GABAA Receptor-Stabilizing Protein Ubqln1 Affects Hyperexcitability and Epileptogenesis after Traumatic Brain Injury and in a Model of In Vitro Epilepsy in Mice.

Posttraumatic epilepsy (PTE) is a major public health concern and strongly contributes to human epilepsy cases worldwide. However, an effective treatment and prevention remains a matter of intense research. The present study provides new insights into the gamma aminobutyric acid A (GABAA)-stabilizing protein ubiquilin-1 (ubqln1) and its regulation in mouse models of traumatic brain injury (TBI) and in vitro epilepsy. We performed label-free quantification on isolated cortical GABAergic interneurons from GAD67-GFP mice that received unilateral TBI and discovered reduced expression of ubqln1 24 h post-TBI. To investigate the link between this regulation and the development of epileptiform activity, we further studied ubqln1 expression in hippocampal and cortical slices. Epileptiform events were evoked pharmacologically in acute brain slices by administration of picrotoxin (PTX, 50 µM) and kainic acid (KA, 500 nM) and recorded in the hippocampal CA1 subfield using Multi-electrode Arrays (MEA). Interestingly, quantitative Western blots revealed significant decreases in ubqln1 expression 1-7 h after seizure induction that could be restored by application of the non-selective monoamine oxidase inhibitor nialamide (NM, 10 µM). In picrotoxin-dependent dose-response relationships, NM administration alleviated the frequency and peak amplitude of seizure-like events (SLEs). These findings indicate a role of the monoamine transmitter systems and ubqln1 for cortical network activity during posttraumatic epileptogenesis.

Tonic activation of GABAB receptors via GAT-3 mediated GABA release reduces network activity in the developing somatosensory cortex in GAD67-GFP mice.

The efficiency of neocortical information processing critically depends on the balance between the glutamatergic (excitatory, E) and GABAergic (inhibitory, I) synaptic transmission. A transient imbalance of the E/I-ratio during early development might lead to neuropsychiatric disorders later in life. The transgenic glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) mouse line (KI) was developed to selectively visualize GABAergic interneurons in the CNS. However, haplodeficiency of the GAD67 enzyme, the main GABA synthetizing enzyme in the brain, temporarily leads to a low GABA level in the developing brain of these animals. However, KI mice did not demonstrate any epileptic activity and only few and mild behavioral deficits. In the present study we investigated how the developing somatosensory cortex of KI-mice compensates the reduced GABA level to prevent brain hyperexcitability. Whole-cell patch clamp recordings from layer 2/3 pyramidal neurons at P14 and at P21 revealed a reduced frequency of miniature inhibitory postsynaptic currents (mIPSCs) in KI mice without any change in amplitude or kinetics. Interestingly, mEPSC frequencies were also decreased, while the E/I-ratio was nevertheless shifted toward excitation. Surprisingly, multi-electrode-recordings (MEA) from acute slices revealed a decreased spontaneous neuronal network activity in KI mice compared to wild-type (WT) littermates, pointing to a compensatory mechanism that prevents hyperexcitability. Blockade of GABAB receptors (GABABRs) with CGP55845 strongly increased the frequency of mEPSCs in KI, but failed to affect mIPSCs in any genotype or age. It also induced a membrane depolarization in P14 KI, but not in P21 KI or WT mice. MEA recordings in presence of CGP55845 revealed comparable levels of network activity in both genotypes, indicating that tonically activated GABABRs balance neuronal activity in P14 KI cortex despite the reduced GABA levels. Blockade of GABA transporter 3 (GAT-3) reproduced the CGP55845 effects suggesting that tonic activation of GABABRs is mediated by ambient GABA released via GAT-3 operating in reverse mode. We conclude that GAT-3-mediated GABA release leads to tonic activation of both pre- and postsynaptic GABABRs and restricts neuronal excitability in the developing cortex to compensate for reduced neuronal GABA synthesis. Since GAT-3 is predominantly located in astrocytes, GAD67 haplodeficiency may potentially stimulate astrocytic GABA synthesis through GAD67-independent pathways.

Repetitive and compulsive behavior after Early-Life-Pain associated with reduced long-chain sphingolipid species.

BACKGROUND: Pain in early life may impact on development and risk of chronic pain. We developed an optogenetic Cre/loxP mouse model of "early-life-pain" (ELP) using mice with transgenic expression of channelrhodopsin-2 (ChR2) under control of the Advillin (Avil) promoter, which drives expression of transgenes predominantly in isolectin B4 positive non-peptidergic nociceptors in postnatal mice. Avil-ChR2 (Cre +) and ChR2-flfl control mice were exposed to blue light in a chamber once daily from P1-P5 together with their Cre-negative mother. RESULTS: ELP caused cortical hyperexcitability at P8-9 as assessed via multi-electrode array recordings that coincided with reduced expression of synaptic genes (RNAseq) including Grin2b, neurexins, piccolo and voltage gated calcium and sodium channels. Young adult (8-16 wks) Avil-ChR2 mice presented with nociceptive hypersensitivity upon heat or mechanical stimulation, which did not resolve up until one year of age. The persistent hypersensitivy to nociceptive stimuli was reflected by increased calcium fluxes in primary sensory neurons of aged mice (1 year) upon capsaicin stimulation. Avil-ChR2 mice behaved like controls in maze tests of anxiety, social interaction, and spatial memory but IntelliCage behavioral studies revealed repetitive nosepokes and corner visits and compulsive lickings. Compulsiveness at the behavioral level was associated with a reduction of sphingomyelin species in brain and plasma lipidomic studies. Behavioral studies were done with female mice. CONCLUSION: The results suggest that ELP may predispose to chronic "pain" and compulsive psychopathology in part mediated by alterations of sphingolipid metabolism, which have been previously described in the context of addiction and psychiatric diseases.