Opto-kindling in CA1 Layer Induces Impairments in the Inhibitory Function of Hippocampus
Abstract number :
3.188
Submission category :
3. Neurophysiology / 3F. Animal Studies
Year :
2022
Submission ID :
2204843
Source :
www.aesnet.org
Presentation date :
12/5/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:26 AM
Authors :
Fabio Tescarollo, PhD – Rutgers University; John-Sebastian Mueller, MBS, BS – Neurosurgery – Rutgers University; Omar Akel, MD – Neurosurgery – Rutgers University; Spencer Chen, PhD – Senior Research Associate, Neurosurgery, Rutgers University; Hai Sun, MD, PhD – Associate Professor & Assistant Director of Research, Neurosurgery, Rutgers University
Rationale: The loss of GABAergic interneurons and impairment to the chloride (Cl-) cotransporters (NKCC1 and KCC2) have been proposed to be key mechanisms of epileptogenesis in temporal lobe epilepsy. Impaired functioning of NKCC1 and KCC2 lead to increased permeability of Cl- in neurons, resulting in GABAAR-mediated depolarized activity. Here we employed a novel optogenetic kindling paradigm in the hippocampal CA1 of mice to assess for the inhibitory signaling impairments associated with the kindling progression.
Methods: Homozygous PV-cre mice were stereotaxically injected in the left dorsal hippocampus with an AAV encoding for ChR2 and implanted with an optic fiber above left CA1 layer. Our kindling protocol consists of laser-stimulation sweeps (30 seconds each, 50 Hz) delivered every 30 minutes, 6 times a day until each animal reached its designated experimental milestone: (1) Partially kindled with 5x optogenetic after-discharges (“5oADs” group, n=4); (2) Fully kindled with 3x consecutive generalized tonic-clonic + jumping seizures (adapted Racine scale class 7 seizure – “3C7” group, n=4); or (3) Rekindled to 3x consecutive class 7 seizures after 14 days of no-stimulation following being fully kindled (“Rek” group, n=4). To assess for pathologic alterations to the inhibitory system, we evaluated the hippocampal expression levels of KCC2, NKCC1 and PV (parvalbumin) among the different experimental groups.
Results: Optogenetic kindling evoked focal to bilateral tonic-clonic seizures with increasing seizure severity and duration. Seizures were reliably evoked after 14 days of no-stimulus (Figure 1). Immunofluorescence analysis of NKCC1 and KCC2 levels in the hippocampus revealed that in the acute stage of opto-kindling (5oADs and 3C7 groups), expression of both cotransporters decreased compared to control animals (NKCC1: 5oADs=-14%, 3C7=-18%; KCC2: 5oADs=-28%, 3C7=-32%; One-way ANOVA, P< 0.0001). After rekindling, KCC2 expression surprisingly exhibited recovery (-16%), while NKCC1 expression reversed trend and increased by 20% above control levels (P< 0.0001). Rekindled animals also showed decreased expression levels of PV (-22%) and decreased number of PV+ cells in the hippocampus compared to control animals.
Neurophysiology