K-Cl Cotransporter 2–mediated Cl− Extrusion Determines Developmental Stage–dependent Impact of Propofol Anesthesia on Dendritic Spines

Abstract

Background General anesthetics potentiating γ-aminobutyric acid (GABA)–mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)–mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)–mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results The authors found a robust effect of the developmental up-regulation of KCC2–mediated Cl− transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions The KCC2–dependent developmental increase in the efficacy of GABAA–mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

Depolarizing GABA Transmission Restrains Activity-Dependent Glutamatergic Synapse Formation in the Developing Hippocampal Circuit

“…Based on these results, increasing GABA A transmission was not sufficient to decrease glutamatergic synapse number or function, suggesting depolarizing GABA A transmission can only limit synapse formation up to a certain point at this stage of development. However, our results do not rule out the possibility that enhancing immature GABA A transmission on different timescales or in other systems decreases glutamatergic synapse formation (Puskarjov et al, 2017).…”

“…Next, we investigated if increasing GABA A transmission over the 3-5 DIV period would have the opposite effect to GABA-blockade and reduce excitatory synapses. Previous work has demonstrated that propofol, a positive allosteric modulator of GABA A Rs, decreases spine density in developing layer 2/3 principal cells of the somatosensory cortex when administered to rat pups over a 6h period at postnatal day 10, when GABA A transmission is still depolarizing (Puskarjov et al, 2017). To test this in CA1 pyramidal cells, we increased depolarizing GABA A transmission by administering muscimol (MUS) or diazepam (DZP) from 3 to 5DIV.…”

Isoflurane Modulates Hippocampal Cornu Ammonis Pyramidal Neuron Excitability by Inhibition of Both Transient and Persistent Sodium Currents in Mice

“…43,44 Since isoflurane may also potentiate chlorine permeable γ-aminobutyric acid type A receptors, the level of intracellular chlorine has important consequences for its actions in a manner similar to that recently shown for propofol. 45,46 We provide evidence that isoflurane at clinically relevant concentrations inhibits transient sodium channel currents, persistent sodium currents, and resurgent sodium currents at physiologic holding potentials in mouse hippocampal cornu ammonis pyramidal neurons. For transient sodium…”

KCC2-Mediated Cl- Extrusion Modulates Spontaneous Hippocampal Network Events in Perinatal Rats and Mice

“…To assess the efficacy of Clextrusion in neonatal CA3 pyramidal neurons, we utilized our standard assay where a constant Clload is imposed on the neuron via a pipette (5-6 MU tip resistance) patched onto the soma in whole-cell configuration (Khirug et al, 2005;Li et al, 2007;Puskarjov et al, 2015;Puskarjov et al, 2017;Spoljaric et al, 2017). In the presence of active Clextrusion in the dendritic compartment, the deviation of the measured E GABA from the E GABA-GHK predicted by the GHK voltage equation for Cland HCO 3 -(the two anions permeating GABA A Rs), becomes more negative with increasing distance from the somatic load (Khirug et al, 2005;Chamma et al, 2013).…”