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

No financial relationships to disclose Disclosure No financial relationships to disclose

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

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.

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.

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

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

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

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

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

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

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

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

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

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

+ 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.

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

Newly Sprouted Axonal Connections IML GC Hilus Glutamate Excitation GABA Inhibition

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 (1.6-4.2% with mild injury, up to 53% with severe injury) Cortical contusion associated with increased risk (11-22%) vs no contusion

Progression of TBI Pathology

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

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.

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

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

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

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

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

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)

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)

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

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

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)

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)

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

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

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

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

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

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

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

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).

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

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

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