Dysregulation of Neuropilin-2 Expression in Inhibitory Neurons Impairs Hippocampal Circuit Development Leading to Autism-Epilepsy Phenotype

Dysregulation of development, migration, and function of interneurons, collectively termed interneuronopathies, have been proposed as a shared mechanism for autism spectrum disorders (ASDs) and childhood epilepsy. Neuropilin-2 (Nrp2), a candidate ASD gene, is a critical regulator of interneuron migration from the median ganglionic eminence (MGE) to the pallium, including the hippocampus. While clinical studies have identified Nrp2 polymorphisms in patients with ASD, whether dysregulation of Nrp2-dependent interneuron migration contributes to pathogenesis of ASD and epilepsy has not been tested. We tested the hypothesis that the lack of Nrp2 in MGE-derived interneuron precursors disrupts the excitation/inhibition balance in hippocampal circuits, thus predisposing the network to seizures and behavioral patterns associated with ASD. Embryonic deletion of Nrp2 during the developmental period for migration of MGE derived interneuron precursors (iCKO) significantly reduced parvalbumin, neuropeptide Y, and somatostatin positive neurons in the hippocampal CA1. Consequently, when compared to controls, the frequency of inhibitory synaptic currents in CA1 pyramidal cells was reduced while frequency of excitatory synaptic currents was increased in iCKO mice. Although passive and active membrane properties of CA1 pyramidal cells were unchanged, iCKO mice showed enhanced susceptibility to chemically evoked seizures. Moreover, iCKO mice exhibited selective behavioral deficits in both preference for social novelty and goal-directed learning, which are consistent with ASD-like phenotype. Together, our findings show that disruption of developmental Nrp2 regulation of interneuron circuit establishment, produces ASD-like behaviors and enhanced risk for epilepsy. These results support the developmental interneuronopathy hypothesis of ASD epilepsy comorbidity.


Introduction
Autism spectrum disorders (ASD) and epilepsy are highly comorbid conditions that are proposed to share common pathophysiological mechanisms.Anomalies in the development, migration, and function of interneurons, collectively termed interneuronopathies, are closely associated with ASD and epilepsy (1)(2)(3)(4).In particular, the development of seizures and behavioral impairments observed in ASD are associated with altered function of inhibitory neurons.GABAergic interneurons play a pivotal role in the organization and function of the hippocampus, a brain region frequently associated with ASD and epilepsy (5,6).
Disruptions in the establishment of interneuron circuits can compromise hippocampal network function, potentially serving as a common factor contributing to the observed high comorbidity between ASD and epilepsy (7)(8)(9)(10)(11)(12).Here we report that developmental dysregulation of a classical guidance cue receptor Neuropilin-2 (Nrp2) speci cally in developing interneurons compromises hippocampal circuit function and predisposes the network to seizures and behavioral de cits consistent with ASD.
During embryonic development, the class 3 secreted semaphorins and their obligate binding receptors, the neuropilins, are key regulators of neuronal migration, axonal guidance, dendritic morphology, and synaptic speci city of various cell types (13)(14)(15).Notably, Nrp2 is a candidate ASD gene on SFARI (Score 2) and polymorphisms in Nrp2 gene have been reported in patients with autistic syndromes (16,17).In excitatory neurons, Nrp2 expression contributes to pruning of synapses, spines and axons, whereas, in inhibitory neurons, Nrp2 regulates migration of interneurons from the medial ganglionic eminence (MGE) to the pallium, including the hippocampus (18)(19)(20).During embryonic development, the expression of Nrp2 in interneuron progenitors is tightly regulated by the transcription factor Nkx2.1 (21), and allows for migration of parvalbumin (PV+), somatostatin (SOM+) and neuropeptide-Y (NPY+) expressing interneurons to the cortex and hippocampus (22).We previously found that global constitutive knockout of Nrp2 leads to loss of hippocampal interneurons, produces behavioral phenotypes consistent with ASD, and increases seizure susceptibility (23,24).However, whether selective deletion of Nrp2 in interneurons alone and during their time of migration to the cortex and hippocampus could result in ASD/epilepsy phenotypes is currently not known.We hypothesize that loss of Nrp2 during embryonic ages (E12.5-13.5)speci cally in interneuron precursors derived from the MGE will result in fewer inhibitory neurons in the hippocampus.We predict that the ensuing altered inhibition and related circuit plasticity will disrupt hippocampal function, increase seizure susceptibility, and impair hippocampal-dependent behaviors.To test these hypotheses, we deleted Nrp2 in MGE-derived interneuron precursors by crossing the Nrp2 ox conditional mouse with the Nkx2.1-CreERT2driver line, where cre recombinase expression is induced by tamoxifen administered to pregnant dams on E12.5 and E13.5.The resulting genotype Nrp2 f/f ;Nkx2.1-Cre+ (iCKO) animals and littermate controls were used to examine hippocampal CA1 circuit functions and evaluated whether the lack of Nrp2 in interneuron precursors enhances the risk for development of seizures and behavioral de cits consistent with ASD.

Animals
All experiments were performed in accordance with IACUC protocols approved by Rutgers University, Newark, NJ, and the University of California at Riverside, CA and in keeping with the ARRIVE guidelines.
The Nrp2 oxed mouse (25), which contains an IRES-GFP-polyA sequence inserted immediately downstream of the 3' loxP in the targeting construct that allows the expression of GFP following cre recombination in all conditional mutant (-/-) neurons, was crossed with the Nkx2.1-CreERT2mouse (stock #014552, The Jackson Laboratory), to selectively target MGE-derived interneuron progenitors during embryonic development(21) (See Fig. 1A).Cre recombinase was induced by administering tamoxifen (5 mg, by oral gavage) to the pregnant dams at E12.5 and E13.5, during the peak timeline for developmental interneuron migration (Fig. 1B).Deletion of Nrp2 in the progeny (Nrp2 f/f ;Nkx2.1Cre+ ) resulted in inhibitory conditional knockout (iCKO) mice and was veri ed by visualization of GFP expression in the hippocampus in adult mice (Fig. 1C&D).All appropriate littermates, such as Cre negative Nrp2 +/f ;Cre − or Nrp2 f/f ;Cre − and Nrp2 +/+ ;Cre + , were used as controls.In immunostaining and behavioral studies, data from Nrp2 +/f ;Cre + mice were not statically different from Cre negative controls or Nrp2 +/+ ;Cre + mice and were pooled for analysis.Mice used in these studies were backcrossed for at least 10 generations to the C57BL/6NTac background strain.Nrp2 genotypes were con rmed by using polymerase chain reaction (PCR).

Immunostaining and cell count:
Immunostaining, cell counts, and photo-documentation are as detailed previously in Eisenberg et al 2021 (24) and described in detail in Supplementary Methods.

Ex vivo physiology:
Whole-cell patch clamp recordings of CA1 pyramidal cells (CA1 PCs) were obtained from horizontal hippocampal slices (350µm) of littermate controls (Nrp2 +/f ;Cre − or Nrp2 f/f ;Cre − ) and iCKO mice (Nrp2 f/f ;Nkx2.1Cre+ ; n = 3 animals/group, 12 months old).Slice preparation and recording methods are as detailed previously (24,26).Voltage and current clamp recordings were obtained using MultiClamp 700B ampli ers, digitized at 10kHz using DigiData 1440A or DigiData 1550B and recorded using pClamp10 software (Molecular Devices, Sunnyvale, CA).Active and passive properties were recorded in current clamp from a holding potential of -70mV using K-Gluconate based internal solution.Voltage clamp recording from CA1 PCs held at -70mV and 0mV were used to isolate glutamatergic and GABAergic synaptic inputs, respectively, using a Cesium-based internal solution.Action potential independent miniature currents were isolated by tetrodotoxin (TTX, 1µM).Intrinsic properties were analyzed using pClamp software 10.7 (Molecular Devices, Sunnyvale, CA) and synaptic currents were detected using template search feature in Easy Electrophysiology (version 2.6).

In vivo electrophysiology:
Eight mice (5 Nrp2 +/f ;Cre − or Nrp2 f/f ;Cre − littermate controls and 3 Nrp2 f/f ;NkxCre + ; average age of 8.75 ± 0.70 months, ve males and three females) were surgically implanted a tungsten wire depth electrode (50 µm, California Fine Wire company) in the CA1 sub eld (AP:2 mm, ML: 1.5 mm, DV: 1.2mm from bregma) and a cortical screw electrode.Two additional screw electrodes (Invivo1, Roanoke, VA) on the contralateral hemisphere served as ground and reference.After 3-5 days of recovery, mice were connected to a tethered video-EEG monitoring system.Signals were sampled at 10 kHz, ampli ed (preampli er: 8202-SE3, gain -x100, Pinnacle Technologies, Lawrence, KS), digitized (Powerlabs16/35, AD Instruments, Colorado Springs, CO), and recorded using LabChart 8.0 (AD instruments).Following 30 minutes of baseline recordings, mice received a single high dose of KA (20 mg/kg), and their latency to electrographic seizure, seizure duration, and mortality were quanti ed.Seizures were scored by a blinded investigator (S.K) based on a modi ed Racine scale.Seizure severity scores in the rst 30 min were averaged over 5 min epochs.

Behavior testing
The behavior methods are fully described in the supplemental methods and in previous publications (23)

Social novelty test
We assessed social behavior in a three-chambered arena.In the rst phase, one chamber contained a caged mouse, the opposite chamber contained an empty cage, and the test mouse was able to explore both chambers.During the test for social novelty, which was conducted immediately following the rst phase, one chamber contained the familiar mouse used previously, and the opposite chamber contained a novel mouse.In both phases, the test mouse explored the arena for 5 minutes.We assessed time spent sni ng the novel and familiar mouse from video footage.

Novel object recognition test
The test mouse explored two identical objects in an open eld arena for 10 minutes.After a 30-min retention interval, the mouse reentered the arena, which contained an object they previously encountered (familiar object) and a novel object.Two observers blind to experimental conditions assessed time spent sni ng the novel and familiar objects from video footage.

Instrumental goal directed behavior test
We tested mice on an instrumental goal-directed learning task.Food-restricted mice pressed levers to obtain food pellets.Responses on one lever delivered chocolate-avored pellets, whereas responses on the opposite lever delivered grain pellets.Mice underwent 36 instrumental training sessions, two sessions per day, with each lever trained in separate sessions.Mice then underwent a selective-satiety outcome devaluation procedure, in which they had free access to one avor of food pellets in their home cage for 1 hour.In the choice test that followed, both levers were available, and mice could respond for 5 minutes, with no pellets delivered during the test.After 4 retraining sessions, we repeated the test with the opposite outcome devalued prior to the choice test.

Statistical analysis
Unpaired t-tests, two-way ANOVA and post hoc Bonferroni's, Sidak's or Tukey's multiple comparison correction were used to compare cell count and behavioral data.Kolmogorov-Smirnov test for cumulative distributions was used to evaluate differences in distribution of synaptic parameters.Unpaired students ttest was used to compare differences in intrinsic properties, latency to convulsive seizures and the total time spent in seizures.Mantel-Cox test was used for survival analysis.All statistical tests were conducted in GraphPad Prism.
The difference between two dependent means (matched pairs) was used to determine the sample size requirement of behavioral tests using G*Power 3.1 software.A sample size requirement of three to seven animals for cell counts and 5 to 8 for behavior testing was estimated by using 80% power and an effect size found in previous work and literature.Exclusion criteria of three standard deviations from the mean was pre-established for behavior tests.One mutant mouse performed over three standard deviations lower than the mean and was therefore eliminated from behavior analysis.Signi cance was set to p < 0.05.Data are shown as mean ± SEM or median and interquartile range (IQR), as appropriate.

Results
Developmental deletion of Nrp2 in interneuron progenitors reduces hippocampal interneuron density.
Previously, Nrp2 was shown to be expressed in MGE derived interneuron precursors and is critical for cortical and hippocampal migration of interneurons (20,28).To examine the impact of Nrp2 expression in inhibitory neuron precursors on hippocampal circuit formation and function, we generated the Nrp2 f/f ;Nkx2.1-CreERT2+ (iCKO), where cre recombinase expression is induced by feeding the pregnant dams with tamoxifen on E12.5 and E13.5 (Fig. 1A-B).Induction of cre-recombinase was con rmed by GFP expression in the hippocampus of the iCKO mice, as the Nrp2 ox contained an IRES-GFP-pA sequence that will be shifted in frame to be transcribed upon cre recombination, but not in littermate controls (Fig. 1C-D).
To understand the effects of Nrp2 deletion on the distribution of different MGE derived hippocampal inhibitory neuron subtypes, we quanti ed the population of PV+, SOM + and NPY + expressing interneurons in hippocampal sub elds of iCKO mice.We particularly focused on the CA1 sub eld, a region closely associated with storing social memories and pathologically implicated in ASD (29,30).Soma targeting PV + interneurons are estimated to account for 21% of MGE-derived interneurons (31).
Together, our results show a signi cant decrease in PV+, SOM + and NPY + interneuron population in the CA1 following developmental deletion of Nrp2 in MGE-interneuron precursors.This reduction in interneuron populations supporting perisomatic and dendritic feedback inhibition is likely to have functional consequences at a neuronal and network level which we examined further.Developmental Nrp2 deletion in interneurons alters inhibitory and excitatory synaptic transmission in CA1.
Nrp2 deletion in interneurons does not affect intrinsic physiology of CA1 PCs.
Selective deletion of Nrp2 in interneurons increases risk for seizures.
Since our experiments consistently show a decrease in interneuron population, network inhibition and increased excitatory neurotransmission, we examined if this could result in increased seizure susceptibility.Interestingly, we noticed an increase in mortality speci cally in iCKO mice after electrode implant (n = 3 of 6).Nevertheless, we did not observe spontaneous seizures or epileptiform activity in iCKO mice during the brief 30 min baseline recording period.iCKO mice injected with a single convulsive dose of KA (20mg/kg, i.p) showed a signi cantly shorter latency to develop convulsive seizures and reached status epilepticus more rapidly (Fig. 5A-B; latency in minutes, Controls: 27.7 ± 3.27, n = 5; iCKO: 4.8 ± 1.95, n = 5 vs 3, p = 0.0025, t (6) = 4.993 unpaired t-test).At 30 minutes post injection, all iCKO mice reached stage 4 seizures, classi ed using a modi ed Racine scale whereas, only 1 control mouse exhibited stage 4 seizure.Moreover, iCKO mice showed a 100% mortality within the rst 60 minutes of KA induction whereas all control animals survived this period (Fig. 5C; Mean latency to mortality in iCKO mice: 27.02 ± 12.22 minutes, p = 0.022, Mantel-Cox Test).Importantly, iCKO mice spent ~ 60% of their time after KA injection exhibiting behavioral seizures compared to controls (Fig. 5D, measured as total time spent in convulsive seizures/total time from induction to death or 60 minutes: Controls: 19.78 ± 4.42%; iCKO: 60.69 ± 3.12%, n = 5 vs 3, p < 0.001, t (6) = 6.483, unpaired t-test).Together, our experiments demonstrate iCKO mice exhibit greater seizure susceptibility than controls.Social novelty and goal directed behaviors are impaired in mice with interneuron targeted Nrp2 deletion.Given Nrp2's association with ASD in humans, and our previous results showing social, learning, and sensorimotor alterations in Nrp2-null mice, we examined ASD-relevant behaviors in iCKO mice.We found that preference for social novelty, measured as the proportion of time spent investigating a novel versus a familiar mouse, was impaired in iCKO mice compared to littermate controls (Fig. 6A).Unlike control mice, which showed a signi cant preference for the novel mouse, iCKO mice showed no preference for social novelty (Fig. 6B; two-way ANOVA interaction F (1,36) = 6.388, p = 0.0110; n = 11 controls and 9 iCKO mice).
Further analysis failed to reveal genotype speci c differences in overall investigation time (in sec, control = 110 ± 21 sec; iCKO = 94 ± 25 sec) indicating that the difference in responses is not due to an overall lack of interest in social interactions.
Since social preference test relies on memory of previous encounters, we tested whether de cits in episodic memory could have contributed to de cits in social novelty preference in iCKO mice.However, iCKO mice did not differ from control mice in an object recognition memory test (Fig. 6C), indicating that the lack of preference for social novelty re ects a speci c change in social behavior in iCKO mice.
In addition to changes in social behavior, iCKO mice differed from control mice in goal-directed behavior (Fig. 6D) in an instrumental lever press task with two actions associated with two distinct food outcomes.Devaluation of one instrumental outcome signi cantly shifted the choice of the control mouse to the action (lever press) associated with the valued outcome, whereas the actions of iCKO mice showed no sensitivity to changes in outcome value (Fig. 6E; two-way ANOVA interaction F (1,26) = 8.265 p < 0.0001; n = 7 controls and 8 iCKO).Importantly, iCKO mice and controls did not differ in acquisition of the instrumental response (Fig. 6F) demonstrating iCKO mice were capable of acquiring and performing a lever press action and showed no learning de cits.Similarly, locomotor measures in the open eld and accelerating rotarod were not different between iCKO mice and controls (Supplementary Fig. 2A-B) indicating lack of motor impairments.Moreover, control and iCKO mice did not differ in the time spent in open arm of the elevated-zero maze or grooming behavior (Supplementary Fig. 2C-D) revealing iCKO mice were not more anxious than controls.Therefore, the inability to adjust actions to changes in outcome value in iCKO mice strongly suggest a selective impairment in control of goal-directed actions.
Together, these ndings suggest that Nrp2-dependent interneuron circuit development is a critical factor determining proper social behavior and cognitive exibility in mice, and de cits in Nrp2 during development can signi cantly impair these functions.

Discussion
Dysregulated excitatory/inhibitory (E/I) balance is often considered a unifying pathology underlying Autism-Epilepsy comorbidity.Altered functional connectivity, loss of interneurons or hypoactive interneurons have been proposed to underlie this shift in functional E/I balance (8).The emerging role of interneurons as critical determinants of circuit dysfunction in ASD is evident from studies in animal models and humans (2,11).Despite this knowledge, the molecular determinants of altered inhibitory circuits during neurodevelopment and the impact of such maladaptive circuits on network and behavioral function are unknown.Neuropilin-2 and its interactions with its ligand the secreted semaphorin 3F are crucial in establishing normal migration of interneuron to the cortex, the lack of which has been shown to result in increased NPY + expressing interneurons in the striatum (20).Importantly, Nrp2 is a candidate ASD gene on SFARI (Score 2) and polymorphisms in Nrp2 gene have been reported in patients with autistic syndromes (16,17).We previously demonstrated that global Nrp2 deletion results in a signi cant decrease in density of several MGE-derived interneuron subtypes in hippocampus and reduced hippocampal inhibition (24).However, the effects of selective embryonic Nrp2 deletion in MGE-derived interneuron precursors and its impact on hippocampal interneuron population and inhibition was unknown.The current studies directly address this gap in knowledge and demonstrate that embryonic deletion of Nrp2 selectively in MGE-derived interneuron precursors leads to a signi cant reduction in PV+, SOM + and NPY + interneurons in the hippocampus.The reduction in interneuron numbers in the hippocampus could result from mislocalization and result in altered interneuron numbers in other brain regions, such as the striatum or cortex.Alternatively, failure of MGE-derived precursors to their nal destination could result in cell death, which future experiments, beyond the scope of this study, can examine.
At a circuit level, CA1 PCs from iCKO mice received a less frequent synaptic inhibition compared to control mice.This nding is similar to our observations in global Nrp2 KO mice and could directly be attributed to the decrease in interneuron population in CA1.The absence of a GABAergic inhibitory tone is believed to reduce signal to noise ratio and disrupt signal processing in individuals with ASD (7,8,36).We identify reductions in three major interneuronal classes in the iCKO mice; the soma-targeting fastspiking PV + basket cells and dendrite targeting SOM + interneurons which are crucial regulators of neuronal spiking and integration of information (37), and NPY + interneurons which are critical regulators of excitatory synaptic transmission and spiking frequency(38).Therefore, the combined reduction in their populations is likely to have a cumulative effect on network function leading to alterations in excitatory synaptic transmission, neuronal oscillations and theta/gamma coupling.Together, these changes could disrupt information processing and lead to behavioral de cits consistent with ASD (39).Despite the reduction in inhibition, we observed a signi cant increase in excitatory synaptic transmission in CA1 PCs from iCKO mice suggesting potential maladaptive plasticity.Moreover, while the de cits in inhibition in the iCKO mice are similar to those reported in the global Nrp2 KO, the changes in CA1 PC excitability and resting membrane potential observed in the global Nrp2 KO were not observed in the iCKO mice.Thus, our ndings support the proposal that interneuron speci c knockout of Nrp2 undermines E/I balance at a circuit level predominantly through disruption of the ne-tuned, layer-speci c inhibitory control of CA1.
Enhanced excitability and de cient inhibitory control are fundamental to seizures.iCKO mice exhibited higher susceptibility to evoked seizures compared to age matched controls.Importantly, the severity of evoked seizures in iCKO mice was high, leading to 100% mortality.Our ndings that the iCKO mice also spent ~ 60% of their time post-induction in seizures highlights their enhanced susceptibility to recurrent epileptic episodes.
Altered social behavior and impaired action control are core features of ASD.In iCKO mice, we observed a lack of preference for social novelty and an impairment in goal-directed behavior, consistent with an ASD phenotype.Preference for social novelty depends, in part, on hippocampal CA2 and its output to ventral CA1 (40,41).Furthermore, studies have shown that disrupted E/I balance in CA2 interferes with social novelty preference and increases seizure susceptibility (42,43).While the trend is not signi cant in the iCKO animals, we previously found in the Nrp2 global KO mice a signi cant decrease of both PV + and SOM + interneurons in CA2 compared to the WT controls.Therefore, loss of social novelty preference in iCKO mice may re ect dysregulated interneuron migration to CA2 or re ect changes in interneuron number and function in ventral CA1 that receives CA2 input.Interestingly, iCKO mice showed no de cits in episodic memory as assessed in the novel object recognition test.These data indicate that the lack of preference for social novelty was not due to an impairment in episodic memory, or a lack of interest in novelty per se.The dependency of novel object recognition on hippocampus is debated, with more evidence in support of entorhinal cortex underlying performance in the novel object recognition task (44).We previously reported that Nrp2 global KO animals were impaired in novel object recognition (23); however, global Nrp2 KO mice also displayed increases in cortical pyramidal neuron dendritic spine number and excitability (18, 27), which may have affected entorhinal cortex processing and produced a de cit in the novel object recognition task.These ndings further underscore the functional distinctions between effects of global versus inhibitory neuron speci c Nrp2 deletion.
Goal-directed action was also impaired in iCKO mice.In an instrumental outcome devaluation task, mice are given the choice of actions associated with either a valued or devalued outcome.Unlike control mice that chose the action associated with the valued outcome, iCKO mice showed no preference.The impairment in goal-directed response in iCKO mice may be attributable to altered hippocampal activity.The dorsal hippocampus is transiently involved in the formation of action-outcome associations (45,46), which could have prevented mice from forming the associations necessary to guide their behavior in the devaluation test.Another possibility is that the altered interneuron migration induced in iCKO mice misplaced striatal interneurons (20).Disruption of striatal GABAergic interneurons impairs goal-directed behavior (47,48), suggesting striatal de cits as a source of impaired goal-directed behavior in iCKO mice.Future studies will address Nrp2's role in striatal interneuron migration and its effects on behavior.
Taken together, our ndings provide a novel insight into the circuit and behavioral effects of embryonic Nrp2 deletion in interneuron precursors.Our studies using interneuron speci c Nrp2 deletion combined with circuit and behavioral analyses support a role for developmental interneuronopathy in comorbidity of ASD-epilepsy syndromes.

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