Reduced Hippocampal Inhibition and Enhanced Autism-Epilepsy Comorbidity in Mice Lacking Neuropilin 2

Abstract

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2 deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have reduced number of parvalbumin, somatostatin and Neuropeptide Y cells, mainly in CA1. Whole cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2 deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/-, but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

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

“…2A-F; cell count, control: 84.03±7.88; iCKO: 52.83±7.50, p=0.0426; t (6) =2.565 unpaired t-test). In the CA1 we observed a substantial 46.41% decrease in SOM+ neurons (cell count: control: 36.90±3.97; iCKO: 19.78±3.14 in n=5 mice each p=0.0004; t (24) =4.659 by two-way ANOVA with Bonferroni multiple comparison correction). Interestingly, there was an apparent reduction in SOM+ cells in the DG, but the difference did not reach statistical significance (p=0.385).…”

“…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 significant 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.…”

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

“…Whole-cell patch clamp recordings of CA1 pyramidal cells (CA1 PCs) were obtained from horizontal hippocampal slices (350µm) of littermate controls (Nrp2 +/f ;Creor 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 amplifiers, digitized at 10kHz using DigiData 1440A or DigiData 1550B and recorded using pClamp10 software (Molecular Devices, Sunnyvale, CA).…”

“…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.…”