PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure
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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2021, Progress in NeurobiologyCitation Excerpt :Consistent with prior studies, Pten immunohistochemistry and reporter gene expression (GFP, Arch-GFP, tdTomato) revealed Gli1-driven Pten deletion among olfactory neurons, hippocampal granule cells and small numbers (<1%) of glial cells (Pun et al., 2012; data not shown). In the hippocampus, Pten deletion produced grossly abnormal granule cells, with enlarged somas, apical dendrites with impaired branch self-avoidance and aberrant hilar-projecting basal dendrites (Fig. 1A; (Arafa et al., 2019; LaSarge et al., 2015; Pun et al., 2012)). Pten KO granule cells tended to be found in close proximity to the subgranular zone, where Gli1-expressing progenitors reside, but smaller numbers were also found close to the dentate molecular layer, consistent with reports showing that the KO cells migrate deeper into the granule cell body layer (Fig. 1B) (Getz et al., 2016).
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2019, British Journal of AnaesthesiaCitation Excerpt :These presynaptic terminals are unusually large in size and contain large quantities of glutamate-containing vesicles and multiple release sites. Changes in mossy fibre bouton size and structure occur under healthy15 and disease conditions.19–21 Larger boutons are indicative of increased numbers, size of synaptic release sites, or both.15
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2019, Experimental NeurologyCitation Excerpt :The mTOR pathway mediates aspects of activity-dependent growth (Switon et al., 2017), so increasing KO cell load – and corresponding increases in network excitability – could further enhance mTOR mediated cellular growth. In addition, in high PTEN KO animals granule cell mossy fiber axons project new collaterals into the dentate molecular layer (Pun et al., 2012; LaSarge et al., 2015). More effective recruitment of these new afferents by KO cells (Skelton et al., 2019) could further enhance their growth.
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2019, Experimental NeurologyCitation Excerpt :For morphological measures of biocytin-filled PTEN KO granule cells, a range of 1–3 cells/mouse was analyzed for a total of 57 cells. Cells were labeled with biocytin in acute hippocampal slices, which were prepared as previously described (LaSarge et al. 2015). All cells were injected with 0.2% biocytin using a “blind” approach with a patch clamp electrode (Pinault, 1996).