so the observation that PTEN deletion is sufficient to cause epilepsy suggests enhanced mTOR signaling may play a critical role in epileptogenesis. The mechanisms by which increased mTOR signaling in dentate granule cells (DGCs) might promote epilepsy, however, are unclear. One possibility is that increased mTOR activation in DGCs induces structural changes in their mossy fiber axons, supporting increased signaling to CA3. Increased DGC ≫ CA3 connectivity would facilitate seizure spread through the hippocampus. To explore this possibility, the mossy fiber axon structure was examined in GFP-expressing PTEN-knockout (KO) and control mice.

Section snippets

Animals

All procedures were approved by the CCHMC Animal Board (IACUC) and followed NIH guidelines. Three transgenic lines were used for these studies: Gli1-CreERT2 mice, CAG-CAT-enhanced green fluorescent protein (GFP) reporter mice, and Ptentm1Hwu/J mice (Jackson Laboratory). Gli1-CreERT2 expressing mice have a cDNA encoding CreERT2 inserted into the 5′UTR of the first coding exon of the Gli1 locus (Ahn and Joyner, 2004, Ahn and Joyner, 2005). GFP reporter mice possess a CAG-CAT-EGFP reporter

PTEN deletion among a subset of granule cells

In control and PTEN KO mice, GFP-expressing dentate granule cells were located close to the hilar border, as predicted for post-natally generated neurons; although a subset of cells were located ectopically in the hilus in PTEN KO mice. GFP expression was detected throughout the cell body, dendrites, and mossy fiber axons (Fig. 1). Expression was robust, allowing for the morphological characterization of mossy fiber axons and terminals at high resolution (Fig. 1G and H). PTEN deletion was

Discussion

Mice with PTEN deleted from a subset (> 9%) of adult-born dentate granule cells develop spontaneous seizures as early as four weeks after gene deletion (Pun et al., 2012). The mechanism by which PTEN KO cells promote epilepsy, however, remains unclear. It has been previously shown that PTEN KO cells have morphological abnormalities indicative of increased excitatory input, including de novo basal dendrites and greater dendritic spine density (Pun et al., 2012). Here, we explored whether PTEN KO

Acknowledgments

This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and R01NS062806; CLL F32NS083239, NRSA F32NS083239). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. We would also like to thank Raymund Y. K. Pun for his work injecting dentate granule cells with biocytin and Keri

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