Inhibition of α5 GABAA receptors has preventive but not therapeutic effects on isoflurane-induced memory impairment in aged rats

Abstract

The α5 subunit-containing gamma-amino butyric acid type A receptors (α5 GABAARs) are a distinct subpopulation that are specifically distributed in the mammalian hippocampus and also mediate tonic inhibitory currents in hippocampal neurons. These tonic currents can be enhanced by low-dose isoflurane, which is associated with learning and memory impairment. Inverse agonists of α5 GABAARs, such as L-655,708, are able to reverse the short-term memory deficit caused by low-dose isoflurane in young animals. However, whether these negative allosteric modulators have the same effects on aged rats remains unclear. In the present study, we mainly investigated the effects of L-655,708 on low-dose (1.3%) isoflurane-induced learning and memory impairment in elderly rats. Young (3-month-old) and aged (24-month-old) Wistar rats were randomly assigned to receive L-655,708 0.5 hour before or 23.5 hours after 1.3% isoflurane anesthesia. The Morris Water Maze tests demonstrated that L-655,708 injected before or after anesthesia could reverse the memory deficit in young rats. But in aged rats, application of L-655,708 only before anesthesia showed similar effects. Reverse transcription-polymerase chain reaction showed that low-dose isoflurane decreased the mRNA expression of α5 GABAARs in aging hippocampal neurons but increased that in young animals. These findings indicate that L-655,708 prevented but could not reverse 1.3% isoflurane-induced spatial learning and memory impairment in aged Wistar rats. All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Academy of Military Medical Science of China (approval No. NBCDSER-IACUC-2015128) in December 2015.

Keywords: M2 type microglia; Wnt/Frizzled signaling pathway; early brain injury; inflammatory cytokines; nerve regeneration; neural regeneration; nuclear factor-κB; peroxisome proliferator-activated receptor-γ; subarachnoid hemorrhage.

Figure 1

Experimental paradigm timeline. Following the place navigation trials for 4 consecutive days, young and aged rats were randomly divided into five groups and received the corresponding treatment. Either 24 or 72 hours after anesthesia, the spatial probe trials were conducted to test the retention of spatial reference memory. After the probe trials, all rats were sacrificed, and RT-PCR was conducted to measure hippocampal α5 GABAAR mRNA expression level. h: Hour(s); ISO: isoflurane; RT-PCR: reverse transcription-polymerase chain reaction; GABAAR: gamma-amino butyric acid type A receptor; L: L-655,708.

Figure 2

Performance of young (3 months old) and aged (24 months old) rats in the place navigation trials. Data are expressed as the mean ± SD (n = 40, repeated measures one-way analysis of variance). Escape latency: The time spent to discover the platform.

Figure 3

Performance of young (3 months old) rats in the Morris maze test. (A, B) Effects of isoflurane (ISO) on the escape latency (A) and time c (B) of young rats in the spatial probe trials. (C, D) Effects of L-655,708 on the escape latency (C) and time c (D) of young rats in the spatial probe trials. (D) Effects of L-655,708 on the time c of young rats in the spatial probe trials. **P < 0.01, ***P < 0.001, vs. control group. Data are expressed as the mean ± SD (8 rats in each group, one-way analysis of variance with Turkey’s post hoc test). Escape latency: The time spent before the first arrival at the original platform location; time c: the time spent in quadrant c.

Figure 4

Performance of aged (24 months old) rats in the Morris maze test. (A, B) Effects of isoflurane on the escape latency (A) and time c (B) of aged rats in the spatial probe trials. (B) Effects of isoflurane on the time c of aged rats in the spatial probe trials. (C, D) Effects of L-655,708 on the escape latency (C) and time c (D) of aged rats in the spatial probe trials. **P < 0.01, ***P < 0.001, vs. control group. Data are expressed as the mean ± SD (8 rats in each group, one-way analysis of variance with Turkey’s post hoc test). Escape latency: The time spent before the first arrival at the original platform location. Time c: The time spent in quadrant c.

Figure 5

Hippocampal α5 GABAAR mRNA expression determined by RT-PCR. (A) Effects of isoflurane anesthesia on hippocampal α5 GABAAR expression in young and aged rats. (B) Effects of L-655,708 on hippocampal α5 GABAAR expression in young and aged rats. *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SD (8 rats in each group, one-way analysis of variance with Turkey’s post hoc test). α5 GABAARs: α5 subunit-containing gamma-amino butyric acid type A receptors; RT-PCR: reverse transcription-polymerase chain reaction.