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. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

TREK-1 and TREK-2 Knockout Mice Are Not Resistant to Halothane or Isoflurane

“…Regarding holding currents, our interest in the TREK-1 channel was initially the result of a norfluoxetine-inhibitable increase in leak current in noncholinergic neurons of the ventral spinal cord of Ndufs4 23 upon exposure to low concentrations of isoflurane, coupled with a report that a Trek-1 mouse was resistant to anesthetics. 13 Since norfluoxetine blocks both TREK-1 and TREK-2 channels, 15,16 we considered whether TREK-1 or TREK-2 might be up-regulated in Ndufs4 mice.…”

“…It is noteworthy that since TASK channels affect current in cholinergic neurons, we did not see any rise in holding currents when we measured signal from cholinergic cells of the spinal cord in Ndufs4 . 23 This makes a role for TASK channels unlikely in mediating an isoflurane-induced hyperpolarizing current in Ndufs4.…”

“…The cells could be identified as cholinergic or noncholinergic in some genotypes ( Ndufs4 and Ndufs4;Trek-1 ) that incorporated a fluorescent tag (Ai14) in cholinergic neurons as described previously. 23 This signal could be seen with fluorescence microscopy and was used in Ndufs4 -containing slices to ensure use of noncholinergic neurons. This selection was used since previous studies showed that the increased holding currents at 0.6% isoflurane in Ndufs4 slices were seen only in noncholinergic cells.…”

“…Slices of lumbar spinal cord were isolated and studied as previously described. 23 They were first held in the superfusate for 30 min without isoflurane for baseline, unexposed measurements. Isoflurane was then applied in the superfusate at equilibrated concentrations delivered by passing carbogen (a mixture of 95% O 2 and 5% CO 2 ) through a calibrated isoflurane vaporizer.…”

Volatile Anesthetics and O2 Activate TASK-1/3 by Weak H-Bonding with X-Gate Uncharged Arg-245: The Major Molecular Mechanism for Carotid Body Hypoxic Sensitivity and Further Insights into Fighter Pilot +Gz-Induced LOC

“…VA activation of TASK-1,3 in supraspinal channels has been implicated in analgesia 105 and immobility 106 [Reviewed: Steinberg et al 107 ] However, more recent work indicates that TREK channels appear to be playing a greater role in VA induced immobility. 108 It has recently been suggested that VA activation of hippocampal TASK-3 channels could affect memory. 109 There is therefore some evidence that VA effects on TASK channels are contributing to the anesthetic state.…”