Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures

Abstract

Neto2 is a transmembrane protein that interacts with the neuron-specific K+-Cl− cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl− gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2−/− neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2−/− neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition

“…However, the greater magnitude of uIPSC property changes observed in Neto mutants compared to KAR antagonism leaves open the possibility that Netos may influence CCK/CB1 interneuron presynaptic function independent of KARs. Though Neto2 associates with and regulates surface expression of KCC2 (Ivakine et al, 2013; Mahadevan et al, 2015) any potential confounding influence by such regulation on sIPSC/uIPSC properties was avoided by directly controlling postsynaptic chloride gradients and providing large chloride driving forces to maximize signal to noise ratios for synaptic currents. Moreover, the normal gamma oscillations observed in Neto2KOs argue against a catastrophic breakdown in postsynaptic GABAergic signaling.…”

Ionotropic and metabotropic kainate receptor signalling regulates Cl− homeostasis and GABAergic inhibition

“…, Mahadevan et al . ), we have previously demonstrated that changes in E GABA can be detected using whole‐cell recordings (Ormond and Woodin, , Ormond and Woodin, , Takkala and Woodin, , Mahadevan et al . ).…”

“…IPSCs were evoked with a stimulating electrode placed in the stratum lucidum/radiatum when recording in whole-cell mode with a holding potential of −70 mV. Although gramicidin-perforated patch clamp recordings are often used to record E GABA , including in our own studies (Acton et al 2012, Ivakine et al 2013, Mahadevan et al 2014, Mahadevan et al 2015, we have previously demonstrated that changes in E GABA can be detected using whole-cell recordings (Ormond and Woodin, 2009, Ormond and Woodin, 2011, Takkala and Woodin, 2013. The use of the whole-cell technique was necessary in the present study as a result of the longer-term nature of the recordings, which required the washing-in and -out of pharmacological agents, and the need to include a pharmacological agent in the recording pipette.…”

“…, Mahadevan et al . ). KCC2 also functionally interacts with several other glutamate receptors, including NMDA receptors (Lee et al .…”

“…KCC2 is highly expressed at excitatory synapses (Gulyás et al 2001, Li et al 2007, Llano et al 2015, where it plays an important role in dendritic spine formation and maintenance, and influences AMPA receptor (AMPAR) content and diffusion out of the synapse (Li et al 2007, Gauvain et al 2011, Llano et al 2015. KCC2 has also been shown to physically interact with several excitatory receptors and proteins , including the GluK2 subunit of kainate receptors (KARs) (Mahadevan et al 2014), and the KAR auxiliary subunit Neto-2 (Ivakine et al 2013, Mahadevan et al 2015. KCC2 also functionally interacts with several other glutamate receptors, including NMDA receptors (Lee et al 2011), and metabotropic glutamate receptors (mGluRs) (Banke and Gegelashvili, 2008).…”