# Minimally-invasive manipulation of spared and hypoactive interneurons reduces CA1 synchronization and nonspatial behavior alterations in epilepsy models

Célanie Matringhen, Alexandre Vigier, Nikoleta Bourtouli, François J Michel, Thomas Marissal

<figure><img src="image_1.png" /></figure>

## To cite this version:

Célanie Matringhen, Alexandre Vigier, Nikoleta Bourtouli, François J Michel, Thomas Marissal. Minimally-invasive manipulation of spared and hypoactive interneurons reduces CA1 synchronization and nonspatial behavior alterations in epilepsy models. Neurobiology of Disease, 2025, pp.107155. 10.1016/j.nbd.2025.107155. hal-05333890

HAL Id: hal-05333890

https://amu.hal.science/hal-05333890v1

Submitted on 27 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
Journal Pre-proof

Minimally-invasive manipulation of spared and hypoactive interneurons reduces CA1 synchronization and nonspatial behavior alterations in epilepsy models

Célanie Matringhen, Alexandre Vigier, Nikoleta Bourtouli, François J. Michel, Thomas Marissal

PII: S0969-9961(25)00372-9

DOI: https://doi.org/10.1016/j.nbd.2025.107155

Reference: YNBDI 107155

To appear in: *Neurobiology of Disease*

Received date: 13 May 2025

Revised date: 15 October 2025

Accepted date: 21 October 2025

<figure><img src="image_3.png" /></figure>

Please cite this article as: C. Matringhen, A. Vigier, N. Bourtouli, et al., Minimally-invasive manipulation of spared and hypoactive interneurons reduces CA1 synchronization and nonspatial behavior alterations in epilepsy models, *Neurobiology of Disease* (2024), https://doi.org/10.1016/j.nbd.2025.107155

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
# Minimally-invasive Manipulation of Spared and Hypoactive Interneurons reduces CA1 Synchronization and Nonspatial Behavior alterations in Epilepsy models

Célanie Matringhen<fnref n="1" />, Alexandre Vigier<fnref n="1" />, Nikoleta Bourtouli<fnref n="2" />, François J. Michel<fnref n="1" />, Thomas Marissal<fnref n="1" />*

**Author affiliations:**

*Correspondence to: Thomas Marissal

Full Postal Address: INMED, Parc Scientifique de Luminy, 163 avenue de Luminy, 13009
Marseille

E-mail Address: thomas.marissal@inserm.fr

**Keywords:** Temporal lobe epilepsy; Hippocampus; Interneurons; Network dynamics; Seizures; Behavioral alterations; Memory deficits;

<footnote n="1">¹ INMED, INSERM, Aix-Marseille University, France</footnote>

<footnote n="2">² National and Kapodistrian University of Athens, Greece</footnote>
Abstract

Temporal lobe epilepsy (TLE) is associated with seizures and severe cognitive impairments, including memory deficits. The dysfunction of a large population of hippocampal interneurons, composed of various subtypes, is proposed as a key mechanism and a therapeutic target. However, the nature and extent of alterations in hippocampal inhibitory neurons remain unclear, as does their impact on TLE pathology. To address this issue, we combined immunolabeling, calcium imaging, electrophysiology, chemogenetic tools, and behavioral assays in mouse pilocarpine TLE models to investigate the survival and changes in the activity of a large population of Dlx-expressing interneurons and test the effect of their manipulation on seizures and cognitive impairments. We observed that a large population of Dlx-expressing interneurons, which includes Cholecystokinin-, Parvalbumin-, and Somatostatin-positive cells, was overall spared from histological damage occurring in CA1 from TLE mice, despite subtype-specific vulnerability. In addition, Dlx-expressing interneurons exhibited reduced activity in vitro, correlated to hypersynchrony in the CA1 network. Enhancing CA1 interneuron discharge in vitro using a chemogenetic strategy rescued CA1 activity and synchronization. In vivo, a minimally invasive approach to normalize interneuron activity does not rescue the full range of pathological features associated with TLE, but significantly reduces some cognitive impairments, particularly behaviors related to nonspatial learning and memory. Our findings suggest that enhancing local CA1 interneuron activity can restore local network balance and improve cognitive function in TLE.
# 1. Introduction

Temporal lobe epilepsy (TLE) is a prevalent and often incurable disease associated with the generation of spontaneous recurrent seizures as well as severe cognitive impairments, including memory deficits (Marissal, 2021). The dysfunction of hippocampal interneurons is proposed as a key mechanism and a therapeutic target (Liu et al., 2014). However, the nature and extent of alterations in hippocampal interneurons remain controversial despite an abundant literature (Cossart et al., 2001; Dinocourt et al., 2003; Peng et al., 2013; Proddutur et al., 2023; Shuman et al., 2020), as does the validity of interneuron-specific strategies designed to reduce the seizures and cognitive impairments related to TLE. Various methods based on stem cell grafting or reactive glia reprogramming have been tested to replenish the pool of hippocampal interneurons (Bershteyn et al., 2023; Lentini et al., 2021), which are postulated to undergo a subclass-specific loss during the early disease stages according to pioneer studies in the CA1 area from TLE rats (Dinocourt et al., 2003). However, the clinical application of these approaches remains questionable given that recent observations from patients and mouse models of TLE suggest that the loss of CA1 inhibitory neurons can be modest or absent, and unlikely to contribute to behavioral coding deficits (Lehner et al., 2024; Shuman et al., 2020). Other approaches are based on the specific manipulation of a single class of interneurons (e.g., Parvalbumin or PV neurons), using chemogenetic or optogenetic tools, sometimes combined with a closed-loop system with EEG monitoring (Kim et al., 2020; Krook-Magnuson et al., 2013; Lévesque et al., 2019; Wang et al., 2020, 2018). Again, apart from the difficulties of implementing such strategies in patients, these approaches also face several limitations. Firstly, most are based on the excitation of an interneuron subtype postulated to be already hyperactive or hyperexcitable in epileptic conditions (Cossart et al., 2001), and their effect on seizures (Krook-Magnuson et al., 2013; Lévesque et al., 2019; Wang et al., 2020) or behavioral alterations (Kim et al., 2020; Wang et al., 2018) is not always beneficial. Secondly, these studies
overlook the extent of degeneration of the considered interneuron subtype, a factor that could
nonetheless affect the effect of their manipulation. Lastly, these studies rarely take into account
the complementary pathophysiological role played by the different classes of interneurons
expressing PV, Somatostatin (SOM), or Cholecystokinin (CCK) (Amilhon et al., 2015; Dudok
et al., 2022, 2021; Miri et al., 2018; Parrish et al., 2019). These different interneuron subclasses
form interconnected inhibitory microcircuits that regulate CA1 network dynamics (Bocchio et
al., 2024), and activation of a large population of interneurons could control pathological
activity more efficiently than stimulation of a single subpopulation (Ledri et al., 2014). To
tackle these issues, we combined immunolabelling and in vitro calcium imaging in mouse TLE
models to investigate the survival and activity changes of a large population of Dlx-positive
CA1 interneurons, including inhibitory neurons expressing PV, SOM, and CCK. Then, we
tested the effect of manipulating this population of Dlx-expressing CA1 interneurons on TLE-
related seizures and cognitive alterations using behavioral assays, *in vivo* electrophysiology,
and minimally invasive chemogenetic tools that are potentially applicable to humans
(Dimidschstein et al., 2016). We show that a large population of Dlx-expressing interneurons,
that includes CCK-, PV- and SOM-positive cells, was overall spared from CA1 atrophy
observed in the TLE mice, despite differential subtype resistance. Overall, CA1 Dlx-expressing
interneurons are less active in epileptic than in control conditions *in vitro*, resulting in increased
activity and synchronization of the epileptic CA1 network. Enhancing CA1 Dlx-expressing
interneuron discharge using a chemogenetic strategy *in vitro* rescued CA1 activity and
synchronization. *In vivo*, the minimally invasive chemogenetic activation of hippocampal Dlx-
expressing interneurons does not affect seizures or deficits related to spatial learning and
memory, but does reduce impairments in behaviors related to anhedonia or nonspatial learning
and memory. Therefore, we propose that rescuing CA1 local network dynamics using
interneurons as a lever could be sufficient to decrease specific behavioral deficits.
## 2. Materials and Methods

### 2.1. Animals

We used wild-type mice from Swiss/CD-1 background (from Charles River and Janvier Labs). Animals were kept on a normal 12h light/dark cycle at 22-24°C and with ad libitum access to food and water. Control and epileptic mice were single-housed in enriched cages. All experiments conducted on mice were performed in accordance with the directives of the European Community Council (2010/63/UE) and received approval from the French Ministry for Research, after ethical evaluation by the institutional animal care and use committee of Aix-Marseille University (APAFIS number: #23627). Across all experiments, particular care was taken to minimize any stress or suffering in animals, including manipulating them using a tunnel to avoid grabbing them by the tail.

### 2.2. *In vivo pilocarpine-based model of TLE*

To examine the contribution of interneurons in cognitive comorbidities *in vivo*, we used a low-mortality pilocarpine-based procedure to generate mice that exhibit histological and electrophysiological characteristics associated with TLE (Vigier et al., 2021). To avoid the difference due to circadian effects, mice underwent the pilocarpine TLE induction at the same time of the day (i.e., starting from around 10:00 AM). In brief, we placed adult males (2-3 months old) in individual cylindrical tanks made from transparent Plexiglas (diameter: 30 cm, height: 40 cm). Mice were administered an intraperitoneal injection of scopolamine methyl-nitrate (10 mg/kg, IP, ChemCruz) to reduce the peripheral cholinergic effects of pilocarpine. Thirty minutes later, we applied a protocol with a ramp-up pilocarpine dose until the development of *Status Epilepticus* (SE). A first dose of pilocarpine was administered (300 mg/kg, IP, Sigma), and the mouse behavior was monitored. As long as the mice did not develop short seizures with motor manifestation (myoclonus), we injected 150 mg/kg of pilocarpine every 30 minutes. If mice experienced myoclonus, we performed a single injection of caffeine
(40 mg/kg, IP, Sigma), which increased the survival of pilocarpine-treated mice (Vigier et al., 2021). SE generally occurred within the following 60-120 minutes after the first injection of pilocarpine. The onset and severity of status epilepticus (SE) was assessed using a behavioral scale described in (Vigier et al., 2021). All mice undergoing status epilepticus exhibited very similar symptoms from one animal to another (e.g., extreme body tremor), continuously throughout the duration of SE. After 1 hour of SE, all mice were injected with diazepam to interrupt seizure activity (10 mg/kg, IP, Laboratoire TVM). With this protocol based on a 1-hour pilocarpine-induced SE, all mice exhibit recurrent and spontaneous epileptic seizures recorded on EEG several weeks after the SE (Boileau et al., 2023; Vigier et al., 2021). In addition, these animals often display behavioral alterations and histological alterations similar to those observed in patients, such as mossy fiber sprouting (Gröticke et al., 2007; Vigier et al., 2021). After the protocol, the animals were injected with a saline solution to avoid dehydration (0.9%, Osalia, subcutaneous). They were then placed in individual cages containing enrichment and a hyper-nourishing gel (Gel-Safe). The mice were observed daily for 3 weeks, then at least twice a week until death. Pilocarpine-treated chronically epileptic animals were referred to as “Pilo” mice.

## 2.3. *In vitro* pilocarpine-based model of TLE

To investigate the relationship between the recruitment of interneurons and the CA1 epileptic network dynamics *in vitro* as in (Călin et al., 2018), we used long lifespan mouse hippocampal organotypic slices (referred to as “slices” for simplicity), which display, after application of pilocarpine, histological and network dynamics alterations that are similar to those observed in the mouse *in vivo* pilocarpine-based model of TLE that we use, as well as tissues from patients with TLE (Boileau et al., 2023; Goirand-Lopez et al., 2023; Peret et al., 2014). 5-7-day-old mice of both sexes were decapitated, and their hippocampi were removed and unfolded in PBS + 30mM Glucose. Transverse hippocampal slices (400 µm thickness) were made using a
chopper (McIlwain). Slices were placed on Millicell-CM Biopore membranes (Millipore) in culture dishes containing 1 ml of the following medium: MEM 50%, HS 25%, HBSS 25%, HEPES 15 mM, glucose 6.5 mg/ml, and insulin 0.1 mg/ml. Slices were maintained in an incubator at 37 °C/5% CO2, and the culture medium was changed every 2-3 days. Viral infection of the slices was performed the day after the preparation of the culture (i.e., day *in vitro* 1, DIV1), by applying approximately 300 nl of adeno-associated-virus (AAV-)containing solution onto the surface of the hippocampus. To induce the histological damages and network reorganization similar to those observed in mouse TLE models and patients (Boileau et al., 2023; Goirand-Lopez et al., 2023; Peret et al., 2014), 0.5 µM of pilocarpine was added to the culture medium from *in vitro* day 3 to 6 (DIV3-6). Calcium imaging experiments were conducted a minimum of one week after the pilocarpine treatment, i.e., from DIV10 to DIV14. Pilocarpine-treated slices were referred to as “Pilo” slices.

## 2.4. Surgeries

Surgery was performed in a subgroup of mice to inject AAVs that enabled the manipulation of inhibitory neurons and/or to record ictal activity. In both cases, mice were subcutaneously injected with an analgesic (Buprenorphine, 45 µg/kg) and an anti-inflammatory drug (Carprofen, 10 mg/kg) 20-30 minutes prior to the surgery. Anesthesia was induced at 5% in a transparent chamber (Minerve) and maintained at 0.5-3% isoflurane (w/v) using a mouse gas mask (Minerve) in a stereotaxic apparatus (Stoelting Co.) coupled to a binocular microscope (SZ61, Olympus). Mice were maintained at 36.5-38 °C using a heating pad and controlled via a rectal thermometer (Stoelting). Mouse heads were secured using ear bars, a jaw bar, and a nose clamp. Eyes were covered with ointment (Ocry-Gel). Lurocaïne (Lidocaine, 7 mg/kg), a local analgesic, was injected below the skin overlaying the skull. Then, the two procedures differed:
## Viral injections

AAVs were injected bilaterally in the dorsal and ventral hippocampus of chronically epileptic Pilo animals (minimum 3 weeks after TLE induction) or age-matched controls. We mainly used AAV constructs enabling the expression of the excitatory chemogenetic “DREADD” receptor hM3D and the red reporter tdTomato under the control of the interneuron-specific hDlx enhancer element (pAAV-hDlx-GqDREADD-tdTomato-Fishell-4, AAV9, titer > 7×10¹² vg/mL; these AAVs were a gift from Gordon Fishell, Addgene, 162375-AAV9) (Dimidschstein et al., 2016). Some mice were injected with a control virus (e.g., pAAV-mDlx-GFP-Fishell-1, retrograde AAV, titer > 7×10¹² vg/mL; these AAVs were a gift from Gordon Fishell, Addgene, 83900-AAVrg). The coordinates for the dorsal and ventral hippocampi were: AP -1.8, ML ± 1, DV -2, and AP -3.3, ML ±2.3, DV -2.5 from the bregma, respectively. In brief, the scalp of the anesthetized animals was shaved using electric clippers and then incised using a scalpel blade. The skull bone was gently cleaned and dried. The position of the craniotomies was then defined with a fine marker. The skull was pierced using a dental drill (Fordedim), without damaging the meninges. Then, 500 nL of AAV was injected using a Nanoject III injector (Drummond) and glass capillaries (rate: 20 nL/seconds, 10 cycles separated by 1 second). After injections, the skin was sutured with surgical stitches (Ethilon 5-0 polyamide thread), then anesthesia was discontinued. An infection was considered successful when robust labeling of tdTomato (expressed by AAV constructs) was detected in the hippocampi (particularly in the CA1 region), and expressed in a substantial number of interneurons. Expression of genes of interest was also reported in the cortex above the hippocampus, which was traversed by the injection capillary.

## Implantation of the EEG recording device

A minimum of two weeks after AAV injection, the scalp and the back of the Pilo animals were shaved using electric clippers. The skin was cleaned with antiseptic (Vetadine) and sterile saline
solution (0.9%, Osalia). The skull was exposed using a scalpel blade. Three craniotomies were made using a drill (Foredom): two in an intermediate position between the two AAV injection sites in both hemispheres (approximately AP -2.55 mm, ML +1.65 mm relative to Bregma), the last above the cerebellum as in (Boileau et al., 2023). Then, the mouse's back was incised, and a subcutaneous pouch was created by blunt dissection. A radio-frequency transmitter (ETA-F10, DSI) was implanted inside the pocket close to the ventral abdominal region, under the left flank, with wires oriented towards the head. The two emitter wires were carefully slipped underneath the mouse's neck skin. The tip of the first wire was inserted into the hole above the right hippocampus (~1.5 mm from the skull surface). The second wire, placed above the cerebellum (~2 mm from the skull surface), served as the ground electrode. To fix the wires, a screw was put in each of the three holes and covered with dental cement (Paladur). Finally, the cranial and flank incisions were sutured using 5-0 polyamide filaments (Ethilon, Ethicon). Mice were injected with saline solution (0.9%, 0.35 mL) and allowed to recover from the surgery on a heating pad until fully awakened. The animal's weight was then monitored. Buprenorphine (45µg/kg) was injected 24 hours after surgery, and Carprofen (10 mg/kg) and saline solution (0.9%, 0.35 mL) were injected 24 and 48 hours after surgery, all subcutaneously.

**2.5. Behavioral testing**

We performed various tests to measure mouse behavior in different experimental conditions.
Thus, we used Control- and Pilo-VEH mice (injected with AAV9-hDlx-GqDREADD-
tdTomato and treated with vehicle), Pilo-CNO animals (injected with AAV9-hDlx-
GqDREADD-tdTomato and treated with CNO), and Pilo-ShamCNO mice (injected with a
control virus and treated with CNO). CNO (clozapine-N-oxide, Tocris) was diluted in the 4%-sucrose drinking water (5 mg/200 mL). The administration of CNO in water is a less invasive
method than daily intraperitoneal injections (Zhan et al., 2019). Although sucrose was used to
cover the taste of CNO, which is postulated to be slightly bitter (Milosavljevic et al., 2016), it
was added to the drinking water of all groups of mice, even those not treated with CNO. Animal assignment to the various experimental groups was randomized. The administration of CNO or VEH started one week before the behavior test (i.e., 1-2 weeks after viral injection) and ended at its completion. Drinking water consumption was measured regularly to calculate the average CNO intake per animal per day. Particular care was taken when manipulating the bottles to limit waste of treated water. Mice were handled by the experimenter every day for a week and habituated to the experimental room the day before behavioral testing. Tests were performed under indirect room lighting conditions (120 lux) and at similar times of the day (i.e., starting from 10:00 AM) to avoid differences due to circadian effects. Mouse behavior was continuously videotaped by a video camera placed above the apparatus, tracked using the Ethovision 11.5 software, and the detection was manually corrected if necessary. The apparatuses were cleaned with 30% ethanol before each trial to eliminate olfactory cues. When a mouse experienced a seizure with motor manifestation before or during the behavioral testing, it was returned to its home cage, then tested again at least 30 minutes later. However, in the absence of EEG monitoring during behavior, we cannot exclude that epileptiform activities without obvious motor signs occurred during the experiment. All criteria used for including and excluding animals were defined prior to analysis. Across all experiments, we discarded any mouse that presented ulcerative dermatitis, a skin condition that can be observed in pilocarpine-treated animals after repetitive scratching. We also excluded from analysis animals that presented abnormal locomotion, i.e., stereotyped movements, or inability to move normally. For exclusion criteria in specific tests, see below for details.

### Barnes circular maze (BCM)

BCM (Gawel et al., 2019) consisted of an elevated circular platform made of metal (diameter = 100 cm; elevation from the ground = 90 cm, color: grey) containing 20 holes equally spaced around the periphery (diameter = 4 cm). Under one hole was placed a magnetic escape room
(black) in which mice could hide. Under the remaining 19 holes were placed black caps, which, from the surface of the platform, appeared indistinguishable from the escape room. Distal visual cues (length/width = 30 cm) surrounded the platform. BCM was performed while a loudspeaker played white noise (50-60 dB). 24h prior to the test, mice were allowed to freely explore the maze during 1-2 habituation sessions of 3 minutes, after which they were gently guided to the escape room. Then, mice underwent the acquisition phase of the test (during which spatial learning-related abilities were assessed). This acquisition phase consisted of 2 trials per day for 4 consecutive days, during which the position of the escape room remained constant. For each trial, mice were placed in a start chamber in the middle of the maze ("release zone"). After 15 seconds, the chamber was lifted, and the animals were allowed to explore the maze. The trial concluded when the mice entered the escape room, or after 2 minutes had elapsed. If a mouse failed to enter the escape room within the 2-minute period, it was gently guided to the escape room by the operator and left there for 15 seconds prior to removal. For each day of habituation and acquisition, several parameters were quantified to assess mouse performance, including: the success and failure rate (i.e., the % of mice per experimental group that identified or not the escape room location at least once per day of acquisition), the latency to locate the escape room ("primary latency"), along with the number of incorrect holes checked prior to finding the escape room or the completion of the test ("primary errors"). Some pilocarpine-treated animals jumped off the apparatus and were considered to have failed the test session. To ensure correct learning, they were quickly returned to the table to complete the test under the same conditions as the other animals. For quantification of primary latencies and errors, we calculated the average of the non-null values for each trial per day of the BCM acquisition phase. We also classified the mouse exploration strategy as direct (when mice explored less than 3 distinct holes prior to finding the escape room), serial (when mice explored consecutive holes separated by up to 2 holes in a clockwise or counterclockwise manner) or random (when mice crossed
the maze center, ran circularly on the periphery with no or few hole inspections, used mixed strategies, jumped off the apparatus or poorly explored the apparatus during the duration of a trial, i.e., less than 5 distinct holes explored) (Gawel et al., 2019). To assess behaviors related to spatial memory, animals underwent the probe trial 72 hours after the last acquisition day. During the probe trial, the escape room was removed and replaced by a false escape box (identical to the cap placed underneath the other 19 holes). The probe trial was performed similarly to the acquisition trials (15 seconds in the start chamber, 2 minutes of free exploration). The success rate, the latency prior to reaching to the previous escape room location, the number of incorrect holes checked, and exploration strategies were measured, along with the time spent in the quadrant where the escape room was previously localized ("target zone"). We measured the distance covered by the mice (giving an indication of locomotory activity) only during the BCM probe trial, given that it is the only stage of the test during which the animals remained on the apparatus for the same amount of time, except when epileptic mice jumped off the apparatus (which we reported in Supplementary data). Given the extent and variability in the altered BCM-related behavior of epileptic mice, and in order to avoid missing or aberrant values, we performed a min-max normalization for latency and number of primary errors, as well as time in the target area, a score of 0 being assigned to the best possible performance (e.g., the mouse quickly finds the escape room without making any error during the acquisition phase, or moves into the target zone during the whole probe trial) and 1 to the worst possible performance. Thus, a score of 1 was assigned for primary latency if a mouse failed to find the escape room location or jumped off the apparatus before finding the escape room. A score of 1 was assigned for primary errors when an animal jumped off the apparatus before finding the escape room location or failed to locate the escape room location after little exploration of the apparatus, i.e., 0-5 distinct holes explored. A score of 1 was assigned for the time in the target area if an animal jumped off the apparatus or did not move
from the release zone for the entire trial period. The original data is shown in the Supplementary
data.

Novel Object Recognition test (NORT)

NORT was performed to measure mouse non-spatial memory (Antunes and Biala, 2012), which
involves the dorsal CA1 (Cohen et al., 2013). NORT was performed in a dark Plexiglas box
(40 cm L x 40 cm W x 40 cm H). After habituation to the arena (10 minutes, 1 day prior to the
familiarization stage), mice were placed into the center of the box, with two identical objects
placed in opposite quarters. During this 5-minute familiarization stage, the time exploring each
object was measured. Mice were defined as exploring the object when their nose was directed
towards the object at a distance of < 2 cm. Mice that remained for >100 seconds in a quarter of
the apparatus without exploratory behavior were excluded from the analysis. 1 day later, during
the testing phase, one of the objects was replaced by a new item of similar size, and the time
exploring the familiar versus the novel object was measured for 5 minutes. To limit bias due to
an animal's preference for a particular location in the apparatus, the new object was
systematically replacing the object explored the least in the previous phase. Used objects
included brick toys, 0.5 L bottles, and needle boxes.

2.6. Histology

The week following the end of behavioral testing, mice were anesthetized (using a mix of
Domitor 1.2mg/kg and Zoletil 80 mg/kg) prior to intracardiac perfusion with PBS
(ThermoFisher)/Heparin (0.1%, Sigma) solution and then with a paraformaldehyde-based
solution (Antigenfix, Diapath). Next, the animals were decapitated, and the brains were kept
for a maximum of 24 h in Antigenfix at 4 °C before being washed in PBS. Brains were placed
overnight in 30% sucrose at 4 °C for cryopreservation. Afterward, brains were embedded in
blocks containing optimum cutting temperature medium (OCT; Tissue-Tek) placed on dry ice,
enabling tissue storage at 80 °C for months if necessary. Finally, 60-µm-thick coronal sections
were prepared at -25 °C from the brain tissue blocks using a Cryostat (Leica CM3050S).
Sections were stored in 24-well plates according to their position in the anteroposterior axis of
the brain at 4 °C in PBS-Azide (0.01%) until immunochemistry was performed. For the
histological analysis, we systematically selected two sections for each animal located 840 and
1200 µm posterior to the most anterior part of the hippocampus, which allowed us to include
interneurons situated in the dorsal part of CA1 as well as more ventral regions of CA1, in which
neuronal damage could be observed (Ekstrand et al., 2011). These sections were permeabilized
with PBS-Triton (1%) containing horse serum (10%) for 2 h at room temperature. Next, sections
were incubated with polyclonal goat primary antibody directed against Parvalbumin (PV,
Swant, 1:1000) and monoclonal mouse primary antibody directed against Somatostatin (SOM,
Santa-Cruz, 1:200), or polyclonal guinea pig primary antibody directed against CCK8 (Synaptic
System, 1:1000) in PBS-Triton (1%) containing horse serum (10%) for 1-2 days at 4 °C under
agitation. Sections were then washed with PBS, placed in PBS containing polyclonal donkey
secondary antibody coupled with Alexa488 directed against goat (Invitrogen, 1:500) and
donkey secondary antibody coupled with Alexa647 directed against mouse (Invitrogen, 1:500),
or with donkey secondary antibody coupled with Cyanine Cy5 directed against guinea pig
(Jackson ImmunoResearch Laboratories, 1:500) for 2 h at room temperature under agitation.
Sections were then washed with PBS, and stained with Hoechst (Sigma, 1:2000) at room
temperature under agitation for 5-10 min before mounting (Fluoromount-G mounting medium,
Invitrogen). 4 images per animal (2 in each hemisphere) were acquired using a confocal
microscope (LSM800, Zeiss equipped with 10x/0.3 and 20x/0.8 objectives).
Immunofluorescence was detected in a field of X638.9 × Y638.9 µm (Z-stacks of 8 planes,
interval 2 µm) covering all sublayers of CA1 from the alveus to the stratum lacunosum-
moleculare. Images were processed, and quantification was performed manually using ImageJ
software. In brief, the area of quantified CA1 was measured, and the number of PV, SOM, CCK, and tdTomato-expressing interneurons per animal was counted in the 4 images on z-stacks plane by plane and not on projections. Then, density per animal was estimated by dividing the number of somata by the volume of quantified CA1, averaged over the 4 images. Additionally, in some control and epileptic animals, low magnification mosaic images of the Hoechst-stained hippocampi (X6172 × Y2695 µm) were acquired to measure the dorsal CA1 area, which is the subregion that shows the most pronounced hippocampal sclerosis under our conditions. For interneuron density quantification, images of epileptic animals presenting a massive sclerosis of CA1 (i.e., stratum pyramidale no longer visible) were excluded from the analysis due to difficulties assessing the borders of CA1 and its subregions properly. In all conditions, we also excluded images presenting damaged tissue attributable to histological processing.

## 2.7. Calcium imaging and analysis

To monitor the activity of a large population of CA1 neurons, slices were transduced with AAV constructs enabling the expression of the genetically encoded calcium sensor jGCaMP8m under the control of the human synapsin promoter (pGP-AAV-syn-jGCaMP8m-WPRE, AAV9, titer > 1×10¹³ vg/mL). pGP-AAV-syn-jGCaMP8m-WPRE was a gift from the GENIE Project (Addgene, 162378-AAV9) (Zhang et al., 2023). To identify and manipulate CA1 interneurons, slices were transduced with AAV9-hDlx-GqDREADD-tdTomato-Fishell-4 (Dimidschstein et al., 2016). On the *in vitro* days 10-14, slices were immersed in an artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 1.3 MgCl₂, 2.0 CaCl₂, and 10 Glucose (pH ~7.4), continuously oxygenated and perfused at ~30 °C using a peristaltic pump. All compounds used for the preparation of the ACSF were purchased from Sigma-Aldrich. Calcium transients were recorded using a multibeam two-photon laser scanning system (TriMScope, LaVision Biotec) mounted on a BX61WI Olympus microscope coupled to
an Ultra II chameleon laser (Coherent). A fluorescent lamp (Olympus U-RFL-T) was used to identify a CA1 field with GCaMP-expressing neurons and tdTomato-expressing interneurons. Slices were imaged using a 20X 0.95NA objective (Olympus) at a ~23 Hz frame rate (i.e., 44 ms per frame) for up to ~7 minutes per recording (i.e., 10,000 frames). The two-photon excitation wavelength was 920 nm. A typical imaging session covered a field of 300 × 150 µm size containing up to 127 individual CA1 neurons. The GCaMP-based calcium activity of CA1 in control versus Pilo conditions was measured under baseline conditions or the continuous presence of 50 µM carbachol (Cch, Sigma). The application of Cch on hippocampal slices *in vitro* notably induces oscillatory activity similar those that occur *in vivo* during navigation, learning, and memory processes (Fisahn et al., 1998; Li et al., 2019; Marissal et al., 2018). To test whether Cch-induced CA1 calcium activity reflects the neuronal action potential discharge, some slices were monitored twice, the first time in Cch only, then the sodium channel blocker tetrodotoxin (TTX, Tocris, 1 µM) was applied in the bath in addition to the Cch for at least 15 minutes, followed by a second calcium imaging of the same slice. In another subset of experiments, to test the effect of interneuron manipulation on the network dynamics, some slices were monitored first in Cch only, then the DREADD ligand Clozapine-N-Oxide (CNO, Tocris, 2 µM) was applied in the bath in addition to the Cch for at least 15 minutes, followed by a second calcium imaging of the same slice. A 15-minute bath application of CNO was shown to be sufficient to elevate the activity of all interneurons expressing excitatory DREADDs (Marissal et al., 2018). The detection of neurons and calcium activity onsets/offsets was performed semi-automatically in each movie using the Matlab-based 'Caltracer' software (Columbia University). Typical calcium events were characterized by a sharp rise and a slow decay, lasting for 1 second and with an amplitude of at least twice the baseline. Then, we calculated the level of calcium activity (i.e., number of active neurons, frequency, amplitude, and duration of calcium transients), and quantified neuronal synchronization as in patients
(Goirand-Lopez et al., 2023; Marissal et al., 2018). Calcium imaging recordings in which the slice displayed signs of degradation (e.g., very bright neurons with fluorescence in the nucleus) were excluded from the analysis.

## 2.8. EEG recordings and analysis

We performed EEG recordings to measure seizure activity in epileptic mice injected with AAV9-hDlx-GqDREADD-tdTomato-Fishell-4 before and after chemo-activation of hippocampal interneurons using CNO. On average, 14 weeks after TLE induction and the week following EEG implantation surgery, individual cages containing implanted animals were placed on telemetric receiver plates, and EEG signals (1000× amplified, filtered at 0.16–97 Hz pass, and acquired at 500 Hz) were monitored 24 hours per day for a minimum of 7 consecutive days. All mice were first recorded during a baseline period with a vehicle in the drinking water (4% sucrose in the drinking water, ad libitum, for 5 days). Then, a group of animals was administered CNO (5 mg in 200 mL of 4% sucrose drinking water, ad libitum, for 5 days). These animals (n = 9) were called “Pilo-CNO”. For comparison, a group of animals (n = 4) was treated with the vehicle throughout the recording. This group was referred to as “Pilo-VEH”. Day 0 corresponds to when CNO was administered to Pilo-CNO mice (and to the sixth day of recording in Pilo-VEH mice). The long duration of recordings was justified by the previous observation that spontaneous ictal events occur in clusters that can be separated by long seizure-free periods (Lim et al., 2018; Vigier et al., 2021). Drinking water consumption was measured regularly to calculate the average CNO intake per animal per day.

EEG was analyzed using the Neuroscore software (DSI). EEG raw signal was visually inspected, and the ‘periodogram’ function of DSI (time-frequency spectrogram of EEG, determined using a Fast Fourier Transform algorithm with a sliding 10 seconds-Hanning window) was used to help detect ictal events. Similar to (Boileau et al., 2023; Vigier et al., 2021), we quantified in each experimental condition the frequency and duration of ictal activity
characterized by rhythmic (4-60 Hz) and prolonged (>10s) spike trains with an amplitude exceeding twice the baseline level. To limit the potential effect of anesthesia and post-surgical care (see Surgery section above), we only analyzed EEG signals recorded from the third day after implantation, as in (Padmasola et al., 2024). Time periods during which we observed artifacts (e.g., due to repeated head scratching by the animal) were removed from the analysis if they did not represent a prominent part of the recordings. Otherwise, the whole recording was excluded from analysis.

## 2.9. Statistical methods

For the comparison of multiple groups of two factors, we used the two-way ANOVA test followed by Sidak's post-hoc test when appropriate. For comparisons between two unpaired groups with normal distribution, the two-tailed Student's t-test was used. For comparisons between two unpaired groups without normal distribution, the two-tailed Mann-Whitney test was used. For comparisons between two paired groups with a normal distribution, the two-tailed paired t-test was used. For comparisons between two paired groups without normal distribution, the two-tailed Wilcoxon test was used. Normality was assessed using the Shapiro-Wilk test. For the analysis of categorical outcomes (e.g., success or failure), we used Fisher's exact test. All statistical tests were performed using GraphPad Prism software and were detailed in the figure legends. Sample size was decided using InVivoStat software.
**3. Results**

**3.1. Dlx-expressing interneurons are overall spared in the CA1 from epileptic mice**

To study the fate of a large population of CA1 interneurons in epileptic mice, we injected non-epileptic control mice and pilocarpine-treated epileptic animals ("Pilo") with AAVs expressing the fluorescent reporter tdTomato into cells expressing the interneuron-specific enhancer Dlx (see **Materials and Methods**) (Dimidschstein et al., 2016). The population of Dlx-expressing interneurons, including PV-, SOM- and CCK-positive inhibitory cells (89.99 ± 3.347% of PV-positive neurons, 86.29 ± 4.196% of SOM-positive neurons, and 74.59 ± 6.697% of CCK-positive neurons in CA1 expressed tdTomato after infection, **Fig. 1A** and **Supplemental Fig. 1A**), showed no significant differences in density or distribution across the different CA1 sublayers in the epileptic mice compared with controls (**Fig. 1B-D**), despite histological alterations similar to those observed in patients (i.e., CA1 atrophy associated with hippocampal sclerosis, **Supplemental Fig. 1B-C**) (Costa et al., 2019). As expected, the overall survival of Dlx interneurons masked differential vulnerability depending on the major inhibitory cell subtype (**Supplemental Fig. 1D-H**). In line with (Shuman et al., 2020), our data did not support a loss of CA1 PV and SOM interneurons in epileptic mice (**Supplemental Fig. 1D, F-G**). In contrast, CCK interneurons were significantly decreased in pilocarpine-treated mice compared with controls (**Supplemental Fig. 1E, H**). The distribution of PV, SOM, and CCK interneurons remained unchanged (**Supplemental Fig. 1F-H**).

In conclusion, our results suggest that a substantial number of Dlx-expressing interneurons survive the epileptic insult despite histological alterations and subtype-specific degeneration in the CA1 region from Pilo mice.
## 3.2. Hypersynchronization of CA1 network in vitro is correlated with hypoactive interneurons and is reversible

Numerous studies based on different models suggest that the different categories of CA1 inhibitory neurons surviving TLE could display altered activity. However, the directionality of these alterations (i.e., hyper- or hypo-activity), and whether their activation could produce a positive or negative effect on the pathological traits associated with TLE, remains unclear (Dudok et al., 2022). To tackle this issue, we monitored the calcium activity of a large number of CA1 neurons as in (Marissal et al., 2018), using *in vitro* GCaMP8m-based calcium imaging in control versus pilocarpine-treated slices (Controls versus Pilo, **Fig. 2A-E**), which exhibit physiological and histological alterations similar to those observed in the hippocampus of pilocarpine-treated mice or even in hippocampal tissues from adult TLE patients (Boileau et al., 2023; Peret et al., 2014). Given that the spontaneous population activity level was very low under baseline conditions (**Supplemental Fig. 2A-C**), the calcium activity of CA1 was recorded under the continuous presence of 50µM carbachol (Cch), which induces in control conditions network activity resembling those that occur *in vivo* during navigation, learning, and memory processes (Fisahn et al., 1998). As expected (Li et al., 2019; Marissal et al., 2018), the Cch-induced CA1 calcium activity reflects the neuronal action potential discharge, given that most calcium transients were abolished in the presence of the sodium channel blocker TTX (**Supplemental Fig. 2A-C**).

While the percentage of active cells was similar between both conditions (**Fig. 2F**), CA1 neuron calcium activity was more frequent and synchronous in Pilo conditions than in control slices, which was reflected by significantly higher values in the frequency of calcium transients (**Fig. 2G**), the number of coactive neurons (**Fig. 2H**) as well as the occurrence of these neuronal coactivations (**Fig. 2I**). We reported no difference between experimental groups in the average amplitude and duration of calcium transients (**Supplemental Fig. 2D-E**).
We also specifically recorded the GCaMP8m-based calcium activity of Dlx-expressing interneurons (see **Materials and Methods, Fig. 2J-M**). Overall, Dlx-expressing CA1 interneurons seemed to be spared from the histological alterations that affect pilocarpine-treated slices (Peret et al., 2014), given that the number of interneurons per calcium imaging field was not different between conditions (Fig. 2K), while the number of GCaMP-positive neurons per field was decreased in Pilo condition (Supplemental Fig. 2F). By contrast, the calcium transients generated by CA1 interneurons were significantly less frequent under Pilo conditions, or even abolished completely (Fig. 2L, M and Supplemental Fig. 2G). Importantly, interneuron calcium activity was not significantly affected by their localization in deep CA1 layers (i.e., strata oriens and pyramidale) where most PV and SOM neuron soma are situated, or superficial CA1 layers (i.e., strata radiatum and lacunosum-moleculare), where most CCK cell bodies are localized (Supplemental Fig. 2G).

In conclusion, our data suggest that while the CA1 neuronal network is more active and synchronous, interneurons are significantly less active in Pilo conditions compared to control slices.

Could an interneuron-targeting chemogenetic strategy restore the activity of CA1 interneurons and re-establish control-like network dynamics? To test this, we compared the *in vitro* calcium activity in the Pilo CA1 neuronal network, including a few tdTomato- and hM3D-expressing interneurons, before ('Pilo-VEH') and after 15 minutes of bath application of CNO (Clozapine-N-Oxide, 2 µM, see **Materials and Methods**). The activation of the excitatory DREADD hM3D using CNO significantly increased the frequency of calcium transient generated by inhibitory neurons (Fig. 3A-B), to levels that are comparable to the Controls (Mann-Whitney test *P* = 0.5148, Fig. 2M).

The chemogenic elevation of CA1 interneuron discharge attenuated the Pilo CA1 network activity and synchronization (Fig. 3C), which was reflected by a significant decrease
in the percentage of active cells (Fig. 3D), the average frequency of calcium transients generated by all neurons of the imaged fields (Fig. 3E), the number of neurons involved in coactivations (Fig. 3F), and the occurrence of these coactivations (Fig. 3G). By contrast, the chemoactivation of hippocampal interneurons affected neither the amplitude nor the duration of calcium transients (Supplemental Fig. 2H, I). Apart from the number of active cells, the *in vitro* calcium activity in pilocarpine-treated CA1 treated with CNO was no longer significantly different from the control slices (Two-tailed unpaired t-test *P* = 0.0331, 0.0820, 0.4058, and 0.2157 for the number of active cells, as well as calcium transient frequency, amplitude and duration, respectively; Mann-Whitney test *P* = 0.4669 and 0.1678 for the number of coactive neurons and the occurrence of coactivations, respectively. Fig. 2F-I and Supplemental Fig. 2D-E). Therefore, our data suggest that rescuing interneuron activity is sufficient to restore local CA1 network dynamics *in vitro*.

**3.3. The *in vivo* chemoactivation of hippocampal interneurons does not rescue the pathology, but attenuates specific cognitive deficits**

Could the chemogenetic stimulation of hippocampal Dlx-expressing interneuron activity decrease the pathological traits associated with TLE, such as seizures and cognitive deficits?

To answer this question, we performed *in vivo* electrophysiological recordings or behavioral assays on separate cohorts of pilocarpine-treated TLE animals while hM3D-expressing hippocampal interneurons were selectively activated through the presence of CNO in the 4%-sucrose drinking water (i.e., ‘Pilo-CNO’ condition), or not (‘Pilo-VEH’ condition, see Materials and Methods). For behavioral testing, we also used a group of vehicle-treated non-epileptic control mice (‘Control-VEHs’). Importantly, the density and distribution of Dlx-positive interneurons (that express hM3D receptors and tdTomato) were similar for all experimental conditions (Supplemental Fig. 3A-C). The average daily consumption of
drinking water was significantly reduced in Pilo-VEH mice compared to Control-VEH mice, which could reflect anhedonia-like behavior (Willner, 1997) typically observed in single-housed epileptic animals (Manouze et al., 2019). Interestingly, consumption of 4%-sucrose drinking water was significantly increased in the Pilo-CNO group compared to Pilo-VEH (Fig. 4A).

In baseline conditions, prolonged ictal activity reminiscent of generalized tonic-clonic seizures has been monitored using EEG recordings in all pilocarpine-treated animals in the hippocampal region distant from injection sites (Fig. 4B, see Materials and Methods). Despite the impact of interneuron chemoactivation on local synchronization in vitro (Fig. 3), we observed no significant difference on the frequency (Fig. 4C) and duration (Supplemental Fig. 3D) of seizures between Pilo-VEH and Pilo-CNO conditions (estimated daily consummation for EEG experiments: 7.6 mg/kg of CNO per mouse and day).

In parallel, we performed behavioral testing on pilocarpine-treated TLE animals with or without chemoactivation of Dlx-expressing hippocampal interneurons. We first used the Barnes Circular maze (BCM) to assess behavior related to spatial learning and memory in Pilo-CNO, Pilo-VEH, and Control-VEH mice. Animals in the Pilo-VEH group show impaired behavior in the BCM compared to Control-VEH mice (Fig. 4D-F, Supplemental Fig. 3E-H, Supplemental Fig. 4A). Notably, the min-max scores related to the latency to locate the escape room at any BCM stage (acquisition days 1-4 and probe trial or PT, Fig. 4E), and the time in the target zone during the PT (Fig. 4F) were significantly higher in Pilo-VEH mice, reflecting poorer performance (Materials and Methods). The proportion of Pilo-VEH animals that identified the location of the escape room during all days of BCM (success rate, Supplemental Fig. 3E-F) and exhibited a direct exploration strategy (Supplemental Fig. 3G) was significantly lower than in Control-VEH animals. Last, Pilo-VEH mice display the highest min-max scores related to the number of primary errors (Supplemental Fig. 3H). The original data
is indicated in **Supplemental Fig. 4A**. Epileptic mice treated with CNO (estimated consumption for behavioral experiments: 6.4 mg/kg per mouse and day) display little enhancement of their performance in the BCM (**Fig. 4D-F, Supplemental Fig. 3E-H, Supplemental Fig. 4A**) despite a slight improvement of their min-max scores related to the latency to locate the escape room (**Fig. 4E**) and success rates (**Supplemental Fig. 3E-F**) during the later phases of the test (e.g., acquisition day 4 and PT).

However, the analysis of BCM was complicated by the multitude of aberrant behaviors exhibited by the epileptic mice (e.g., jumping off the apparatus, no movement from the release zone) (see **Materials and Methods, Supplemental Fig. 4A**), which were not necessarily related to spatial processes, or even to CA1 circuits. To investigate other types of CA1-dependent memory that are altered in TLE, we conducted the novel object recognition task (NORT) on all experimental groups (see **Materials and Methods, Fig. 4G-H**). During the NORT familiarization phase, no experimental group showed a preference for either of the two identical objects (**Supplemental Fig. 4B**). During the test phase, the Control-VEH mice explored the novel object significantly more than the familiar object, while Pilo-VEH mice spent a similar amount of time exploring the two objects in the absence of Dlx-expressing interneuron manipulation. By contrast, preference for the novel object was rescued in Pilo-CNO animals (**Fig. 4H**). Regarding locomotory activity, although Pilo animals displayed a greater variability compared to Controls, we observed no significant group differences in the distance covered during the BCM probe trial, or the NORT familiarization and test phases (**Supplemental Fig. 4C**). Finally, the beneficial effects of chemoactivation of hippocampal Dlx interneurons cannot be explained by CNO alone, since epileptic mice treated with CNO in the absence of hM3D receptor expression (mice injected with a control virus, referred to as “Pilo-shamCNO” animals) show no improvement in their behavior in either BCM or NORT (**Supplemental Fig. 5**).
Our data suggest that chemoactivation of a large population of Dlx-expressing
hippocampal interneurons does not rescue the full range of pathological features associated with
TLE, since seizures and deficits related to spatial learning and memory are unaffected.
However, activation of Dlx-expressing interneurons in the hippocampus does reduce some
cognitive impairments, such as behaviors related to nonspatial learning and memory.

**4. Discussion**

Using immunohistological analysis, we show that pilocarpine-treated TLE mice display no decrease in the density of CA1 Dlx-expressing interneurons, a large population of inhibitory cells that includes PV-, SOM-, and CCK-expressing interneurons. The overall resistance of Dlx-expressing interneurons hides diversity in the vulnerability of inhibitory neuron subtypes. Surprisingly, we showed an increase in CA1 PV- and SOM-expressing interneuron density in pilocarpine-treated TLE mice. Our observations suggest that this phenomenon is more likely explained by the decrease in CA1 area (reflecting CA1 atrophy associated with hippocampal sclerosis) (Costa et al., 2019) than by a plasticity mechanism (Donato et al., 2013). Although in contradiction with some pioneer observations in the epileptic rat (Cossart et al., 2001; Dinocourt et al., 2003), these data are in agreement with recent studies performed on tissues from patients, as well as others performed using the mouse pilocarpine model, which suggests that spatial processing impairment is not correlated to a loss of CA1 PV and SOM interneuron (Lehner et al., 2024; Shuman et al., 2020). In contrast, CCK interneurons were diminished in epileptic mice, as in other models of TLE (Kang et al., 2021). Given that the silencing of CA1 CCK interneurons does not impact mouse performance in a spatial memory test comparable to the BCM (Guerrero et al., 2024), we do not believe that the reduction of their density alone can explain the cognitive alterations displayed by pilocarpine-treated mice.
In line with our *in vitro* calcium imaging data, Dlx-positive interneurons are not diminished in pilocarpine-treated slices compared to controls, despite a smaller number of GCaMP-expressing neurons, which could be the consequence of *in vitro* histological alterations similar to those observed *in vivo* in TLE mice or even in patients (Boileau et al., 2023; Peret et al., 2014). Dlx-expressing interneurons are overall hypoactive under epileptic conditions *in vitro*, while the network presents excessive and hypersynchronous activity. This observation brings new insight to the debate as to whether hippocampal interneurons are less active ("dormant") or rather hyperactive. We did not attempt to decipher the mechanisms that explain the reduced activity of CA1 interneurons *in vitro*. Several studies suggest that the intrinsic and synaptic properties of certain inhibitory neurons may be altered under epileptic conditions, thereby permanently modifying their firing (Dugladze et al., 2007; Lee et al., 2024; Proddutur et al., 2023). However, characterizing the pathophysiological properties of Dlx-expressing interneuron population, which includes various morpho-physiological subtypes (Dimidschstein et al., 2016), would be an entire study on its own. Similarly, future investigations are needed to determine whether the activity of hippocampal interneuron subtypes is also altered *in vivo*, while epileptic mice are behaving. Interestingly, our data shown in **Supplemental Fig. 2G** suggest that the reduction of activity is more marked for interneurons localized in the superficial CA1 layers (i.e., strata radiatum and lacunosum-moleculare), which include CCK-positive cells. In addition, our results suggest that, despite the average decrease of interneuron activity, a small subpopulation of inhibitory cells localized close to the pyramidal layer is hyperactive as in (Cossart et al., 2001).

As this study focused on the activity of Dlx-expressing interneurons in controls or under conditions reproducing the alterations associated with TLE, we did not combine *in vitro* calcium imaging with local field potential recordings to characterize the activity patterns generated by hippocampi treated or not with pilocarpine. Under control conditions, carbachol-induced
GCaMP fluorescence variations in CA1 correlate with physiological activities such as theta
rhythms (Li et al., 2019). In the pilocarpine-treated hippocampus, which shows similar
alterations to those observed *in vivo* or in patients, excessive and hypersynchronous calcium
activity could be explained by the abnormal recruitment of CA1 neurons into altered rhythmic
activities or interictal-like-epileptiform events (Goirand-Lopez et al., 2023). Either way,
chemogenetic activation of Dlx-expressing interneurons restores CA1 network dynamics
towards control levels.

Most, if not all, studies testing the effect of manipulating a subclass of interneurons on
the pathological features of TLE overlook the nature and extent of alterations affecting the
neuronal type of interest. Therefore, strategies based on chemo- or optogenetic excitation of a
subclass of interneurons, a substantial proportion of which may degenerate (Dinocourt et al.,
2003), and the survivors of which may be hyperactive (Cossart et al., 2001; Dugladze et al.,
2007), remain questionable. Furthermore, these strategies are not always beneficial for seizures
(Krook-Magnuson et al., 2013; Lévesque et al., 2019; Wang et al., 2020) or behavioral
alterations (Kim et al., 2020; Wang et al., 2018). In our study, we build on our prior observation
that a large population of Dlx-expressing hippocampal interneurons is overall spared from
histological alterations *in vivo* and displays decreased activity *in vitro*. On these premises, we
used a chemogenetic strategy to enhance their discharge specifically. *In vitro*, this interneuron-
specific manipulation restores the activity and synchronization of the CA1 neuronal network to
levels comparable to control. *In vivo*, the chemoactivation of Dlx-expressing interneurons does
not affect seizures recorded at a distance from the virus injection sites. This lack of effect is
unlikely to be due to the duration of our recordings, since we performed in vivo
electrophysiological recordings 24 hours a day for 5 days of CNO treatment, which is
comparable to other studies that have shown a beneficial effect of pharmacogenetic strategies
targeting other elements of hippocampal circuits on spontaneous epileptic seizures (e.g., 8-hour
recordings per day for 3 days during chemoactivation of PV interneurons in (Wang et al., 2018), 8-hour recordings per day for 7 days during chemogenetic modulation of septohippocampal cholinergic signaling in (Wang et al., 2020)). At the cognitive level, the chemoactivation of Dlx-expressing interneurons does not reduce spatial behavior deficits but does alleviate specific cognitive impairments, such as recognizing a new object or being interested in palatable drinks. This suggests that hippocampal interneuron manipulation does not prevent the emergence of ictal activities, which involve a broad network of brain structures (Toyoda et al., 2013). The persistence of seizures could interfere with cognition, which is only partially rescued in our conditions. However, a local normalization of hippocampal activity *in vivo*, similar to that we observed *in vitro*, could be sufficient to improve some behavior. Such normalization could be reflected by a reduction in *in vivo* epileptiform activities (e.g., interictal discharges) or the restoration of *in vivo* neuronal activities correlated with memory processes (e.g., *theta* rhythms, shown to be disrupted in pilocarpine-treated animals (Chauvière et al., 2009)). This should be the subject of further research.

We focused on the CA1 region of the hippocampus, which is considered an important node in TLE. Highly susceptible to histological damage, CA1 could not only be involved in seizure generation and propagation (Ang et al., 2006; Buckmaster et al., 2022; Wozny et al., 2005), but also in the cognitive alterations associated with TLE (Chauvière et al., 2009; Lenck-Santini and Holmes, 2008; Shuman et al., 2020), which drastically impair the patients' quality of life (Holmes, 2013). In addition, the inhibitory microcircuits have been particularly well described in the CA1 region under physiological (Klausberger and Somogyi, 2008) and pathological conditions (Cossart et al., 2001; Dinocourt et al., 2003; Dudok et al., 2022; Dugladze et al., 2007; Muldoon et al., 2015; Peng et al., 2013). However, interneuron subclasses from other key temporal lobe regions (e.g., dentate gyrus (Proddutur et al., 2023)), are also postulated to be dysfunctional in TLE, and a similar study should be performed in the future to
clarify the nature and extent of their specific alterations to craft the most adequate rescue strategy.

Most, if not all, studies focusing on the role of biochemically-defined inhibitory neuron subtypes in TLE are based on transgenic lines expressing a recombinase under the control of a specific promoter (i.e., PV-Cre) (Krook-Magnuson et al., 2013; Lévesque et al., 2019; Wang et al., 2018). Here, we used a method based on AAV vectors and regulatory sequences to restrict DREADD expression to interneurons. This method, which does not rely on the Cre-Lox system, is effective in all non-genetically tractable vertebrate species, including humans (Dimidschstein et al., 2016). Therefore, this approach could potentially be applied to intractable adult patients with TLE, in whom the genetic modification of the hippocampus, which would be otherwise surgically removed as a last resort, is not out of the question in terms of ethics and biosafety (Ling et al., 2023; Marissal, 2021). In addition, we believe that our method, based on AAV injections, can be described as “minimally invasive” since it limits the number, duration, and severity of invasive procedures. We used a DREADD chemogenetic strategy, which, unlike optogenetics, does not require heavy surgery to implant optical fibers for photostimulation, combined or not with an EEG device for closed-loop seizure detection (Krook-Magnuson et al., 2013; Lévesque et al., 2019). Given that we aimed at manipulating interneuron activity for a long, uninterrupted period and with as few adverse effects as possible, we used a drinking water-based method of DREADD ligand delivery, a technique reputed to be less invasive and stressful than intraperitoneal injections or the use of mini-pumps (Zhan et al., 2019).

Although this method did not rescue all the pathology associated with TLE, it was sufficient to restore non-spatial behavior in epileptic mice, suggesting that it may be valid under certain conditions where the activity of a neuronal subtype needs to be manipulated in a sustained manner, rather than for a specific brief period (e.g., after seizure initiation) (Kim et al., 2020).
**Data Statement**

The data that supports the findings of this study are available from the corresponding author,
upon reasonable request.

CRediT Author Contribution Statement

The list of authors has been established in accordance with INSERM recommendations for the
signature of scientific articles in the field of life and health sciences
(https://pro.inserm.fr/rubriques/recherche-responsable/integrite-scientifique/signature-des-
publications-scientifiques). The author contribution to the present project was the following:
Célanie Matringhen: Conceptualization, Investigation, Formal analysis, Validation, Writing -
Review & Editing; Alexandre Vigier: Investigation, Formal analysis, Methodology, Writing -
Review & Editing; Nikoleta Bourtouli: Investigation, Formal analysis, Writing - Review &
Editing; François J. Michel: Investigation, Formal analysis, Writing - Review & Editing;
Thomas Marissal: Conceptualization, Investigation, Formal analysis, Methodology, Validation,
Resources, Writing - Original Draft, Visualization, Supervision, Project administration,
Funding acquisition.

Acknowledgements

The authors have benefited from some resources and facilities hosted at the INMED as well as
the teams of Valérie Crépel and Ede Rancz, where the project has been initiated and completed,
respectively. The authors want to thank Ma Xiao, Emilie Pallesi, Severine Pellegrino, Philippe
Moudery, Céline Boileau, Catherine Faivre-Sarrailh, Jean-Bernard Manent, Charles Quairiaux,
Pierre-Pascal Lenck-Santini, Elodie Fino, Agnès Baude, and Marie Kurz for having led their
respective facilities, contributed to student supervision, and/or provided technical or
administrative help and useful discussions. Part of the data was presented as a pre-print (DOI:
10.1101/2024.08.02.606307).
**Funding sources**

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and their First Step and International Research Project programs, Aix-Marseille University (AMU), the Agence Nationale de la Recherche, the Foundation A*Midex (AMX-22-RE-AB-161), the Foundation Fyssen, the Neuroschool MSc incoming mobility program, the NeuroMarseille International Collaborative Research program (NM2MARIINM), the Fondation pour la Recherche sur le Cerveau (FRC), the PHC-Germaine-de-Staël program, and the Ligue Française contre l'Epilepsie (LFCE). This work has received support from the French government under the "France 2030" program via A*Midex (Initiative d'Excellence d'Aix-Marseille Université, AMX-19-IET-004) and ANR funding (ANR-17-EURE-002).

**Declaration of interests**

The authors declare no competing interests.
Reference List

Amilhon, B., Huh, C.Y.L., Manseau, F., Ducharme, G., Nichol, H., Adamantidis, A., Williams, S., 2015. Parvalbumin Interneurons of Hippocampus Tune Population Activity at Theta Frequency. Neuron 86, 1277-1289. https://doi.org/10.1016/j.neuron.2015.05.027

Ang, C.W., Carlson, G.C., Coulter, D.A., 2006. Massive and Specific Dysregulation of Direct Cortical Input to the Hippocampus in Temporal Lobe Epilepsy. J. Neurosci. 26, 11850-11856. https://doi.org/10.1523/JNEUROSCI.2354-06.2006

Antunes, M., Biala, G., 2012. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13, 93-110. https://doi.org/10.1007/s10339-011-0430-z

Bershteyn, M., Bröer, S., Parekh, M., Maury, Y., Havlicek, S., Kriks, S., Fuentealba, L., Lee, S., Zhou, R., Subramanyam, G., Sezan, M., Sevilla, E.S., Blankenberger, W., Spatazza, J., Zhou, L., Nethercott, H., Traver, D., Hampel, P., Kim, H., Watson, M., Salter, N., Nesterova, A., Au, W., Kriegstein, A., Alvarez-Buylla, A., Rubenstein, J., Banik, G., Bulfone, A., Priest, C., Nicholas, C.R., 2023. Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy. Cell Stem Cell 30, 1331-1350.e11. https://doi.org/10.1016/j.stem.2023.08.013

Bocchio, M., Vorobyev, A., Sadeh, S., Brustlein, S., Dard, R., Reichinnek, S., Emiliani, V., Baude, A., Clopath, C., Cossart, R., 2024. Functional networks of inhibitory neurons orchestrate synchrony in the hippocampus. PLOS Biology 22, e3002837. https://doi.org/10.1371/journal.pbio.3002837

Boileau, C., Deforges, S., Peret, A., Scavarda, D., Bartolomei, F., Giles, A., Partouche, N., Gautron, J., Viotti, J., Janowitz, H., Penchet, G., Marchal, C., Lagarde, S., Trebuchon, A., Villeneuve, N., Rumi, J., Marissal, T., Khazipov, R., Khalilov, I., Martineau, F., Maréchal, M., Lepine, A., Milh, M., Figarella-Branger, D., Dougy, E., Tong, S., Appay, R., Baudouin, S., Mercer, A., Smith, J.B., Danos, O., Porter, R., Mulle, C., Crépel, V., 2023. GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy. Annals of Neurology 94, 745-761. https://doi.org/10.1002/ana.26723

Buckmaster, P.S., Reyes, B., Kahn, T., Wyeth, M., 2022. Ventral Hippocampal Formation Is the Primary Epileptogenic Zone in a Rat Model of Temporal Lobe Epilepsy. J. Neurosci. 42, 7482-7495. https://doi.org/10.1523/JNEUROSCI.0429-22.2022

Călin, A., Stancu, M., Zagrean, A.-M., Jefferys, J.G.R., Ilie, A.S., Akerman, C.J., 2018. Chemogenetic Recruitment of Specific Interneurons Suppresses Seizure Activity. Frontiers in Cellular Neuroscience 12. https://doi.org/10.3389/fncel.2018.00293

Chauvière, L., Rafrafi, N., Thinus-Blanc, C., Bartolomei, F., Esclapez, M., Bernard, C., 2009. Early Deficits in Spatial Memory and Theta Rhythm in Experimental Temporal Lobe Epilepsy. J. Neurosci. 29, 5402-5410. https://doi.org/10.1523/JNEUROSCI.4699-08.2009

Cohen, S.J., Munchow, A.H., Rios, L.M., Zhang, G., Ásgeirsdóttir, H.N., Stackman, R.W., 2013. The Rodent Hippocampus Is Essential for Nonspatial Object Memory. Current Biology 23, 1685-1690. https://doi.org/10.1016/j.cub.2013.07.002

Cossart, R., Dinocourt, C., Hirsch, J.C., Merchan-Perez, A., De Felipe, J., Ben-Ari, Y., Esclapez, M., Bernard, C., 2001. Dendritic but not somatic GABAergic inhibition is
decreased in experimental epilepsy. Nature Neuroscience 4, 52–62.
https://doi.org/10.1038/82900

Costa, B.S., Santos, M.C.V., Rosa, D.V., Schutze, M., Miranda, D.M., Romano-Silva, M.A., 2019. Automated evaluation of hippocampal subfields volumes in mesial temporal lobe epilepsy and its relationship to the surgical outcome. Epilepsy Research 154, 152–156.
https://doi.org/10.1016/j.eplepsyres.2019.05.011

Dimidschstein, J., Chen, Q., Tremblay, R., Rogers, S.L., Saldi, G.-A., Guo, L., Xu, Q., Liu, R., Lu, C., Chu, J., Grimley, J.S., Krostag, A.-R., Kaykas, A., Avery, M.C., Rashid, M.S., Baek, M., Jacob, A.L., Smith, G.B., Wilson, D.E., Kosche, G., Kruglikov, I., Rusielewicz, T., Kotak, V.C., Mowery, T.M., Anderson, S.A., Callaway, E.M., Dasen, J.S., Fitzpatrick, D., Fossati, V., Long, M.A., Noggle, S., Reynolds, J.H., Sanes, D.H., Rudy, B., Feng, G., Fishell, G., 2016. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nature Neuroscience 19, 1743–1749.
https://doi.org/10.1038/nn.4430

Dinocourt, C., Petanjek, Z., Freund, T.F., Ben-Ari, Y., Esclapez, M., 2003. Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures. Journal of Comparative Neurology 459, 407–425.
https://doi.org/10.1002/cne.10622

Donato, F., Rompani, S.B., Caroni, P., 2013. Paryalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning. Nature 504, 272–276.
https://doi.org/10.1038/nature12866

Dudok, B., Klein, P.M., Hwaun, E., Lee, B.R., Yao, Z., Fong, O., Bowler, J.C., Terada, S., Sparks, F.T., Szabo, G.G., Farrell, J.S., Berg, J., Daigle, T.L., Tasic, B., Dimidschstein, J., Fishell, G., Losonczy, A., Zeng, H., Soltesz, I., 2021. Alternating sources of perisomatic inhibition during behavior. Neuron 109, 997–1012.e9.
https://doi.org/10.1016/j.neuron.2021.01.003

Dudok, B., Klein, P.M., Soltesz, I., 2022. Toward Understanding the Diverse Roles of Perisomatic Interneurons in Epilepsy. Epilepsy Curr 22, 54–60.
https://doi.org/10.1177/15357597211053687

Dugladze, T., Vida, I., Tort, A.B., Gross, A., Otahal, J., Heinemann, U., Kopell, N.J., Gloveli, T., 2007. Impaired hippocampal rhythmogenesis in a mouse model of mesial temporal lobe epilepsy. PNAS 104, 17530–17535.
https://doi.org/10.1073/pnas.0708301104

Ekstrand, J.J., Pouliot, W., Scheerlinck, P., Dudek, F.E., 2011. Lithium pilocarpine-induced status epilepticus in postnatal day 20 rats results in greater neuronal injury in ventral versus dorsal hippocampus. Neuroscience 192, 699–707.
https://doi.org/10.1016/j.neuroscience.2011.05.022

Fisahn, A., Pike, F.G., Buhl, E.H., Paulsen, O., 1998. Cholinergic induction of network oscillations at 40 Hz in the hippocampus in vitro. Nature 394, 186–189.
https://doi.org/10.1038/28179

Gawel, K., Gibula, E., Marszalek-Grabska, M., Filarowska, J., Kotlinska, J.H., 2019. Assessment of spatial learning and memory in the Barnes maze task in rodents—methodological consideration. Naunyn-Schmiedeberg's Archives of Pharmacology 392, 1. https://doi.org/10.1007/s00210-018-1589-y

Goirand-Lopez, L., Moulinier, M., Vigier, A., Boileau, C., Carleton, A., Muldoon, S.F., Marissal, T., Crépel, V., 2023. Kainate receptors modulate the microstructure of
synchrony during dentate gyrus epileptiform activity. Neurobiology of Disease 185, 106260. https://doi.org/10.1016/j.nbd.2023.106260

Gröticke, I., Hoffmann, K., Löscher, W., 2007. Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice. Experimental Neurology 207, 329–349. https://doi.org/10.1016/j.expneurol.2007.06.021

Guerrero, D.K.R., Balueva, K., Barayeu, U., Baracskay, P., Gridchyn, I., Nardin, M., Roth, C.N., Wulff, P., Csicsvari, J., 2024. Hippocampal cholecystokinin-expressing interneurons regulate temporal coding and contextual learning. Neuron 112, 2045–2061.e10. https://doi.org/10.1016/j.neuron.2024.03.019

Holmes, G.L., 2013. EEG abnormalities as a biomarker for cognitive comorbidities in pharmacoresistant epilepsy. Epilepsia 54, 60–62. https://doi.org/10.1111/epi.12186

Kang, Y.-J., Clement, E.M., Park, I.-H., Greenfield, L.J., Smith, B.N., Lee, S.-H., 2021. Vulnerability of cholecystokinin-expressing GABAergic interneurons in the unilateral intrahippocampal kainate mouse model of temporal lobe epilepsy. Exp Neurol 342, 113724. https://doi.org/10.1016/j.expneurol.2021.113724

Kim, H.K., Gschwind, T., Nguyen, T.M., Bui, A.D., Felong, S., Ampig, K., Suh, D., Ciernia, A.V., Wood, M.A., Soltesz, I., 2020. Optogenetic intervention of seizures improves spatial memory in a mouse model of chronic temporal lobe epilepsy. Epilepsia. https://doi.org/10.1111/epi.16445

Klausberger, T., Somogyi, P., 2008. Neuronal Diversity and Temporal Dynamics: The Unity of Hippocampal Circuit Operations. Science 321, 53–57. https://doi.org/10.1126/science.1149381

Krook-Magnuson, E., Armstrong, C., Oijala, M., Soltesz, I., 2013. On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy. Nature Communications 4. https://doi.org/10.1038/ncomms2376

Ledri, M., Madsen, M.G., Nikitidou, L., Kirik, D., Kokaia, M., 2014. Global Optogenetic Activation of Inhibitory Interneurons during Epileptiform Activity. J. Neurosci. 34, 3364–3377. https://doi.org/10.1523/JNEUROSCI.2734-13.2014

Lee, S.-H., Kang, Y.-J., Smith, B.N., 2024. Activation of hypoactive parvalbumin-positive fast-spiking interneurons restores dentate inhibition to reduce electrographic seizures in the mouse intrahippocampal kainate model of temporal lobe epilepsy. Neurobiology of Disease 203, 106737. https://doi.org/10.1016/j.nbd.2024.106737

Lehner, A., Hoffmann, L., Rampp, S., Coras, R., Paulsen, F., Frischknecht, R., Hamer, H., Walther, K., Brandner, S., Hofer, W., Pieper, T., Reisch, L.-M., Bien, C.G., Blumcke, I., 2024. Age-dependent increase of perineuronal nets in the human hippocampus and precocious aging in epilepsy. Epilepsia Open n/a. https://doi.org/10.1002/epi4.12963

Lenck-Santini, P.-P., Holmes, G.L., 2008. Altered Phase Precession and Compression of Temporal Sequences by Place Cells in Epileptic Rats. J. Neurosci. 28, 5053–5062. https://doi.org/10.1523/JNEUROSCI.5024-07.2008

Lentini, C., d'Orange, M., Marichal, N., Trottmann, M.-M., Vignoles, R., Foucault, L., Verrier, C., Massera, C., Raineteau, O., Conzelmann, K.-K., Rival-Gervier, S., Depaulis, A., Berninger, B., Heinrich, C., 2021. Reprogramming reactive glia into interneurons reduces chronic seizure activity in a mouse model of mesial temporal lobe epilepsy. Cell Stem Cell 28, 2104-2121.e10. https://doi.org/10.1016/j.stem.2021.09.002
Lévesque, M., Chen, L.-Y., Etter, G., Shiri, Z., Wang, S., Williams, S., Avoli, M., 2019. Paradoxical effects of optogenetic stimulation in mesial temporal lobe epilepsy. Annals of Neurology 86, 714–728. https://doi.org/10.1002/ana.25572

Li, P., Geng, X., Jiang, H., Caccavano, A., Vicini, S., Wu, J., 2019. Measuring Sharp Waves and Oscillatory Population Activity With the Genetically Encoded Calcium Indicator GCaMP6f. Frontiers in Cellular Neuroscience 13.

Lim, J.-A., Moon, J., Kim, T.-J., Jun, J.-S., Park, B., Byun, J.-I., Sunwoo, J.-S., Park, K.-I., Lee, S.-T., Jung, K.-H., Jung, K.-Y., Kim, M., Jeon, D., Chu, K., Lee, S.K., 2018. Clustering of spontaneous recurrent seizures separated by long seizure-free periods: An extended video-EEG monitoring study of a pilocarpine mouse model. PLOS ONE 13, e0194552. https://doi.org/10.1371/journal.pone.0194552

Ling, Q., Herstine, J.A., Bradbury, A., Gray, S.J., 2023. AAV-based in vivo gene therapy for neurological disorders. Nat Rev Drug Discov 22, 789–806. https://doi.org/10.1038/s41573-023-00766-7

Liu, Y.-Q., Yu, F., Liu, W.-H., He, X.-H., Peng, B.-W., 2014. Dysfunction of hippocampal interneurons in epilepsy. Neurosci. Bull. 30, 985–998. https://doi.org/10.1007/s12264-014-1478-4

Manouze, H., Ghestem, A., Poillerat, V., Bennis, M., Ba-M'hamed, S., Benoliel, J.J., Becker, C., Bernard, C., 2019. Effects of Single Cage Housing on Stress, Cognitive, and Seizure Parameters in the Rat and Mouse Pilocarpine Models of Epilepsy. eNeuro 6. https://doi.org/10.1523/ENEURO.0179-18.2019

Marissal, T., 2021. An inventory of basic research in temporal lobe epilepsy. Revue Neurologique.

Marissal, T., Salazar, R.F., Bertollini, C., Mutel, S., De Roo, M., Rodriguez, I., Müller, D., Carleton, A., 2018. Restoring wild-type-like CA1 network dynamics and behavior during adulthood in a mouse model of schizophrenia. Nature Neuroscience 21, 1412–1420. https://doi.org/10.1038/s41593-018-0225-y

Milosavljevic, N., Cehajic-Kapetanovic, J., Procyk, C.A., Lucas, R.J., 2016. Chemogenetic Activation of Melanopsin Retinal Ganglion Cells Induces Signatures of Arousal and/or Anxiety in Mice. Current Biology 26, 2358–2363. https://doi.org/10.1016/j.cub.2016.06.057

Miri, M.L., Vinck, M., Pant, R., Cardin, J.A., 2018. Altered hippocampal interneuron activity precedes ictal onset. eLife 7, e40750. https://doi.org/10.7554/eLife.40750

Muldoon, S.F., Villette, V., Tressard, T., Malvache, A., Reichinnek, S., Bartolomei, F., Cossart, R., 2015. GABAergic inhibition shapes interictal dynamics in awake epileptic mice. Brain 138, 2875–2890. https://doi.org/10.1093/brain/awv227

Padmasola, G.P., Friscourt, F., Rigoni, I., Vulliémoz, S., Schaller, K., Michel, C.M., Sheybani, L., Quairiaux, C., 2024. Involvement of the contralateral hippocampus in ictal-like but not interictal epileptic activities in the kainate mouse model of temporal lobe epilepsy. Epilepsia. https://doi.org/10.1111/epi.17970

Parrish, R.R., Codadu, N.K., Scott, C.M.-G., Trevelyan, A.J., 2019. Feedforward inhibition ahead of ictal wavefronts is provided by both parvalbumin- and somatostatin-expressing interneurons. The Journal of Physiology 597, 2297–2314. https://doi.org/10.1113/JP277749
Peng, Z., Zhang, N., Wei, W., Huang, C.S., Cetina, Y., Otis, T.S., Houser, C.R., 2013. A Reorganized GABAergic Circuit in a Model of Epilepsy: Evidence from Optogenetic Labeling and Stimulation of Somatostatin Interneurons. J. Neurosci. 33, 14392-14405. https://doi.org/10.1523/JNEUROSCI.2045-13.2013

Peret, A., Christie, L.A., Ouedraogo, D.W., Gorlewicz, A., Epsztein, J., Mulle, C., Crépel, V., 2014. Contribution of Aberrant GluK2-Containing Kainate Receptors to Chronic Seizures in Temporal Lobe Epilepsy. Cell Reports 8, 347-354. https://doi.org/10.1016/j.celrep.2014.06.032

Proddutur, A., Nguyen, S., Yeh, C.-W., Gupta, A., Santhakumar, V., 2023. RECLUSIVE CHANDELIERS: FUNCTIONAL ISOLATION OF DENTATE AXO-AXONIC CELLS AFTER EXPERIMENTAL STATUS EPILEPTICUS. Prog Neurobiol 231, 102542. https://doi.org/10.1016/j.pneurobio.2023.102542

Shuman, T., Aharoni, D., Cai, D.J., Lee, C.R., Chavlis, S., Page-Harley, L., Vetere, L.M., Feng, Y., Yang, C.Y., Mollinedo-Gajate, I., Chen, L., Pennington, Z.T., Taxidis, J., Flores, S.E., Cheng, K., Javaherian, M., Kaba, C.C., Rao, N., La-Vu, M., Pandi, I., Shtrahman, M., Bakhurin, K.I., Masmanidis, S.C., Khakh, B.S., Poirazi, P., Silva, A.J., Golshani, P., 2020. Breakdown of spatial coding and interneuron synchronization in epileptic mice. Nat Neurosci 1-10. https://doi.org/10.1038/s41593-019-0559-0

Toyoda, I., Bower, M.R., Leyva, F., Buckmaster, P.S., 2013. Early activation of ventral hippocampus and subiculum during spontaneous seizures in a rat model of temporal lobe epilepsy. J Neurosci 33, 11100-11115. https://doi.org/10.1523/JNEUROSCI.0472-13.2013

Vigier, A., Partouche, N., Michel, F.J., Crépel, V., Marissal, T., 2021. Substantial outcome improvement using a refined pilocarpine mouse model of temporal lobe epilepsy. Neurobiology of Disease 161, 105547. https://doi.org/10.1016/j.nbd.2021.105547

Wang, Ying, Liang, J., Chen, L., Shen, Y., Zhao, J., Xu, C., Wu, X., Cheng, H., Ying, X., Guo, Y., Wang, S., Zhou, Y., Wang, Yi, Chen, Z., 2018. Pharmaco-genetic therapeutics targeting parvalbumin neurons attenuate temporal lobe epilepsy. Neurobiology of Disease 117, 149-160. https://doi.org/10.1016/j.nbd.2018.06.006

Wang, Ying, Wang, Yi, Xu, C., Wang, S., Tan, N., Chen, C., Chen, L., Wu, X., Fei, F., Cheng, H., Lin, W., Qi, Y., Chen, B., Liang, J., Zhao, J., Xu, Z., Guo, Y., Zhang, S., Li, X., Zhou, Y., Duan, S., Chen, Z., 2020. Direct Septum-Hippocampus Cholinergic Circuit Attenuates Seizure Through Driving Somatostatin Inhibition. Biological Psychiatry, Neurodegeneration 87, 843-856. https://doi.org/10.1016/j.biopsych.2019.11.014

Willner, P., 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134, 319-329. https://doi.org/10.1007/s002130050456

Wozny, C., Gabriel, S., Jandova, K., Schulze, K., Heinemann, U., Behr, J., 2005. Entorhinal cortex entrains epileptiform activity in CA1 in pilocarpine-treated rats. Neurobiology of Disease 19, 451-460. https://doi.org/10.1016/j.nbd.2005.01.016

Zhan, J., Komal, R., Keenan, W.T., Hattar, S., Fernandez, D.C., 2019. Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity. J Vis Exp 10.3791/59439. https://doi.org/10.3791/59439

Zhang, Y., Rózsa, M., Liang, Y., Bushey, D., Wei, Z., Zheng, J., Reep, D., Broussard, G.J., Tsang, A., Tsegaye, G., Narayan, S., Obara, C.J., Lim, J.-X., Patel, R., Zhang, R.,
Ahrens, M.B., Turner, G.C., Wang, S.S.-H., Korff, W.L., Schreiter, E.R., Svoboda, K., Hasseman, J.P., Kolb, I., Looger, L.L., 2023. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature 615, 884–891.
https://doi.org/10.1038/s41586-023-05828-9
Figure Legends

<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

**Fig. 1: Dlx-expressing interneurons are overall spared in TLE mice.** A. Examples of CA1 Dlx-expressing interneurons that co-express tdTomato (top) and PV (bottom left), SOM (bottom middle), or CCK (bottom right) in the hippocampus infected with AAV9-hDLX-GqDREADD-tdTomato-Fishell-4. B. Representative Hoechst staining (top), as well as tdTomato labelling (bottom) in the CA1 region from a control animal (left) or a pilocarpine-treated epileptic mouse (Pilo, right) infected with AAV9-hDLX-GqDREADD-tdTomato-
Fishell-4 in the hippocampus. Neurons with Hoechst and tdTomato co-labelling are indicated by a yellow arrow. C. Number of tdTomato-labelled interneurons per µm³ of CA1. Two-tailed Mann-Whitney test *P* = 0.2107. D. Percentage of tdTomato-labelled interneurons per CA1 layer (two-way ANOVA experimental condition effect *F* (2, 36) = 0.8553, *P* = 0.4336). SO: Stratum Oriens, SP: Stratum Pyramidale, SR: Stratum Radiatum, SLM: Stratum Lacunosum-Moleculare. Data are presented as mean ± s.e.m. Each circle represents a given mouse (The number of mice is specified in parentheses).
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>
**Fig. 2: Epileptic CA1 network is hyperactive and synchronous while Dlx interneurons are less active in vitro.** **A.** Schematic representation of the experimental procedure (left) and photograph of GCaMP8m-expressing neurons in the hippocampal CA1 region (right). **SO:** Stratum Oriens, **SP:** Stratum Pyramidale, **SR:** Stratum Radiatum. **B.** Contours of GCaMP8m-expressing neurons shown in **A**. **C.** Calcium transients recorded in the presence of carbachol (50 µM) from selected neurons identified as 1 to 5 in **B** (represented with red-filled contours). **D-E.** Representative raster plots of calcium transients recorded in control (D, black) or Pilo (E, red) conditions. **F.** Proportions of CA1 neurons displaying at least one calcium transient during the duration of the movie in each condition (Two-tailed unpaired t-test, *P* = 0,9844). **G.** Frequency of calcium transients recorded in all CA1 neurons (Two-tailed unpaired t-test). **H-I.** Percentage of co-active neurons above chance level (H) and occurrence (I) of these co-activations (H, I: Two-tailed Mann-Whitney test). **J.** Photograph of the field represented by a dashed rectangle in **A**, in which a Dlx-expressing interneuron co-expresses GCaMP8m and tdTomato. **K.** Number of interneurons per calcium imaging field (Two-tailed Mann-Whitney test, *P* = 0,3246). **L.** Examples of calcium activity exhibited by GCaMP+ expressing tdTomato-negative CA1 neurons (top) or tdTomato-positive interneurons (bottom) in control (left) or pilo (right) conditions. Each trace represents a different neuron. **M.** Average frequency of calcium transients recorded in CA1 interneurons per slice (Two-tailed Mann-Whitney test). Asterisks indicate significant differences (*P* < 0.05). Data are presented as mean ± s.e.m. Each circle represents the mean for a given slice (the number of slices is specified in parentheses). Slices were prepared from *N* = 7 and 5 animals in control and Pilo conditions, respectively.
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

**Fig. 3: Reestablishing Dlx interneuron activity decreases local CA1 network synchronization in vitro.** A. Photographs of CA1 interneurons that co-express GCaMP8m and tdTomato (fused to hM3D) (top) and an example of calcium activity exhibited by hM3D-expressing CA1 interneurons in the Pilo condition before (red, Pilo-VEH) and after 15 minutes of treatment with CNO (2 µM, blue) (bottom). B. Frequency of calcium transients recorded in hM3D-expressing CA1 interneurons in Pilo condition before and after treatment with CNO (Two-tailed Wilcoxon test). C. Representative raster plots of calcium transients recorded in the same slice before and after treatment with CNO. D-G. Levels of calcium activity (D, E) and synchronization (F, G), reflected by the number of active neurons (D), the frequency of calcium transients recorded in all CA1 neurons (E), the percentage of co-active neurons above chance level (F) and the occurrence of these co-activations (G) in both conditions (Two-tailed paired t-test for D and E, Two-tailed Wilcoxon test for F and G). Data are presented as mean ± s.e.m. Each gray line represents a given slice (the number of slices is specified in parentheses. Slices were prepared from *N* = 3 animals).
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

**Fig. 4: The in vivo chemoactivation of Dlx interneurons does not rescue TLE pathology, but reduces nonspatial behavioral deficits.**

**A.** Average daily consumption of drinking water (with 4% of sucrose) in control or epileptic mice in the absence (Control-VEH, Pilo-VEH) or presence of CNO (Pilo-CNO) to specifically elevate or not the activity of Dlx- and hM3D-expressing hippocampal interneurons.

**B.** Schematic representation of the experimental procedure (top) and example traces of ictal activity in Pilo-VEH or Pilo-CNO animals (bottom).

**C.** Frequency of ictal events in Pilo-VEH versus Pilo-CNO conditions across days of recording (Two-way ANOVA experimental group effect F (1, 11) = 1.338 *P* = 0.2719.

**D.** Schematic representation of the Barnes Circular Maze (BCM) acquisition phase (top) and probe trial
(bottom). **E.** Min-max scores of latencies to find the escape room during the BCM acquisition phase. Data were compared in Control-VEH versus Pilo-VEH groups (Two-way ANOVA experimental group effect F (1, 32) = 23.10 *P* < 0.0001, followed by Sidak's post hoc test, *P* = 0.0251 0.0001, 0.0001, 0.0001, 0.0009 and 0.0030 for habituation, acquisition days 1-4 and PT, respectively), Control-VEH versus Pilo-CNO groups (Two-way ANOVA experimental group and treatment effect F (1, 25) = 29.67 *P* < 0.0001, followed by Sidak's post hoc test, *P* = 0.0371 0.0001, 0.0005, 0.0040, 0.0221 and 0.0715 for habituation, acquisition days 1-4 and PT), and Pilo-VEH versus Pilo-CNO groups (Two-way ANOVA treatment effect F (1, 37) = 0.0161, *P* = 0.8999). Straight horizontal lines across the top of the plots indicate that a mouse has scored 1 for two or more consecutive days. **F.** Min-max scores of times spent in the target zone during the BCM probe trial in various experimental conditions (Two-tailed Mann-Whitney test for the comparison between Control-VEH and Pilo-VEH and between Pilo-VEH and Pilo-CNO *P* = 0.1789, unpaired t-test for the comparison between control-VEH and Pilo-CNO, *P* = 0.0230).

**G.** Schematic representation of the NORT procedure. **H.** Time spent exploring the novel and the familiar object during the NORT test phase, in each experimental condition (two-way ANOVA interaction experimental condition x type of object F (2, 45) = 8.182, *P* = 0.0009, followed by Sidak's post hoc test to compare the time in novel versus familiar object, *P* < 0.0001 in Control-VEH and Pilo-CNO conditions, *P* = 0.0508 in Pilo-VEH condition).

Asterisks indicate significant differences. Data are presented as mean ± s.e.m., unless specified otherwise. Each circle or line represents a given mouse (The number of mice is specified in parentheses).
**Declaration of interests**

The authors declare no competing interests.
Highlights :

* Dlx-expressing interneurons are overall spared from CA1 atrophy in epileptic mice

* Dlx interneurons are less active in epileptic, hypersynchronous CA1 *in vitro*

* Chemoactivation of Dlx interneurons normalizes epileptic CA1 synchrony *in vitro*

* *In vivo*, this strategy does not affect seizures but rescues nonspatial behavior

<figure><img src="image_8.png" /></figure>
A

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

B

Control

<figure><img src="image_15.png" /></figure>

Pilo

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /></figure>

D

<figure><img src="image_18.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>Figure 4</figcaption></figure>