Chemogenetic Activation of Feed-Forward Inhibitory Parvalbumin-Expressing Interneurons in the Cortico-Thalamocortical Network During Absence Seizures

Abstract

Parvalbumin-expressing (PV+) interneurons are a subset of GABAergic inhibitory interneurons that mediate feed-forward inhibition (FFI) within the cortico-thalamocortical (CTC) network of the brain. The CTC network is a reciprocal loop with connections between cortex and thalamus. FFI PV+ interneurons control the firing of principal excitatory neurons within the CTC network and prevent runaway excitation. Studies have shown that generalized spike-wave discharges (SWDs), the hallmark of absence seizures on electroencephalogram (EEG), originate within the CTC network. In the stargazer mouse model of absence epilepsy, reduced FFI is believed to contribute to absence seizure genesis as there is a specific loss of excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at synaptic inputs to PV+ interneurons within the CTC network. However, the degree to which this deficit is directly related to seizure generation has not yet been established. Using chemogenetics and in vivo EEG recording, we recently demonstrated that functional silencing of PV+ interneurons in either the somatosensory cortex (SScortex) or the reticular thalamic nucleus (RTN) is sufficient to generate absence-SWDs. Here, we used the same approach to assess whether activating PV+ FFI interneurons within the CTC network during absence seizures would prevent or reduce seizures. To target these interneurons, mice expressing Cre recombinase in PV+ interneurons (PV-Cre) were bred with mice expressing excitatory Gq-DREADD (hM3Dq-flox) receptors. An intraperitoneal dose of pro-epileptic chemical pentylenetetrazol (PTZ) was used to induce absence seizure. The impact of activation of FFI PV+ interneurons during seizures was tested by focal injection of the "designer drug" clozapine N-oxide (CNO) into either the SScortex or the RTN thalamus. Seizures were assessed in PV^Cre/Gq-DREADD animals using EEG/video recordings. Overall, DREADD-mediated activation of PV+ interneurons provided anti-epileptic effects against PTZ-induced seizures. CNO activation of FFI either prevented PTZ-induced absence seizures or suppressed their severity. Furthermore, PTZ-induced tonic-clonic seizures were also reduced in severity by activation of FFI PV+ interneurons. In contrast, administration of CNO to non-DREADD wild-type control animals did not afford any protection against PTZ-induced seizures. These data demonstrate that FFI PV+ interneurons within CTC microcircuits could be a potential therapeutic target for anti-absence seizure treatment in some patients.

Keywords: DREADDs; GABAergic interneurons; absence seizures; cortico-thalamocortical; feed-forward inhibition; parvalbumin; pentylenetetrazol.

FIGURE 1

(A,C,E) Confocal images showing the expression of HA-tag in PV+ interneurons in the SScortex, the RTN thalamus and the cerebellum of PV^Cre/Gq-DREADD animals, respectively. White arrows in merged images represent co-localized cells. Yellow arrows indicate PV positive neurons, which are immunonegative for HA. (B,D,F) Percentage of co-localization of HA-tag and PV in neurons in the SScortex, the RTN thalamus, and the cerebellum, respectively. Immunolabelled cells in the SScortex and the cerebellum were counted at 10× magnified confocal images whereas in the RTN thalamus cells were counted using 40× images (Het = PV^Cre/Gq-DREADD offspring from homozygous PV-Cre female and heterozygous hM3Dq-flox male; WT = Non-DREADD wild type control animals).

FIGURE 2

(A) Schematic protocol for EEG recordings before and after IP PTZ injection in the pilot study. Representative EEG traces from animals after (B) 10 mg/kg (C) 20 mg/kg (D) 30 mg/kg of PTZ injection. Asterisks (^∗) and hash signs (#) represent absence-like seizures and tonic-clonic seizures, respectively. Each trace represents 5 min of EEG recording. All representative EEG traces were obtained from different animals.

FIGURE 3

(A) Schematic of protocol for EEG recordings before and after PTZ injection on day 1 in experimental animals. Comparison of (B) onset of seizure and (C) last incident of seizure during 1 h of EEG recording in PV^Cre/Gq-DREADD (DREADD) (n = 7) and non-DREADD (n = 5) WT control animals of the SScortex group and the RTN thalamus group after PTZ treatment on day 1. All values in graphs represent mean ± SEM. Comparison between treatment groups was performed using Mann Whitney unpaired rank test.

FIGURE 4

Schematic of protocol for EEG recordings before and after (A) PTZ injection on day 1 and (C) PTZ and CNO injection on day 2. Comparison of the percentage of different types of seizures in PV^Cre/Gq-DREADD (DREADD) (n = 7) and non-DREADD WT control (n = 5) animals of the SScortex and the RTN thalamus group after (B) PTZ injection on day 1 and (D) PTZ and CNO injection on day 2.

FIGURE 5

Representative EEG traces from a PV^Cre/Gq-DREADD animal after i.p. PTZ injection on day 1 and focal (SScortex) CNO and i.p. PTZ injection on day 2. Asterisks (^∗), hash signs (#), and dot signs (•) represent absence-like, tonic-clonic and other types of seizures, respectively. Each trace represents 10 min of EEG recording.

FIGURE 6

Representative EEG traces from a PV^Cre/Gq-DREADD animal after i.p. PTZ injection on day 1 and focal (RTN thalamus) CNO and i.p. PTZ injection on day 2. Asterisks (^∗), hash signs (#), and dot signs (•) represent absence-like, tonic-clonic and other types of seizures, respectively. Each trace represents 10 min of EEG recording.

FIGURE 7

Comparison of the latency to first seizure (any of the two seizure type) in PV^Cre/Gq-DREADD and non-DREADD WT controls of the (A) SScortex group and the (B) RTN thalamus group between day 1 (PTZ only treated) and day 2 (CNO and PTZ treated). Comparisons between the treatment groups were made using log-rank test. (A) *p = 0.0477; (B) **p = 0.0019.

FIGURE 8

(A) Comparison of the latency to first absence seizure in animals which experienced absence seizures on day 1. (B) Comparison of the latency to first absence seizure in all tested PV^Cre/Gq-DREADD and non-DREADD WT controls of the SScortex group and the RTN thalamus group between day 1 (PTZ only treated) and day 2 (CNO and PTZ treated). Comparisons between the treatment groups were made using log-rank test. (A) **p = 0.0024, ***p = 0.0002; (B) *p = 0.0268, ***p = 0.0001.

FIGURE 9

(A) Comparison of the latency to first tonic-clonic seizure in animals, which experienced tonic-clonic seizures on day 1. (B) Comparison of the latency to first tonic-clonic seizure in all tested PV^Cre/Gq-DREADD and non-DREADD WT controls of the SScortex group and the RTN thalamus group between day 1 (PTZ only treated) and day 2 (CNO and PTZ treated). Comparisons between the treatment groups were made using log-rank test. **p = 0.0067.

FIGURE 10

Comparison of mean number of epileptic bursts (discharges/h) and mean length of bursts between PV^Cre/Gq-DREADD (DREADD) (n = 7) and non-DREADD WT (n = 5) animals of (A) the SScortex and (B) the RTN thalamus group after PTZ treatment on day 1 and CNO and PTZ treatment on day 2. All values represent mean ± SEM. Comparisons were performed using Wilcoxon matched-pairs signed-rank test.