This study examines synapse formation between transplanted GABAergic interneurons and newborn dentate granule cells (DGCs) in the hippocampus of mice with temporal lobe epilepsy. The results show that epileptic mice receiving transplants of GABAergic interneurons from the medial ganglionic eminence had fewer seizures than control mice. Newborn DGCs were labeled using retroviruses and showed a range of normal and hyperexcitable morphologies. Confocal microscopy revealed appositions and clusters of synaptic proteins between the dendrites of newborn DGCs and axons of transplanted interneurons, suggesting synapse formation between the cells. Future studies aim to further characterize this synaptic connectivity.

1. SYNAPSE FORMATION BETWEEN NEWBORN GRANULE CELLS AND TRANSPLANTED GABAERGIC INTERNEURONS IN THE
HIPPOCAMPUS OF MICE WITH TEMPORAL LOBE EPILEPSY
E. Paquette1 and J. Radell1, K. Henderson1, B. Luikart2, J. R. Naegele1
1Dept. of Biology, Wesleyan University, Middletown, CT; 2Depts. of Physiology and Neurobiology, Dartmouth College, Hanover, NH
I. Introduction
Temporal lobe epilepsy (TLE) is a disorder characterized by recurrent seizures persisting throughout
life. Medications are available for seizure management, but approximately one third of patients with TLE
do not respond to treatment. TLE is also linked to pathological changes in the brain and cognitive
impairments; furthering the need for more effective treatments.
In TLE, the death of inhibitory GABAergic interneurons and morphological changes to one of the
population’s post-synaptic partners, dentate granule cells (DGCs) lead to hyperexcitability in the
hippocampus (Kobayashi and Buckmaster 2003, Scharfman 2004).
Neural progenitor cells in the Medial Ganglionic Eminence (MGE) of embryonic mice (E13.5) give rise
to GABAergic interneurons in the adult brain (Maisano et al. 2012). By harvesting these cells and
transplanting them into the hippocampus of an epileptic brain, we hope to replace the lost population of
GABAergic interneurons. We have shown that epileptic mice that receive MGE transplants (TX) have
reduced seizure number and severity (Henderson et al. Submitted). However, the mechanism through
which this reduction occurs is in need of further investigation.
The subgranular zone (SGZ) of the dentate gyrus (DG) is one of the few sites in which adult
neurogenesis occurs in mammals. New DGCs are born throughout life and are continually being wired into
existing hippocampal circuitry (Gage 2002). In epilepsy, newborn DGCs sprout mossy fibers, extend hilar
basal dendrites, and ectopically migrate into the hilus, increasing their excitability (Kobayashi and
Buckmaster 2003). This study examines the formation of synapses between transplanted GABAergic
interneurons and newborn DGCs.
II. Experimental Design
Neural progenitor cells are harvested from the Medial Ganglionic Eminence (MGE) (C) of VGAT-Venus
positive embryos (E13.5) ((A) GFP, (B) Brightfield, Scale 1mm) and transplanted into the DG (D) of
epileptic mice via stereotaxic injection using a quintessential stereotaxic injector (100,000 cells in 1µl of
media over 5 minutes).
III. Results
1. Seizure Suppression
Epileptic mice receiving MGE transplants in the
hilus of the DG have significantly fewer
seizures than those receiving media injections
or MGE transplants in the entorhinal cortex
(EC). (* p ≤ .001)
B
2. Retroviral Labeling
The pRubi retrovirus labels only cells in the process of
mitosis. (A) Newborn DGCs are robustly labeled in the
epileptic brain. (Scale 100µm) (B) The retroviral
construct contains an mCherry reporter expressed
under the control of a ubiquitin promoter.
3. Range of Granule Cell Morphologies
Tracings of newborn DGCs from confocal micrographs containing a normotopic DGC (A), a DGC extending
a hilar basal dendrite (B), and an ectopic DGC (C). (B) and (C) are known hyperexcitable cell types thought
to contribute to the development of seizures. (Scale 20µm)
4. Confocal Micrographs of Granule Cell Morphologies
Confocal images of the newborn DGCs depicted in figure 3. (RV: mCherry: magenta, TX: GFP: green, Scale
20µm) The proximity of transplanted interneurons to newborn DGCs suggests the possibility of synaptic
interaction. (ML: Molecular layer, GCL: Granule Cell Layer)
5. Appositions between TX and RV Labeled Newborn DGCs
(A) Low magnification view of a DG with retrovirally labeled DGCs (magenta) in the granule cell layer (GCL)
(outlined in white) and TX cells (green) in the hilus and molecular layer (ML) (Scale 100µm). (B) High
magnification image of the boxed area in A, containing several RV labeled DGCs (Scale 20 µm). (C) Digital
zoom of the boxed area in B, which shows contacts between RV dendrites and TX axons. (Scale 20µm)
Probable appositions are indicated in the Z-stack video.
6. Gephyrin Puncta Indicate Putative Synapses
Appositions between
an RV labeled DGC
dendrite (magenta)
and TX axons (green)
depicted as a confocal
micrograph (A) and a
tracing (B). Clusters of
gephyrin (blue), a
post-synaptic
scaffolding protein,
are localized at
GABAergic synapses.
Probable synapses are
labeled with arrows.
(Scale 20µm)
IV. Conclusions and Future Directions
Newborn DGCs, some with hyperexcitable morphologies, appear to make synaptic contacts with
transplanted GABAergic Interneurons
Future:
• Increase number of cases
• Quantify synapse formation between TX and newborn versus mature DGCs to determine if TX cells
preferentially contact hyperexcitable newborn DGCs with altered morphologies
• Perform electrophysiological recordings of DGCs to confirm inhibitory synaptic input from TX
V. Acknowledgements and References
Thanks to the entire Naegele lab for their constant support and assistance on this project, to Nick Woods
for laying the foundations, and to Jeff Gilarde for sharing his microscopy expertise with us. This work
would not have been possible without generous support from Randy and Lisa Siegel and grants from the
CURE Epilepsy Foundation and the National Institute of Health.
Kobayashi, M. and P. S. Buckmaster (2003). Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. J Neurosci 23(6): 2440-2452
Scharfman, H. E. (2004). Functional implications of seizure-induced neurogenesis. Advances in Experimental Medicine and Biology, 548: 192-212.
Maisano, X., Litvina, E., Tagliatela, S., Aaron, G.B., Grabel, L.B., & Naegele, J.R. (2012). Differentiation and functional incorporation of embryonic stem cell
derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy. The Journal of neuroscience: the official journal of the
Society for Neuroscience, 32(1):46-61.
Henderson, K. W., Gupta, J., Tagliatela, S., Litvina, E., Zheng, X. T., Van Zandt, M. A., Woods, N., Grund, E., Lin, D., Royston, S., Yanagawa, Y., Gloster, A. B.,
Naegele, J. R. (Submitted) Long-Term Seizure Supression and Optogenetic Analyses of Synaptic Connectivity in Epileptic Mice with Hippocampal Grafts
of GABAergic Interneurons
Gage F. H. Neurogenesis in the adult brain. J Neurosci 22: 612– 613, 2002.
mCherry / GFP / Gephyrin