MicroRNA-134 (miR-134) is a brain-enriched noncoding RNA upregulated in temporal lobe epilepsy that regulates the levels of proteins controlling dendritic structure, ion channel expression, and neuronal excitability. We recently demonstrated that miR-134 inhibition using antisense oligonucleotides (antimir-134) reduces spontaneous recurrent seizures (SRS) in epileptic male mice, also attenuating CA1 neuronal epilepsy hallmarks such as intrinsic hyperexcitability, exacerbated synaptic transmission and impaired long-term potentiation (LTP). It remains unproven, however, if increased miR-134 levels alone are sufficient to produce hyperexcitability or epilepsy-associated co-morbidities.
We combined electrophysiological, morphological and behavioral analyses to investigate the effects of increased miR-134 modelled to a level that matches human TLE. Viral vector-directed miR-134 overexpression was achieved in the hippocampi of naïve mice of both sexes, followed by behavioral testing, patch-clamp experiments to principal hippocampal neurons and post-hoc morphological analyses of biocytin-filled CA1 neurons. We also used recordings of local field activity to investigate evoked epileptiform-like activity susceptibility, synaptic strength and plasticity magnitude, and probability of excitatory neurotransmitter release.
Overexpression of miR-134 in naïve mice (AAV.miR134) (+4.85±0.8-fold increase, n=9 mice/group, p=0.015 Welch’s test) to mimic findings in human epilepsy resulted in sex-specific effects on naturalistic behavior, consistent with enhanced exploratory or risk-tolerant behavior. AAV.miR134 was sufficient to increase cell excitability of transduced CA1 pyramidal neurons (n=10 mice/group, p<0.001, LMM), associated with an alteration of action potential afterdepolarization slope (n=10 mice/group, p=0.036, LMM) and delayed voltage-gated potassium peak activation (n=10 mice/group, p=0.04, LMM). Furthermore, morphological analyses of patched CA1 neurons indicated selective reduction of apical (n=10 mice/group, p=0.006, Mann-Whitney), but not basal, dendritic complexity and spine density (n=10 mice/group, p=0.026, unpaired t). This suggests a structural substrate for altered network integration and neuronal excitability[PQ1] . Functionally, synaptic strength, measured as local field response to electrical stimuli, was enhanced in both major CA1 strata in AAV.miR134 animals (n=6 mice/group, p<0.05, 2way ANOVA interaction), whereas neurotransmitter release was selectively enhanced at SC-CA1 synapse (n=8 mice/group, p=0.043, Mann-Whitney), indicating circuit-specific presynaptic modulation. Despite increased synaptic strength and NT release in SC-CA1, TBS-induced LTP magnitude was comparable to controls. In contrast to controls, post-LTP NT release was not further increased, consistent with a reduced dynamic range of presynaptic plasticity. Despite observed CA1 cellular and circuit altered phenotypes, epileptiform-like activity evoked by modified artificial cerebro-spinal fluid (maCSF, 0.25 mM Mg2+, 8mM K+) perfusion trended increased power in area CA3 (n=3 animals/group, p=0.058, Mann-Whitney), but not CA1, on AAV.miR134 slices in an interface chamber setup.
Our findings showcase that miR-134 is a critical regulator of neuronal excitability, dendritic architecture and population synchronization in the hippocampus, and suggest a role of miR-134 overexpression alone to increase seizure susceptibility in ex vivo slices. Future studies should investigate whether miR-134 overexpression is sufficient to manifest in vivo spontaneous seizures, alone or in combination with a second hit. Altogether, our results strengthen the rationale for targeting miR-134 as a disease-modifying therapeutic strategy in epilepsy and provide new mechanistic insight into how microRNAs shape neuronal and circuit-level function.