The changing of α5-GABAA receptors expression and distribution participate in sevoflurane-induced learning and memory impairment in young mice

Abstract

Background: Sevoflurane is a superior agent for maintaining anesthesia during surgical procedures. However, the neurotoxic mechanisms of clinical concentration remain poorly understood. Sevoflurane can interfere with the normal function of neurons and synapses and impair cognitive function by acting on α5-GABAAR.

Methods: Using MWM test, we evaluated cognitive abilities in mice following 1 h of anesthesia with 2.7%-3% sevoflurane. Based on hippocampal transcriptome analysis, we analyzed the differential genes and IL-6 24 h post-anesthesia. Western blot and RT-PCR were performed to measure the levels of α5-GABAAR, Radixin, P-ERM, P-Radixin, Gephyrin, IL-6, and ROCK. The spatial distribution and expression of α5-GABAAR on neuronal somata were analyzed using histological and three-dimensional imaging techniques.

Results: MWM test indicated that partial long-term learning and memory impairment. Combining molecular biology and histological analysis, our studies have demonstrated that sevoflurane induces immunosuppression, characterized by reduced IL-6 expression levels, and that enhanced Radixin dephosphorylation undermines the microstructural stability of α5-GABAAR, leading to its dissociation from synaptic exterior and resulting in a disordered distribution in α5-GABAAR expression within neuronal cell bodies. On the synaptic cleft, the expression level of α5-GABAAR remained unchanged, the spatial distribution became more compact, with an increased fluorescence intensity per voxel. On the extra-synaptic space, the expression level of α5-GABAAR decreased within unchanged spatial distribution, accompanied by an increased fluorescence intensity per voxel.

Conclusion: Dysregulated α5-GABAAR expression and distribution contributes to sevoflurane-induced partial long-term learning and memory impairment, which lays the foundation for elucidating the underlying mechanisms in future studies.

Keywords: changing; expression and distribution; neurotoxic mechanism; partial long‐term learning and memory impairment; sevoflurane; α5‐GABAAR.

FIGURE 1

The evaluation of learning and memory after sevoflurane exposure. (A) Diagrammatic presentation of the mainly procedures leading to the fabrication and identification of learning and memory (a1), and anesthesia time and concentration (a2). (B) Swimming trajectory of mice on the fourth day post‐sevoflurane exposure in SEVO, and CON as Control (b1). Line chart comparing the swimming speed and escape latency of reaching the platform (n = 7) (b2, b3). (C) Swimming trajectory of mice on the sixth day in SEVO and CON (c1). Scatter diagram and histogram comparing of distance and time to first cross the virtual platform (n = 7) (c2, c3), and violin plot showing the percentage of time in target quadrant (n = 7) (c4). ***p < 0.001 versus CON. p = 0.0728 versus CON on the fourth day. In the CON group: ^# p < 0.05, ^## p < 0.01 versus CON on the first day.

FIGURE 2 2

Change of hippocampal transcriptome after anesthesia. (A) 3D principal component analysis showing differences in total mRNA in whole‐hippocampus were analyzed using gene chip method (n = 3) (a1); The differential expressed genes were illustrated using volcano plot (n = 3) (a2). (B) Heatmap of the mRNAs was normalized with the quantile algorithm between CON and SEVO. Significantly enriched downregulated (green) and upregulated (red) GO biological processes (|Fold Change| ≥ 2; p ≤ 0.05). Scale bar explains level of expression (red represents upregulated, green represents downregulated) (n = 3). (C) Histograms showing Normalized RNA expressive value of IL‐6 using gene expression microarray (c1); Histograms show semi‐quantitative analyses for IL‐6 protein conducted by Western blot (n = 3) (c2). *p < 0.05 versus CON.

FIGURE 3

The expression of α5‐GABAAR after anesthesia in hippocampus. (A) Schematic showing the expression of α5‐GABAAR in whole‐hippocampus were analyzed using Western blot and IF (n = 3). (B) Results from Western blot, along with a chart illustrating the expression of α5‐GABAAR, Radixin, P‐ERM, P‐Radixin, and ROCK in the whole hippocampus (n = 3) (b1–b6). (C) The coronal plane image of the hippocampus, immunostained for α5‐GABAAR (pink) and processed using Adobe Illustrator, is shown in c1. The location of neuronal somata in the CA1 area is also illustrated using cartoons (c1). Scale bar, 500 μm. The bar chart showing variation of α5‐GABAAR expression among CA1 subregion in hippocampus (c2–c4) (n = 3). *p < 0.05, **p < 0.01 versus CON. Unit of fluorescence intensity = arbitrary units (a.u.).

FIGURE 4

The spatial variation of α5‐GABAAR in synaptic cleft and extra‐synaptic membrane. (A) Anchoring proteins, gephyrin, were quantified using RT‐PCR and Western blot (a1, a2) (n = 3). (B) The space confinement size of synaptic α5‐GABAAR is displayed using 3D images from cartoon and immunostaining (b1, b2). Column graphs exhibit the degree α5‐GABAAR aggregation after sevoflurane treated. Space confinement size of α5‐GABAAR is displayed using voxel (b6–b8), and fluorescence intensity of α5‐GABAARs per unit area in CA1 synaptic space and extra‐synaptic space in 30 neuronal somata (b3–b5). *p < 0.05, **p < 0.01 versus CON. Unit of fluorescence intensity = arbitrary units (a.u.).

FIGURE 5

Principal mechanism for α5‐GABAAR participated in sevoflurane‐induced learning and memory impairment. An anesthesia model in young mice with a maintained sevoflurane concentration of 2.7%–3.0% (left). Increasing dephosphorylation of Radixin, activating α5‐GABAAR in extra‐synaptic space partially diffusing into synaptic cleft, where α5‐GABAARs anchored in a smaller confinement size and expressed stronger fluorescence intensity per voxel, although α5‐GABAARs also expressed stronger fluorescence intensity per voxel but stayed in unchanged confinement size in extra‐synaptic space. Interestingly, dephosphorylation of Radixin did not change α5‐GABAARs expression level in synaptic cleft. So wrong anchoring of α5‐GABAARs in synaptic and extra‐synaptic space participate in sevoflurane‐induced learning and memory impairment (right).