Authors :
Presenting Author: Gabrielle McGinty, BS – Boston Children's Hospital
Amanda Liebhardt, BSc – Boston Children's Hospital; Sheryl -Anne Vermudez, PhD – Boston Children's Hospital; Ervin Johnson, MD PhD – Pediatric Neurologist, Neurology Center of Toronto/Numinus Toronto; Mustafa Hameed, MD – Boston Children's Hospital; Henry Lee, PhD – Senior Scientist, FM Kirby Neurobiology Center, Preclinical Science Program Manager, RSZ Translational Neuroscience Center, Neurology, Boston Children's Hospital; Alexander Rotenberg Rotenberg, MD PhD – Professor of Neurology, Joseph J. Volpe Chair in Neurology, Harvard Medical School, Neurology, Boston Children's Hospital
Rationale:
Post traumatic epilepsy (PTE) is characterized by a loss of cortical inhibitory tone during a seizure-free latent period after the initial injury. Our previous studies in rodent traumatic brain injury (TBI) models identified that a progressive loss of cortical parvalbumin (PV)-expressing interneurons underlies PTE. Specifically, anatomical decline in PV expression correlates with functional impairment of intracortical inhibition and increased seizure susceptibility post-TBI. Here, we test the hypothesis that PV cell activation leads to a lasting increase in PV expression with inhibitory tone mitigating seizure progression. We utilize the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) chemogenetic system to globally activate PV cells in vivo. We use GABA blocker pentylenetetrazol (PTZ) to test whether seizure threshold is modifiable upon DREADD-mediated PV activation. In parallel, we check whether DREADD-mediated PV activation results in a lasting increase in PV expression. We aim to establish a functional-anatomical relationship upon PV activation which might be used to alleviate seizure burden before rodent TBI testing.
Methods:
We cross PV-Cre (JAX#017320) and R26-hM3Dq mice (JAX#026220) to form PV-DREADD mice, enabling global PV-specific expression of hM3Dq, an activating DREADD responsive to clozapine-N-oxide (CNO). Adult PV-DREADD mice (postnatal day P80-150) received a daily intraperitoneal (IP) injection of CNO (up to 1mg/kg) or saline vehicle for up to four days. Twenty-four hours after the last CNO injection, CNO/vehicle-treated mice (n=3-6 per group) received PTZ (70mg/kg) via IP followed by a 30-minute continuous video recording capturing generalized tonic-clonic (GTC) seizure progression. Another cohort of CNO/vehicle-treated PV-DREADD mice (n=3-7 per group) was transcardially perfused with 4% paraformaldehyde and brains harvested for PV immunostaining.
Results:
After PTZ injection, 0% of CNO-treated PV-DREADD mice (n=3) developed GTC seizures with wild jumping (Racine stage 6), but 100% of saline-treated PV-DREADD mice (n=6) did (p=0.0119, Fisher exact test, Fig 1A,B). The average terminal Racine stage reached post-PTZ was 3.7±0.7 in CNO-treated mice and 6.7±0.2 in saline-treated mice (p=0.0008, unpaired t-test, Fig 1C). PTZ-induced mortality was 0% in CNO-treated mice and 67% in saline-treated mice. Total ictal time post-PTZ was 6.7±6.7s in CNO-treated mice and 90.5±40.63s in saline-treated mice (p=0.0476, Mann-Whitney test, Fig 1D). Immunostaining revealed increased cortical PV expression after 4 days of 1mg/kg CNO injection, and to a lesser extent with lower dose (0.25mg/kg) (Fig 2A,B). Notably, body weight was reduced by 14.3±1.9% in CNO-injected mice but not in saline control mice (1.8±2.6%) (p=0.0043, Mann-Whitney test, Fig 2C,D).
Conclusions:
Sub-chronic chemogenetic PV cell activation leads to lasting seizure dampening effects correlating with increased PV expression in a dose dependent manner. The same PV activation paradigm also leads to body weight loss, revealing additional global metabolic mechanisms potentially relevant for seizure suppression.Funding:
NFL Player Association, NINDS