Selective deletion of PTEN from postnatally-generated granule cells
Deletion of PTEN from a subset of postnatally-generated neurons was achieved by treating 14-day-old triple transgenic Gli1-CreERT2
, GFP reporter+/−
(PTEN KO; see figure S1
for breeding strategy) mice with tamoxifen. Effective PTEN deletion was confirmed by simultaneous NeuN and PTEN immunostaining in brain sections from PTEN KO mice (n=30). In these animals, numerous PTEN negative, NeuN-positive neurons were evident in the neurogenic regions of the postnatal brain; the granule cell layer () and olfactory bulb (Fig. S2
). Despite careful analyses of NeuN/PTEN/GFP triple immunostained sagittal sections through the medial-lateral extent of the brain, no other neuronal subtypes exhibited either loss of PTEN or expression of GFP (Fig. S2
). In littermate control animals, 100% of NeuN-positive granule cells (2 dentate gyri/mouse, n=23 mice) co-labeled with PTEN antibodies (). Normal patterns of PTEN immunoreactivity were also present in PTEN KO mice that were not treated with tamoxifen (not shown). In addition to neuronal recombination, recombination in non-neuronal cells was evident among astrocytes in midbrain and neocortex (Fig. S3
), oligodendrocytes in corpus callosum (Fig. S3
) and Bergmann glial cells in cerebellum (not shown). No animals exhibited gross congenital defects or tumors in the brain.
Figure 1 Confocal optical sections of NeuN staining (green) reveal the dentate granule cell layer (boxed region in inset, top left panel) in cre control and PTEN KO mice treated with tamoxifen on P14. Sections were double-immunostained for PTEN (red), and in control (more ...)
PTEN deletion reproduces hallmark pathologies of temporal lobe epilepsy
PTEN KO granule cells exhibited numerous morphological abnormalities characteristic of granule cells from rodents with temporal lobe epilepsy (Parent et al., 2006
; Jessberger et al., 2007
; Walter et al., 2007
; Kron et al., 2010
; Murphy et al., 2011
; Pierce et al., 2011
; Murphy et al., 2012
), including neuronal hypertrophy, de novo
appearance of basal dendrites, increased dendritic spine density and ectopically located somata. For illustrative purposes, a small number of PTEN KO animals were crossed into the Thy1-GFP expressing mouse line (Feng et al., 2000
; Vuksic et al., 2008
; Danzer et al., 2010
), which labels a subset of granule cells with GFP regardless of PTEN expression. GFP expression within adjacent wildtype and PTEN KO cells in these animals revealed the dramatic morphological impact of PTEN deletion (). Quantification of these changes in PTEN KO animals crossed to GFP reporter mice revealed increases in mean soma area from 59.3±3.5 μm2
in control animals to 176.2±12.1 μm2
in PTEN KO animals (p<0.001, t-test; control n=4 mice [40 cells]; PTEN KO n=5 mice [36 cells]). The percentage of GFP-expressing granule cells ectopically located in the hilus () increased from 0.3±0.3% in controls to 3.3±1.0% in PTEN KO mice (p=0.049, t-test; control n=5 mice [519 cells examined]; PTEN KO n=8 mice [1544 cells]). The number of apical dendrites increased from 1 [range 1.0–1.1] in control mice to 1.8 [1.4–2.3] in PTEN KO mice (p=0.016, Mann-Whitney rank sum test [RST]; control n=4 [40 cells], PTEN KO n=5 mice [36 cells]). Spine density along these dendrites more than doubled (), increasing from 2.9±0.4 spines/μm to 7.5±0.5 spines/μm (control, n=4 mice [12 cells]; PTEN KO, n=4 mice [12 cells], p<0.001, t-test). The number of basal dendrites/cell increased from an animal median of 0 [range 0–0] in controls to 0.8 [0.4–1.0] in PTEN KO mice (; p=0.016, RST; control n=4 mice, [40 cells]; PTEN KO n=5 mice, [36 cells]). Basal dendrites, normally lacking in control rodents, are common in several models of temporal lobe epilepsy. 58.1±6.9% (12 dendrites from 3 mice) of dendritic spines coating these hilar basal dendrites were apposed to puncta immunoreactive for zinc transporter-3 (). Zinc transporter-3 (ZnT-3) labels granule cell mossy fiber terminals (McAuliffe et al., 2011
), and the apposition of presynaptic and postsynaptic components implies that PTEN KO cells receive recurrent excitatory input from neighboring granule cells. Further immunostaining experiments revealed the presence of PSD-95 in GFP-expressing basal dendrite spines apposed to ZnT-3 immunoreactive puncta (), supporting the conclusion that these inputs are functional.
Figure 2 A–D: Disrupted development of PTEN-immunonegative granule cells in Gli1-CreERT2 X PTENflox/flox X Thy1-GFP mice. In these animals, the Thy1 promoter drives GFP expression in a subset of wildtype and PTEN KO granule cells. A: PTEN immunostaining (more ...)
Figure 3 A: Confocal maximum projection showing aberrant hilar basal dendrites on a PTEN KO granule cell. Single optical sections of these basal dendrites reveal dendritic spines (A.1) apposed to ZnT-3 immunoreactive puncta (arrowheads, A.2 and merged image in (more ...)
PTEN KO mice exhibit spontaneous seizures
Simultaneous video/EEG recordings were made from either hippocampus or cortex of control (n=9) and PTEN KO mice (n=14) using 2-lead wireless EEG transmitters beginning at 6–8 weeks of age. Four additional animals (n=1 control, 3 PTEN KO) were recorded simultaneously from hippocampus and ipsilateral motor cortex using 4-lead wireless transmitters. A total of 96 days of video/EEG data was collected from control animals and 112 days collected from PTEN KO animals. Strikingly, 82% of PTEN KO mice exhibited spontaneous seizures. Two of the three animals that did not exhibit seizures died after only 4 days of recording, so whether they would have exhibited seizures eventually is not known. Many animals exhibited seizures during the first week of recording (≈6–8 weeks of age), indicating that epilepsy can develop in these mice in as little as four weeks after tamoxifen injection. No seizures were observed in any control animals. In PTEN KO animals followed over a period of weeks, the seizure phenotype was progressive. Initially (≈8 weeks), animals exhibited relatively frequent epileptiform activity (e.g. brief spike trains with no change in frequency) and occasional stereotypical seizures, characterized by progressive changes in frequency and amplitude before terminating after about 30–60 seconds (). In animals recorded from hippocampus and cortex simultaneously, epileptiform activity and seizures were consistently observed in hippocampus hours to days before abnormalities were evident in the cortical EEG (). Animals were typically immobile during these focal hippocampal seizures. As the animals became older, EEG abnormalities became more severe. Some animals exhibited more frequent stereotypical seizures, which were associated with convulsions when they spread to cortex (). Other animals exhibited fewer overt seizures but begin to exhibit increasing amounts of abnormal background activity and interictal spikes. Over time abnormal activity typically devolved to intermittent bursting () or burst suppression patterns (), and manifested in both hippocampus and cortex. Latencies between bursts ranged from about 1–60 seconds. Periods of burst suppression could persist for 20–30 minutes or longer, during which animals were largely immobile. Normalization of the EEG in these animals was followed by a return to normal behavior. PTEN KO animals exhibiting burst suppression patterns exhibited poor grooming and declining health. Ten of 17 PTEN KO mice became moribund and were euthanized or died prematurely, compared to only one of ten EEG implanted control mice. Mean age for morbidity/mortality among PTEN KO mice was 2.2 months. Many of these animals exhibited some variation of a burst suppression pattern prior to death. While most of the animals we observed exhibiting this pattern had cortical electrodes only, the animal shown in developed a hippocampal seizure lasting for almost 30 minutes while exhibiting cortical burst suppression. The animal was moribund during this event, raising the possibility that death in some animals might result from underlying non-convulsive status epilepticus.
Figure 4 PTEN KO mice treated with tamoxifen on P14 develop epileptic seizures. For all panels, cortical traces (ctx) are shown in black and hippocampal traces (hp) in blue. A: Typical electrographic seizure recorded using hippocampal depth electrodes. B: Dual (more ...)
The extent of CreERT2-mediated recombination varied among PTEN KO animals, so studies were undertaken to determine whether the percentage of granule cells in which PTEN was deleted correlated with epileptogenesis. Among the eight EEG-recorded PTEN KO mice (aged 3–7 months) for which good histology was available (e.g. mice that survived and could be perfusion-fixed), the percentage of dentate granule cells with no detectable PTEN immunoreactivity varied between <1 to 24%. Seizure activity was confirmed in seven of these mice, with PTEN deletion measures of 9–24%. No seizures were observed in the remaining animal which exhibited few PTEN KO granule cells (<1%).
PTEN deletion from olfactory neurons
One limitation of the Gli1 promoter used to target hippocampal granule cell progenitors is that subventricular zone progenitors, which populate olfactory bulb via the rostral migratory stream, are also targeted. Cells produced by this pathway differentiate into inhibitory olfactory granule cells (≈95%; OGCs) or olfactory periglomerular cells (≈5%), the majority of which are also GABAergic (Whitman and Greer, 2009
). Although excess growth of inhibitory interneurons may, in principle, be less likely to promote epileptogenesis, it is a formal possibility.
To explore this possibility, we first assessed the morphology of OGCs in olfactory bulb from wildtype and PTEN KO mice (). Initially, GFP expression was used to identify OGCs for morphological characterization in the olfactory bulb. Surprisingly, in olfactory bulb, neither soma area (control, n=5 [77 cells], 41.1±2.2 μm2; PTEN KO, n=4 [44 cells], 47.2±6.8; p=0.381, t-test) nor primary dendrite number (control, n=5 [77 cells], 3.2±0.2 dendrites/cell; PTEN KO, n=4 [44 cells], 3.7±0.6; p=0.353, t-test) differed between GFP-expressing OGCs in wildtype and PTEN KO mice. Given the robust morphological impact of PTEN deletion from hippocampal granule cells, we queried whether poor recombination efficiency among GFP expressing OGCs might account for this lack of effect. Analysis of PTEN/GFP double immunostaining revealed that only 44.6±6.5% of GFP-expressing OGCs in PTEN KO mice were also immunonegative for PTEN. By contrast, 84±6.1% of GFP-expressing dentate granule cells in PTEN KO mice were PTEN-immunonegative (p=0.007, t-test). These data suggest that cre recombinase is more effective at inducing GFP expression and deleting PTEN from hippocampal granule cells relative to OGCs, although the mechanism of this phenomenon is not clear. Given these findings, we reexamined OGC soma area in PTEN KO mice, comparing GFP-expressing, PTEN-immunopositive cells to GFP-expressing, PTEN-immunonegative cells. This analysis revealed a clear difference between the two populations, with the latter being significantly larger (GFP+,PTEN+, 37.5±2.1 μm2; GFP+,PTEN−, 65.4±4.5; p<0.001, t-test). Interestingly, the 75% increase in OGC soma area was less than half the almost 200% increase observed among hippocampal granule cells, suggesting that the hippocampal granule cells may respond more robustly to PTEN deletion.
Figure 5 Confocal maximum projections showing GFP-expressing olfactory granule neurons from control (A) and PTEN KO (B, C, D) mice. Olfactory granule cells from a PTEN KO animal immunostained for GFP and PTEN are shown in C and D. The cell in C (arrow) was immunoreactive (more ...)
To confirm that olfactory bulb was not the source of the seizure activity in PTEN KO mice, dual EEG recordings were made from olfactory bulb and hippocampus of four PTEN KO animals. In these animals, numerous episodes of epileptiform activity and seizures were observed in hippocampal EEG traces. During these events, EEG traces from olfactory bulb were qualitatively normal (). No examples of seizure activity originating in olfactory bulb and spreading to hippocampus were observed during four weeks of continuous video/EEG monitoring. These findings strongly suggest that olfactory bulb is not driving seizure activity in these animals, and support the conclusion that hippocampus is the source of the seizures.
PTEN KO mice exhibit minimal reactive gliosis
Deletion of the mTOR inhibitor Tsc1 primarily from astrocytes leads to the development of epilepsy in mice( Uhlmann et al., 2002
; Erbayat-Altay et al., 2007
). The mechanism underlying epileptogenesis in this model is still being explored; however, a recent study suggests that decreased expression and function of astrocyte glutamate transporters may be important (Zeng et al., 2010
). Glial changes are also implicated in other animal models of epilepsy as well as humans with the condition (for review see Vezzani et al., 2011
). We queried, therefore, whether astrocytic changes might be an important feature in PTEN KO animals by staining brain sections from wildtype and PTEN KO mice with the astrocytic marker GFAP. Hippocampi from five wildtype and five PTEN KO animals were examined, with the latter exhibiting PTEN deletion from 14–24% of the granule cell population. While a couple PTEN KO animals showed some evidence of reactive astrocytosis, such as enlarged glial cell bodies, thicker astrocytic processes and brighter GFAP labeling (Fig. S4
), quantitative measures of astrocyte cell body area (based on GFAP labeling) did not reveal a significant difference between groups (wildtype, 36.7±4.3 μm2
; PTEN KO, 51.6±6.2 μm2
; p=0.085, t-test). Similarly, no difference was observed in the density of labeled astrocytes (wildtype, 49.5±11.6 astrocytes × 103
; PTEN KO, 46.8±14.0 × 103
; p=0.886, t-test), with values being roughly similar to published reports for C57BL/6 mice (Ogata and Kosaka, 2002
The lack of a glial phenotype in PTEN KO animals likely reflects the low recombination rates among these cells. GFP-expressing astrocytes were virtually absent from hippocampus (on average 5.7±3.1 astrocytes/hippocampus; n=6 PTEN KO animals) and were rare throughout cortex (Fig. S3
; 0.8±0.4% of glial cells expressed GFP, n=3 PTEN KO mice). Quantification of the number of GFP-expressing astrocytes in the lateral posterior thalamic nucleus, a region showing comparatively large numbers of recombined astrocytes (Fig. S3
, boxed region), produced only slightly larger recombination rates (2.7±0.8%, n=3 PTEN KO mice). Finally, in contrast to neurons, in which PTEN immunoreactivity was readily detectable in wildtype cells and clearly absent in knockout cells (), PTEN immunoreactivity was undetectable among GFP-expressing (recombined) astrocytes from both wildtype and KO animals (Fig. S4
). Comparatively low levels of endogenous PTEN protein in this astrocyte population lead us to speculate that PTEN deletion from these cells may have relatively minimal effects.
Epileptogenesis in PTEN KO mice is mediated by enhanced mTOR activation
PTEN deletion is predicted to lead to increased phosphorylation of the mTOR effector S6. To determine whether the mTOR pathway was disrupted in PTEN KO mice, sections from six control and nine PTEN KO mice were immunostained for phospho-S6 (pS6). pS6 immunostaining intensity was significantly higher within the dentate gyrus of PTEN KO mice relative to controls (control, 77% [25–171] over background; PTEN KO, 160% [105–526] over background; p=0.022, RST), consistent with previous studies (Amiri et al., 2012
). These findings are indicative of enhanced mTOR signaling in these animals.
To confirm that the seizure phenotype was mediated by the mTOR pathway, PTEN KO animals were treated with the mTOR antagonist rapamycin. Rapamycin treatment significantly reduced seizure frequency in PTEN KO animals (n=5) relative to vehicle treated PTEN KO animals (n=4). Specifically, 100% of vehicle-treated PTEN KO animals developed epilepsy, with a median seizure frequency of 0.69/day (range: 0.40 – 2.60). Only 2 of 5 rapamycin treated KO mice exhibited any seizures at all, leading to an overall median seizure frequency of 0.06/day (range: 0.00 – 0.17; p=0.016, RST). These findings likely underestimate the effect of rapamycin on seizures in this model, as rapamycin also reduced the growth rate of treated mice, making it necessary to delay electrode implantation until animals reached criterion weight (18–20g) for implantation of wireless EEG devices. Vehicle-treated PTEN KOs reached 18g at a mean age of 8.3 ± 0.5 weeks, while rapamycin-treated KOs didn’t reach this weight until they were 13.8 ± 1.2 weeks-old. Advantageously, rapamycin also appeared to mitigate progression in this model and prolonged animal survival, so despite the greater age of rapamycin-treated PTEN KOs during EEG recording, they still exhibited fewer seizures than their younger vehicle-treated PTEN KO siblings.
The number of granule cells immunoreactive for pS6 was significantly reduced in PTEN KO animals treated with rapamycin relative to vehicle treated KOs (, vehicle, 17.5 [15–35] cells/field; rapamycin 1 [0–14]; p=0.029, RST), confirming the efficacy of the drug. None of the rapamycin treated PTEN KOs exhibited mossy fiber axon sprouting in the inner molecular layer, while all 4 vehicle treated KOs had obvious mossy fiber sprouting (). Taken together, these finding strongly implicate excess activation of the mTOR pathway in mediating epileptogenesis and granule cell pathology in these animals.
Figure 6 Confocal maximum projections of NeuN (green) and PTEN (red) double-immunostaining reveal PTEN KO granule cells in animals treated with vehicle (V) or rapamycin (RAP). PTEN KO cells are shown in gray in the merged image. Confocal maximum projections showing (more ...)
Neuronal hypertrophy precedes and mossy fiber sprouting follows epileptogenesis in PTEN knockouts
Mossy fiber sprouting occurs when granule cell axons sprout into the dentate inner molecular layer and form excitatory synaptic connections with other granule cells. The creation of these recurrent excitatory circuits is hypothesized to be a contributing factor in the development of temporal lobe epilepsy (Sutula and Dudek, 2007
). To assess mossy fiber sprouting among PTEN KO animals, brain sections were immunostained for ZnT-3. A significant positive correlation was found between the percentage of PTEN KO granule cells in the dentate and the extent of mossy fiber sprouting in the inner molecular layer (; R=0.757, p=0.007, Pearson product moment correlation). Essentially, mice with >16% PTEN KO granule cells (n=5) exhibited robust mossy fiber sprouting () and exhibited spontaneous seizures. By contrast, animals with PTEN deletions from <15% of their granule cells populations exhibited no mossy fiber sprouting. Interestingly, three of these animals with 9–15% recombination rates were confirmed as epileptic by video/EEG monitoring. This implies that mossy fiber sprouting is not required for epileptogenesis in this model. Granule cell soma area, on the other hand, was dramatically increased in all PTEN KO animals examined, regardless of whether they had seizures (). These cells also possessed basal dendrites (not shown). Taken together, these finding suggest the neuronal hypertrophy may be important for epileptogenesis in this model, while mossy fiber sprouting may be a consequence of recurrent seizures rather than a cause.
Figure 7 Confocal maximum projections of hippocampi from tamoxifen-treated control and PTEN KO mice immunostained for GFP (red) and ZnT3 (cyan) are shown. ZnT3-labeling reveals the normal mossy fiber axon terminal field (hilus and stratum lucidum) in the control (more ...)
Wildtype granule cells contribute to mossy fiber sprouting in PTEN KO mice
Three animals with robust mossy fiber sprouting were selected to determine the relative contribution of PTEN KO cells to this phenomenon. Mossy fiber terminals in the inner molecular layer were identified by ZnT-3 immunolabeling, and the percentage of these terminals co-labeled with GFP was determined. In these animals, 25.2±2.3% of ZnT-3 immunoreactive puncta in the inner molecular layer were GFP positive (), indicating that about a quarter of the mossy fiber sprouting is due to PTEN KO cells. This corresponded roughly to the total number of PTEN KO cells in these animals, at 20.9±2.0%.
Figure 8 Confocal optical sections of GFP-expressing mossy fiber axons in the dentate inner molecular layer from PTEN KO cells are shown in red. Double-immunostaining for the presynaptic granule cell terminal marker ZnT-3 is shown in cyan. White arrows denote (more ...)