Fig. 1.
Developmental stage–dependent effects of propofol on dendritic spines temporally correlate with up-regulation of cortical potassium-chloride (K-Cl) cotransporter 2 (KCC2) expression and neuronal Cl− extrusion capacity in rat somatosensory cortex (SSC). Propofol exposure of slices potentiates γ-aminobutyric acid (GABA)–mediated changes in membrane potential of layer 2/3 pyramidal neurons (arrow: timing of 5-ms-long GABA uncaging flash), facilitating spiking in a postnatal day (PND) 10 neuron and enhancing membrane hyperpolarization in a PND 15 neuron (A). Representative immunoblots (B) and quantitative analysis of protease-treated SSC slices (C) showing the developmental expression of surface (~100 kDa tryptic band) and total (cytosolic plus surface) KCC2 pools (n = 10 slices, 10 animals [PND 5]; 18 slices, 9 animals [PND 10]; 12 slices, 6 animals [both PNDs 15 and 20]). (D) Sample recordings of GABA uncaging–induced currents (IGABA) at the soma and at a distance 50 μm away along the apical dendrite. Bar indicates timing of a 10-ms uncaging flash. (E) Mean somatic and apical dendritic EGABA values demonstrate an anti-KCC2 shRNA (shKCC2)–sensitive developmental increase in Cl− extrusion capacity. EGABA [GHK]: level predicted by the Goldman–Hodkin–Katz voltage equation. Premature expression of KCC2 using in utero electroporation (IUE) of a full-length KCC2 (KCC2-FL) construct hyperpolarizes EGABA values recorded at PND 10 close to levels observed at PND 15 (n = 9 neurons, 5 slices [naive PND 5]; 10 neurons, 5 slices [naive PND 10]; 10 neurons, 3 slices [naive PND 15]; 11 neurons, 3 slices [shKCC2 PND 20]; 10 neurons, 4 slices [KCC2-FL PND 10]; 3 animals per group). (F, I) Representative confocal images and quantitative analysis of spine densities (G, J) and head diameters (H, K) show the qualitatively opposite effects of propofol anesthesia at PND 10 (F–H) versus anesthesia at PND 15 (I–K) on spines of layer 2/3 pyramidal neurons post hoc filled with Lucifer Yellow (n = 4 animals per group). A total of 2,922 spines in the control and 2,116 spines in the propofol groups were counted. Results are expressed as mean ± SD. Scale bar: 6 μm. (C) One-way ANOVA with repeated measures followed by Bonferroni post hoc test were used in comparing both the total and the surface signal levels of KCC2. (E) Two-way ANOVA with Bonferroni post hoc testing was performed to compare the naive groups, while two-tailed Student’s t tests were conducted in the shKCC2 as well as in the KCC2-FL groups. (G, J) Two-tailed Student’s t tests were conducted. (H, K) Two-way ANOVA with Bonferroni post hoc testing was performed. *P < 0.05. **P < 0.01. ***P < 0.001. ns = not significant.