Mitochondrial Function and Anesthetic Sensitivity in the Mouse Spinal Cord

Abstract

Background: Ndufs4 knockout (KO) mice are defective in mitochondrial complex I function and hypersensitive to inhibition of spinal cord-mediated response to noxious stimuli by volatile anesthetics. It was hypothesized that, compared to wild-type, synaptic or intrinsic neuronal function is hypersensitive to isoflurane in spinal cord slices from knockout mice.

Methods: Neurons from slices of the vestibular nucleus, central medial thalamus, and spinal cord from wild-type and the global Ndufs4 knockout were patch clamped. Unstimulated synaptic and intrinsic neuronal characteristics were measured in response to isoflurane. Norfluoxetine was used to block TREK channel conductance. Cholinergic cells were labeled with tdTomato.

Results: All values are reported as means and 95% CIs. Spontaneous synaptic activities were not different between the mutant and control. Isoflurane (0.6%; 0.25 mM; Ndufs4[KO] EC95) increased the holding current in knockout (ΔHolding current, 126 pA [95% CI, 99 to 152 pA]; ΔHolding current P < 0.001; n = 21) but not wild-type (ΔHolding current, 2 7 pA [95% CI, 9 to 47 pA]; ΔHolding current, P = 0.030; n = 25) spinal cord slices. Knockout and wild-type ΔHolding currents were significantly different (P < 0.001). Changes comparable to those in the knockout were seen in the wild type only in 1.8% (0.74 mM) isoflurane (ΔHolding current, 72 pA [95% CI, 43 to 97 pA]; ΔHolding current, P < 0.001; n = 13), the control EC95. Blockade of action potentials indicated that the increased holding current in the knockout was not dependent on synaptic input (ΔHolding current, 154 pA [95% CI, 99 to 232 pA]; ΔHolding current, P = 0.506 compared to knockout without blockade; n = 6). Noncholinergic neurons mediated the increase in holding current sensitivity in Ndufs4 knockout. The increased currents were blocked by norfluoxetine.

Conclusions: Isoflurane increased an outwardly rectifying potassium current in ventral horn neurons of the Ndufs4(KO) mouse at a concentration much lower than in controls. Noncholinergic neurons in the spinal cord ventral horn mediated the effect. Presynaptic functions in Ndufs4(KO) slices were not hypersensitive to isoflurane. These data link anesthetic sensitivity, mitochondrial function, and postsynaptic channel activity.

Figure 1.

Spontaneous synaptic activity in ventral horn cells of the spinal cord of wildtype control (circles) and mutant (triangles) adult mice. A. Representative whole-cell patch recording of a control cell voltage-clamped at −60 mV. 0.6% isoflurane applied to the slice from minute 5 to 15 of the recording (black bar). Two sections of the recording shown at enlarged scale to illustrate kinetic properties of the synaptic events (red lines to bottom traces). B-G. Synaptic event properties of frequency, amplitude, and decay from the spinal cord are quantified and compared between Ndufs4(KO) and control with and without isoflurane exposure. Spontaneous excitatory postsynaptic currents are shown in B-D; Spontaneous inhibitory postsynaptic currents are shown in E-G. Black and white symbols are mean absolute values of the five minutes before isoflurane is applied to the slice. Colored symbols are mean normalized values of the last five minutes of isoflurane exposure. Red = .6% isoflurane, green = 1.8% isoflurane. Normalized mean values were calculated for each cell by dividing its exposed mean by its unexposed mean for the final five minutes prior to isoflurane exposure. The mean values for each group were then calculated from the individual cell ratios. Crossbars are mean and boxes represent the 95% confidence intervals. B-D; n=24 (wildtype; 11M, 13F), n=22(Ndufs4(KO); 9M, 13F): E-G; n=23 (wildtype;11M, 12F), n=21(Ndufs4(KO), 8M, 13F).

Figure 2.

A-D. Holding currents required to voltage clamp wildtype control (circles) or mutant (triangles) cells at −60 mV. Black and white symbols are mean absolute values of the five minutes before isoflurane is applied to the slice. Colored symbols are mean normalized values for each cell during the last two minutes of isoflurane exposure. Red = 0.6% isoflurane, green = 1.8% isoflurane. Normalized mean values in this figure and in Figures 3, 5, and 6 and S7 were calculated for each cell by subtracting the unexposed mean for the last five minutes prior to isoflurane exposure from the exposed mean for the last five minutes of exposure. In the CA1 hippocampus (A), the central medial thalamus (B), and the vestibular nucleus (C), holding current in Ndusf4(KO) cells does not change with exposure to 0.6% isoflurane. D. In the spinal cord holding current in the mutant rises at a lower concentration (0.6%) of isoflurane than control. E. Differences in holding current over time in controls (circles) or mutant (triangles) cells with no isoflurane (blue) or exposure to either 0.6% (red) or 1.8% (green) isoflurane. Black bar indicates when isoflurane was applied to the slice. Lines and shaded ribbons are mean and 95% confidence interval. F. Representative whole-cell patch recording of a Ndusf4(KO) cell voltage-clamped at −60 mV. 0.6% isoflurane applied to slice from minute 5 to 15 of the recording (black bar). Two sections of the recording shown at enlarged scale to illustrate kinetics of the synaptic events (red lines to bottom traces). Note the shift in the baseline indicating an increase in holding current necessary to maintain resting membrane potential at −60mV. A; n=12(wildtype; 5M, 7F), n=23(Ndufs4(KO); 18M, 5F): B; n=7(wildtype;4M, 3F), n=7(Ndufs4(KO), 3M, 4F): C; n=10(wildtype;2M, 8F), n=16(Ndufs4(KO), 5M, 11F): D; Unexposed: n=25(control;11M, 14F), n=26(Ndufs4(KO); 9M, 17F). Isoflurane exposed: N=13(wildtype; 7M, 6F), n=21(Ndufs4(KO); 8M, 13F).

Figure 3.

A. Mean holding current in wildtype control (circles) and mutant (triangles) cells in the five minutes before application of norfluoxetine (left) and in the last two minutes of application of norfluoxetine (right). The differences plotted on the right are the mean change in holding current for each cell compared to its pre- norfluoxetine value. B. Time course of action of norfluoxetine on holding current. The bar shows time of norfluoxetine application. C. Changes in holding current when isoflurane is applied in the presence of norfluoxetine. The values on the left are the mean holding current in control and mutant cells in norfluoxetine in the last five minutes before application of isoflurane. The values on the right represent the mean difference in holding current for each cell from its value before isoflurane exposure in the presence of norfluoxetine to its value in the last two minutes of isoflurane exposure in norfluoxetine (purple or brown symbols.) D. Differences in holding current over time of wildtype controls in 1.8% isoflurane (circles, purple plot) and mutant cells in 0.6% isoflurane (triangles, brown plot) in the presence of norfluoxetine. Red and green plots of controls and mutants are of isoflurane exposure alone from Figure 2E. A,B; n=8(wildtype; 5M, 3F), n=8(Ndufs4(KO); 3M, 5F): C,D; n=13(wildtype;7M, 6F), n=21(Ndufs4(KO), 8M, 13F).

Figure 4.

Spontaneous synaptic activity in in the ventral horn of spinal cord cells from wildtype control (circles) and mutant (triangles) mice. A. Confocal images of a spinal cord slice from ChAT;Ai14 mice. Left image shows the entire transverse slice. Right image shows an enlarged portion of the slice (indicated by the white dotted-line box in the left image). White scale bars in upper right corners are 50 μm. Red color indicates cholinergic cells. Blue color indicates nuclear stain. B- G. Synaptic event properties of frequency, amplitude, and decay quantified as in Figure 1. Cholinergic cells are labeled as ChAT⁺; noncholinergic cells are labeled ChAT^-. Spontaneous excitatory postsynaptic currents are shown in B-D; Spontaneous inhibitory postsynaptic currents are shown in E-G. Black and white symbols are mean absolute values of the five minutes before isoflurane is applied to the slice. Colored symbols are mean normalized values of the last five minutes of isoflurane exposure. Red = .6% isoflurane, green = 1.8% isoflurane. Normalized mean values were calculated for each cell by dividing its exposed mean by its unexposed mean for the final five minutes prior to isoflurane exposure. The mean values for each group were then calculated from the individual cell ratios. Crossbars are mean and boxes represent the 95% confidence intervals. Symbols as in Figure 1. B-G; n=19(wildtype; 10M, 9F), n=7(Ndufs4(KO); 5M, 2F).

Figure 5.

Holding current in cholinergic (ChAT⁺) cells. A. At baseline, mutant cholinergic cells (triangles) require less holding current than control cholinergic cells (circles) to voltage clamp at −60 mV (p=0.001); however, their holding current does not rise with 0.6% isoflurane. Control cholinergic cells show a rise in holding current with 1.8% isoflurane (p=0.0005). B. Differences in holding current over time in wildtype control (circles) or mutant (triangles) cholinergic cells in either 0.6% (red) or 1.8% (green) isoflurane. Black bar indicates when isoflurane was applied to the slice. Lines and shaded ribbons are mean and 95% confidence interval. A; n=18(wildtype; 10M, 8F), n=7(Ndufs4(KO); 5M, 2F).

Figure 6.

Holding current in non-cholinergic (ChAT⁻) cells. A. At baseline, non-cholinergic cells for both genotypes require similar holding current to voltage clamp at −60 mV. Holding current of control noncholinergic cells did not reach a significant change with either concentration of isoflurane. Mutant cells increase their holding currents with 0.6% isoflurane (red triangles) (p=0.001). B. Differences in holding current over time in control (circles) or mutant (triangles) non-cholinergic cells to either 0.6% (red) or 1.8% (green) isoflurane. Black bar indicates when isoflurane was applied to the slice. Lines and shaded ribbons are mean and 95% confidence interval. A; unexposed; n=12(wiltype; 7M, 5F), n=6(Ndufs4(KO); 3M, 3F): exposed n=6(3M, 3F; in all cases).