Knockdown of <i>Ndufs4</i> in the CMT.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the CMT (Co-ordinates: ML = ± 0.32; AP = -1.2; DV = 3.85, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. **indicates p-values <0.005, *** indicates p-values <0.001.</p

Knockdown of <i>Ndufs4</i> in the DMT.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the DMT (Co-ordinates: ML = ± 0.32; AP = -1.2; DV = 3.7, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. *** indicates p-values <0.001.</p

Regional knockdown of NDUFS4 implicates a thalamocortical circuit mediating anesthetic sensitivity

<div><p>Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an <i>Ndufs4</i>^lox/lox gene, adeno-associated virus expressing Cre recombinase was injected into regions of the brain postulated to affect sensitivity to volatile anesthetics. These injections generated otherwise phenotypically wild type mice with region-specific, postnatal inactivation of <i>Ndufs4</i>, minimizing developmental effects of gene loss. Sensitivities to the volatile anesthetics isoflurane and halothane were measured using loss of righting reflex (LORR) and movement in response to tail clamp (TC) as endpoints. Knockdown (KD) of <i>Ndufs4</i> in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. <i>Ndufs4</i> loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of <i>Ndufs4</i> only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (<i>Ndufs4(KO)</i>). Excitatory synaptic transmission in the parietal association cortex in slices from <i>Ndufs4(KO)</i> animals was hypersensitive to isoflurane compared to control slices. We identified a direct neural circuit between the parietal association cortex and the central thalamus, consistent with a model in which isoflurane sensitivity is mediated by a thalamic signal relayed through excitatory synapses to the parietal association cortex. We postulate that the thalamocortical circuit is crucial for maintenance of consciousness and is disrupted by the inhibitory effects of isoflurane/halothane on mitochondria.</p></div

Knockdown of <i>Ndufs4</i> in the MPTA.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the MPTA (Coordinates: ML = ± 0.70; AP = -4.6; DV = 4.1, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 7, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 7, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. **indicates p-value <0.01.</p

Electrophysiological field recordings of the excitatory synaptic transmission.

<p><b>A.</b> Amplitudes of fEPSPs decreased with 0.6% (248 μM) ISO exposure in control and <i>Ndufs4(KO)</i> brain slices. The decrease of fEPSPs was significantly greater for the <i>Ndufs4(KO)</i> than the control (*** indicates p-value <0.001). <b>B.</b> Fiber volleys of both genotypes were not significantly affected by the isoflurane exposure. <b>C.</b> Representative traces of field recordings from control and KO slices before and during exposure to 0.6% ISO (equivalent to 248 μM). 0.6% ISO exposure led to a significantly larger decrease in the amplitudes of fEPSP in the KO when compared to the respective decrease in controls. The amplitudes of the fiber volleys were decreased (statistically tending to significance, p = 0.015) by 0.6% ISO exposure in both genotypes.</p

The thalamocortical circuit.

<p><b>A.</b> Fluorescent image of a brain slice from a mouse injected with Δ-Cre virus into the PAC (Magnification X40). <b>B.</b> Magnified confocal image of the white-boxed region within the thalamus in <b>A</b> (Magnification X200)<b>. C.</b> Magnified confocal image of white boxed region in <b>B</b> showing viral-GFP expressing cells in the thalamus following injection into the PAC (Magnification X1000). Scale bars: A-1mm, B-50μm, C-10μm.</p

EC₅₀s for ISO and HAL for the WT-Cre and Δ-Cre virus-injected mice in the LORR and TC assays.

<p>EC₅₀s for ISO and HAL for the WT-Cre and Δ-Cre virus-injected mice in the LORR and TC assays.</p

Knockdown of <i>Ndufs4</i> in the VN.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus into the VN (Coordinates: ML = ± 1.25; AP = -5.8; DV = 4.3, Magnification X40). Virus-infected cells appear green due to the viral GFP co-expression B, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] (Image credit: Allen Institute) depicting the antero-posterior (<b>B</b>), mediolateral (<b>C</b>) and dorso-ventral (<b>C</b>) viral spread (green boxed regions in <b>C</b>). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 7, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 7, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. **indicates p-values <0.005, *** indicates p-values <0.001. The error bars in all bar graphs indicate standard deviation.</p

Knockdown of <i>Ndufs4</i> in the PAC.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the PAC (Co-ordinates: ML = ± 0.80; AP = -1.75; DV = 0.9, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 5, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 5, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. *** indicates p-values <0.001.</p

Viral injections and characterization.

<p><b>A</b>. Schematic diagram depicting the five regions which were injected with either rAAV/WT-Cre GFP (knockout virus expressing functional Cre recombinase) or rAAV/Δ-Cre GFP (sham virus expressing nonfunctional Cre recombinase). <b>B</b>. Schematic showing the mechanism of gene deletion. Exon 2 of genomic <i>Ndufs4</i> is excised by the virus expressing active Cre recombinase, resulting in the loss of NDUFS4 protein as described by Kruse <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref006" target="_blank">6</a>]. <b>C</b>. Loss of <i>Ndufs4</i> expression in active Cre virus infected cells in the PAC. Infected cells appear green under the confocal microscope due to the viral GFP co-expression (Magnification X1000). In the Δ-Cre sham virus infected cells of the PAC, mitochondrial NDUFS4 fluorescence (red) is seen, which is absent in the WT-Cre infected cells. <b>D</b>. Representative quantitation of NDUFS4 expression in the virus infected cells, shown as a percentage of total infected cells. 20 image fields were quantified per injected locus. Scale bar: 10μm. *** indicates p-values <0.001.</p