1. Epilepsy-Associated Neuroplasticity in the Dentate Gyrus
Bret N. Smith, Ph.D.
Epilepsy Center
Department of Physiology
University of Kentucky

2. No financial relationships to disclose
Disclosure
No financial relationships to disclose

3. Educational Need
Need = To better understand the relationship between epileptogenesis and reactive synaptic reorganization in the brain.

4. Objectives Describe models of TLE and PTE;
Identify biological similarities and/or differences between models
Construct cellular hypotheses to study epileptogenesis;
Demonstrate experimentally a set of functional changes in brain circuitry involved in epileptic seizure generation;
Identify effects of mTOR inhibition on reactive synaptic reorganization
Construct a working model of local-circuit neural plasticity associated with epileptogenesis.

5. Expected Outcome
Prompt/reinforce a “translational” mindset regarding development of new therapies for refractory epilepsy, including an appreciation of how mechanistic analyses might influence clinical outcomes.

6. Acknowledgements Laura Haselhorst Jennifer Rios-Pilier Corwin Butler
Kathleen Schoch, PhD
Robert F Hunt III, PhD
Deepak Bhaskaran, MD, PhD
Jeffrey Boychuk, PhD
Tim Kubal , MD
Heather Shibley, MD
Ron Winokur, MD
Eva Bach, PhD
Katalin Cs Halmos, MPH
Dan Liu
Stephen Scheff, PhD
Kathryn Saatman, PhD
Epilepsy Foundation
NINDS
DoD

7. Temporal Lobe Epilepsy Models
Premise: To study epilepsy preclinically, we need a model
that is “epileptic”
EPILEPSY
(a tendency toward recurrent seizures unprovoked by systemic or neurologic insults)
SEIZURE GENERATION
(the clinical manifestation of an abnormal and
excessive excitation of a population of cortical neurons) vs

8. Characteristics of epilepsy models
In rodents, chemical or electrical induction of status epilepticus (SE) or traumatic brain injury (TBI) leads to TLE.
Characteristics in common with patients (from resected tissue):
Insult (e.g., kindling, SE, TBI) followed by a “silent period”, then spontaneous seizures;
Cellular correlates include selective cell loss, axon sprouting, increased local excitability.
Injury Epilepsy
Latency
SE, TBI Epileptogenesis permanent structure/function change

9. General Hypothesis - Models:
Trauma or period of repetitive seizures leads to the functional destabilization of the temporal lobe and consequent epileptogenesis.
Inhibition (too little)
-Ionic—inward Cl-, outward K+ currents
-Neurotransmitter—GABA
Excitation (too much)
-Ionic—inward Na+, Ca++ currents
-Neurotransmitter—glutamate
Glu
GABA

10. Specific Hypothesis: Pilocarpine injection in mice leads to SE and
subsequent spontaneous seizures,
with cell loss and mossy fiber sprouting
Time
(weeks)
+ pilocarpine injection SE
Spontaneous sz +
MF sprouting

11. Behavioral Seizures
Seizure: temporary period of excessive and synchronous neuronal activity
Category 2
Category 3
Category 4
Category 5

12. Relationship Between SE and TLE
The number of pilocarpine-induced
seizures at injection time (SE) “predicts”
the success of the model in inducing
TLE by 8 weeks.
Shibley and Smith, 2002

13. The Hippocampus & Dentate Gyrus
Mouse
Database Center for Life Sciences (DCLS)

14. The Hippocampus & Dentate Gyrus
Hilus
Granule cell layer
Pyramidal cell layer
CA3
CA1 PP MF
Damaged after TBI
Damaged in epilepsy

15. C57BL/6 or CD-1 Mouse: Timm Stain
(weeks)

16. + pilocarpine injection SE Spontaneous sz + MF sprouting
time
+ pilocarpine injection SE
Spontaneous sz +
MF sprouting
Pyramidal cell layer
CA1
CA3 MF PP
Hilus
Granule cell layer
Mg2+-free, 30 mM bicuculline to block GABAA receptors and enhance any glutamatergic responses that might be present.

17. Responses to Hilar Stimulation
…often of long duration and with long and variable latency following the antidromic spike.
Antidromic population spike followed by afterdischarge...
Winokur et al., 2004
Adapted from Winokur et al., 2004

18. Newly Sprouted Axonal Connections
IML GC
Hilus
Glutamate
Excitation
GABA
Inhibition

19. TBI and PTE Traumatic brain injury (TBI):
Sudden and direct insult to the brain by mechanical force that disrupts the normal function of the brain
Posttraumatic epilepsy (PTE):
Recurrent, unprovoked seizures weeks to years after TBI
TBI is estimated to be the cause of >20% of symptomatic epilepsy
Commonly manifests as TLE or neocortical epilepsy
Severity of TBI positively correlated to risk of PTE ( % with mild injury, up to 53% with severe injury)
Cortical contusion associated with increased risk (11-22%) vs no contusion

20. Progression of TBI Pathology

21. Controlled Cortical Impact (CCI) Injury
Closed-head TBI
Focal injury
Models cortical
contusion/ impact
injury

22. Behavioral Seizures After CCI Injury
Category 2
Category 3
Category 4
Category 5
Hunt et al., J Neurophysiol. (2010)
CCI in mice leads to spontaneous seizures with cell loss, mossy fiber sprouting, and synaptic reorganization.

23. Regionally Localized Mossy Fiber Sprouting (MFS) Ipsilateral to Contusion Injury Within 6 Weeks After CCI
Ipsilateral + No MFS
Ipsilateral + MFS
Hunt et al., 2012

24. mTOR Inhibition to Modify Posttraumatic Epileptogenesis
Vol 8, e6408, 2013

25. mTOR Inhibition to Modify Posttraumatic Epileptogenesis
TBI
Electrophysiology
Histology
Daily rapamycin treatment
Wks
post-TBI 8 10 13
Seizure monitoring

26. Specific Hypothesis:
Rapamycin treatment reduces mossy fiber sprouting and increased excitability in the dentate gyrus after CCI
IML GC
Hilus
GABA
Inhibition
GABA
Inhibition
Glutamate
Excitation
Glutamate
Excitation

27. Butler et al., Front Sys Nsci (2015)
Rapamycin Treatment Reduces Mossy Fiber Sprouting Ipsilateral to Contusion Injury
Butler et al., Front Sys Nsci (2015)

28. Butler et al., Front Sys Nsci (2015)
Neurogenesis Ipsilateral to Contusion Injury is Suppressed by Rapamycin Treatment
Neuron 75, 1022–1034, 2012
Butler et al., Front Sys Nsci (2015)

29. Increased Excitability After TBI
Hilus
Granule cell layer
Pyramidal cell layer
CA3
CA1 PP MF
Hunt. et al., Exp. Neurol, (2009)
Butler et al., Frontiers Sys Nsci (2015)

30. Conclusions - Recurrent Excitatory Synaptic Reorganization in the DG is Suppressed by Rapamycin After CCI
Continuous treatment with rapamycin after focal brain injury reduces:
Spontaneous seizure prevalence (11-12% vs 40-50%)
Mossy fiber sprouting
Increased EPSC frequency
Evoked population burst after antidromic stimulation
Rapamycin modifies post-injury epileptogenesis

31. Traumatic Brain Injury
(too little)
(too much)
Excitation
Inhibition
Increased
activity
New recurrent
excitatory
connections
Excitatory inputs to surviving interneurons
Inhibition of granule cells
Adult
neurogenesis
Perisomatic Dendritic 31

32. Butler et al, Exp Neurol (2016)
Altered Inhibition Part 1: Loss of Hilar Inhibitory Interneurons After CCI is Not Attenuated by Rapamycin
Butler et al, Exp Neurol (2016)

33. Butler et al, Exp Neurol (2016)
Altered Inhibition Part 1: Reduced GABA Release Onto Granule Cells After CCI is Further Reduced by Rapamycin
1-2 weeks post-injury
8-13 weeks post-injury
Hunt et al., J Neurosci. (2011)
Butler et al, Exp Neurol (2016)

34. Specific Hypothesis:
Mossy fiber sprouting correlates with increased excitability in surviving interneurons after CCI and rapamycin treatment eliminates this “compensation”
Glutamate
Excitation
GABA
Inhibition
GABA
Inhibition
Glutamate
Excitation

35. Hilar Somatostatin-positive Inhibitory Neurons (eGFP+)
Ipsilateral
DAPI eGFP
Hilar Interneurons associated with the Perforant Pathway (HIPP)

36. Butler et al., unpublished
Altered inhibition part 2: Increased APs In Cell Attached Recordings from eGFP+ Interneurons
Hunt et al., J Neurosci. (2011)
Butler et al., unpublished

37. From Where Do These Increased Inputs Arise?
Increased synaptic contacts (miniature EPSC frequency)
Increased network-driven input (spontaneous activity & mEPSC/sEPSC ratio)
Hypothesis:
Rapamycin treatment inhibits new network-driven input to HIPP cells arising from dentate gyrus or CA3 after CCI
Mossy Fibers
CA3 pyramids

38. 0Mg2+ ACSF + 100μM PTX + “caged” glutamate
Photolysis of Caged Glutamate
“caged”
glutamate
“cage”
glutamate
0Mg2+ ACSF + 100μM PTX + “caged” glutamate

39. Butler et al., unpublished
Altered Inhibition Part 2: Glutamate Photostimulation of Dentate Granule Cells Ipsilateral to Injury
Hunt et al., J Neurosci. (2011)
Butler et al., unpublished

40. Butler et al., unpublished
Altered Inhibition Part 2: Glutamate Photostimulation of CA3 Pyramidal Cells Ipsilateral to Injury
Hunt et al., J Neurosci. (2011)
Butler et al., unpublished

41. Excitability of HIPP Cells After CCI
Increased excitatory synaptic activity in HIPP cells is increased after CCI and is reduced, but not normalized after rapamycin treatment;
Increased synaptic input from dentate granule cells after CCI is suppressed by rapamycin treatment, but the increased input from CA3 pyramidal cells is unaffected.
Rapamycin treatment reduces DGC-related components of excitatory synaptic reorganization after CCI, but not those related to other areas (CA3).

42. X Normal Epileptic? Epileptic? IML GC Hilus Glutamate Excitation GABA
Inhibition
Glutamate
Excitation
GABA
Inhibition X
GABA
Inhibition
Glutamate
Excitation

43. Does synaptic reorganization cause TLE?
“Those guys are out of breath and they
need more oxygen. So they’re bending
over because it helps them breathe
if they’re closer to the grass...
…’cause there’s oxygen in the grass”
--John Madden

44. HIPP cell synaptic reorganization
Normal
Epileptic
inactivation
Seizure
Excessive
depolarization
GABA
Inhibition
Glutamate
Excitation