Inactivation of Prefrontal Cortex Delays Emergence From Sevoflurane Anesthesia

Abstract

Studies aimed at investigating brain regions involved in arousal state control have been traditionally limited to subcortical structures. In the current study, we tested the hypothesis that inactivation of prefrontal cortex, but not two subregions within parietal cortex-somatosensory barrel field and medial/lateral parietal association cortex-would suppress arousal, as measured by an increase in anesthetic sensitivity. Male and female Sprague Dawley rats were surgically prepared for recording electroencephalogram and bilateral infusion into prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9). After at least 10 days of post-surgical recovery, 156 μM tetrodotoxin or saline was microinjected into one of the cortical sites. Ninety minutes after injection, rats were anesthetized with 2.5% sevoflurane and the time to loss of righting reflex, a surrogate for loss of consciousness, was measured. Sevoflurane was stopped after 45 min and the time to return of righting reflex, a surrogate for return of consciousness, was measured. Tetrodotoxin-mediated inactivation of all three cortical sites decreased (p < 0.05) the time to loss of righting reflex. By contrast, only inactivation of prefrontal cortex, but not somatosensory barrel field or medial/lateral parietal association cortex, increased (p < 0.001) the time to return of righting reflex. Burst suppression ratio was not altered following inactivation of any of the cortical sites, suggesting that there was no global effect due to pharmacologic lesion. These findings demonstrate that prefrontal cortex plays a causal role in emergence from anesthesia and behavioral arousal.

Keywords: anesthesia; consciousness; parietal cortex; prefrontal cortex; rat; righting reflex; sevoflurane.

FIGURE 1

Schematic illustrating the experimental design and timeline. After a baseline wake period of 30 min, tetrodotoxin (TTX) or saline were injected bilaterally into prefrontal cortex (PFC), somatosensory barrel field (S1BF), or medial/lateral parietal association cortex (M/LPtA). Ninety minutes after the TTX injection, sevoflurane administration (2.5%) was started and the time to loss of righting reflex (LORR) was measured. Sevoflurane anesthesia was stopped after 45 min and the time to return of righting reflex (RORR) was measured. The EEG data were collected for the entire duration of the experiment.

FIGURE 2

Histological verification of sites of microinjection. The sites of microinjections were confirmed in cresyl violet stained coronal brain sections (40 μm) through prefrontal cortex, somatosensory barrel field (S1BF), and medial/lateral parietal association cortex (M/LPtA). (A) Shows microinjection locations (green dots) plotted onto stereotaxic diagrams through prelimbic region (PrL) of the prefrontal cortex, and a representative PrL section is shown in (B). (C) Shows the microinjection locations in S1BF (red dots) and medial (MPtA) and lateral parietal association cortex (LPtA) (blue triangles). Representative brain sections for S1BF and M/LPtA are shown in (D) and (E), respectively. Each symbol (dots or triangles) represents one rat. The numbers below the stereotaxic diagram are the anteroposterior distance from Bregma. Positive numbers show distance anterior to Bregma while the negative numbers show distance posterior to Bregma. The stereotaxic diagrams are modified from the atlas (The Rat Brain in Stereotaxic Coordinates) by Paxinos and Watson (2007). Fmi; forceps minor of the corpus callosum; IL, infralimbic cortex.

FIGURE 3

Effect of TTX-mediated inactivation of prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9) on the time to loss of righting reflex after sevoflurane anesthesia. Inactivation of prefrontal cortex (A), somatosensory barrel field (B), and medial/lateral parietal association cortex (C) decreased the time to loss of righting reflex after sevoflurane anesthesia. The group data are shown as mean ± standard deviation. Individual rat data are shown as colored dots with red dots representing female rats and blue dots representing male rats. The TTX condition in (A,B) show the data points (dots) from both TTX sessions (i.e., TTX1 and TTX2). The significance symbol (*) denotes p < 0.05 and shows statistical comparison with the saline injection using a linear mixed model. The actual p-values are provided in the text in the results section.

FIGURE 4

Effect of TTX-mediated inactivation of prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9) on the time to return of righting reflex after sevoflurane anesthesia. Inactivation of prefrontal cortex (A) increased the time to return of righting reflex after sevoflurane anesthesia. In contrast, inactivation of neither somatosensory barrel field (B) nor medial/lateral parietal association cortex (C) had a significant effect on the time to return of righting reflex after sevoflurane anesthesia. The group data are shown as mean ± standard deviation. Individual rat data are shown as colored dots with red dots representing female rats and blue dots representing male rats. The TTX condition in (A,B) show the data points (dots) from both TTX sessions (i.e., TTX1 and TTX2). The significance symbol (*) denotes p < 0.05 and shows statistical comparison with the saline injection using a linear mixed model. The actual p-values are provided in the text in the results section. ns, not significant.

FIGURE 5

Representative EEG traces showing burst suppression pattern. The EEG traces show the pattern of burst suppression after saline and tetrodotoxin (TTX) microinjection into prefrontal cortex, somatosensory barrel field, and medial/lateral parietal association cortex. The horizontal bar on the lower right is the scale for time. The vertical bar on the lower right is the EEG amplitude scale.

FIGURE 6

Effect of TTX-mediation inactivation of prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9) on burst suppression ratio during sevoflurane anesthesia. Statistical comparisons using a linear mixed model showed that as compared to saline controls, inactivation of prefrontal cortex (A), somatosensory barrel field (B) or medial/lateral parietal association cortex (C) did not affect the burst suppression ratio during sevoflurane anesthesia. Two rats in the prefrontal cohort were excluded from analysis for a lack of burst suppression across all experiments. The group data are shown as mean ± standard deviation. Individual rat data are shown as colored dots with red dots representing female rats and blue dots representing male rats. The TTX condition in (A,B) show the data points (dots) from both TTX sessions (i.e., TTX1 and TTX2). ns, not significant.