Role Of Hippocampal NRG1-ErbB4 Signaling In The Cognitive Impairments Induced By Isoflurane Exposure

Objective:Isoflurane exposure is reported to induce cognitive impairments in generous rodent experiments. However the underlying mechanisms are not totally understood. Neuregulin 1 (NRG1), a member of the epidermal growth factor family, acts by promoting the tyrosine kinase of ErbB receptor. The NRGl-ErbB4 signaling plays a key role in the hippocampal neural development and the synaptic plasticity. NRG1 regulates the neurotransmission largely via ErbB4 in parvalbumin (PV) interneurons and then controls the pyramidal neuron activity. In the ErbB4 novel mice, the levels of PV and glutamic acid decarboxylase (GAD) 67 are significantly decreased. Therefore, we hypothesized that the cognitive impairments induced by the isofiurane exposure in aged mice may be attributed to the disruption of hippocampal NRGl-ErbB4 signaling probably in the PV-positive interneurons by down-regulating the levels of PV and GAD67.Methods:The first part:seven days after the intracerebroventricular cannulation, twenty fourteen-month-old C57BL/6 mice were randomly divided into five groups (n=4):0 h group,0.5 h group,1 h group,12 h group, and 24 h group. We harvested the hippocampus before the NRG1-β1 injection,0.5 h,1 h,12 h, and 24 h after the NRGl-β1 injection separately and assessed the levels of hippocampal ErbB4 and p-ErbB4 by the Western Blotting. The second part:ninety fourteen-month-old C57BL/6 mice were randomly divided into five groups (n=18):Control group, Vehicle group, Isoflurane group, Isofiurane+NRG1-β1 group, and Isoflurane+ NRGl-β1+AG1478 group. They received the following treatment separately:100% O2 exposure for 2 h,100% O2 exposure for 2 h,1.5%isoflurane exposure for 2 h, 1.5% isoflurane exposure for 2 h, and 1.5% isoflurane exposure for 2 h. Ten mice in each group were exposed to open field test (OFT) and fear conditioning test (FCT) 24 h after the O2 and/or isoflurane expose and the mice in Vehicle group, Isoflurane group, Isoflurane+NRG1-β1 group, and Isoflurane+NRG1-β1+AG1478 group received dimethyl sulfoxide (DMSO), DMSO, NRG1-β1, and AG1478+NRG1-β1 by intracerebroventricular injection 1 h before the behavior test every day. The rest eight mice in each group had the same drug administration with no behavioral test and we harvested the hippocampus 48 h after the O2 and/or isoflurane expose. ELISA was used to determine the level of hippocampal NRG1, Western Blotting was employed to evaluate the levels of hippocampal ErbB4, p-ErbB4, PV, and GAD67, and immunofluorescence was employed to evaluate the expression of hippocampal ErbB4, PV, and GAD67.Results:The hippocampal p-ErbB4 level increased significantly 0.5 h and peaked 1 h after the injection of NRG1-β1, and the increase lasted to 24 h after the injection of NRG1-β1 (P< 0.05). In the OFT, there was no significant different in the total ambulatory distance traveled and the time spent in the center among the five groups (P>0.05). In the FCT, the freezing time to context decreased significantly in the Isoflurane group compared with the Control group (P< 0.05); NRG1-β1 increased the freezing time to context in the Isoflurane+NRG1-β1 group compared with that in the Isoflurane group (P< 0.05), which was abolished by AG1478 in the Isoflurane+NRG1-β1+AG1478 group (P<0.05); there was no significant different in the freezing time to tone among the five groups (P> 0.05). The expression of NRG1 reduced significantly in the Isoflurane group detected by ELISA (P< 0.05). The double-immunofluorescence result indicated that ErbB4 was primarily expressed in the GAD67-immunoreactive interneurons and the average coexpression of PV and ErbB4 in PV-positive interneurons was 82.73±11.37% and in ErbB4-positive neurons was 63.73±10.05% across the hippocampus. Compared with Control group, the levels of hippocampal p-ErbB4, PV, and GAD67 decreased in the Isoflurane group (P< 0.05); NRG1-β1 reversed these reductions in the Isoflurane+NRG1-β1 group (P <0.05) compared with that in the Isoflurane group which was abolished by AG1478 in the Isoflurane+NRG1-β1+AG1478 group (P<0.05).Conclusions:Disruption of hippocampal NRGl-ErbB4 signaling in the parvalbumin-positive interneurons might contribute to the isoflurane-induced hippocampus-dependent cognitive impairment in aged mice, and it may be related to the decrease of PV and GAD67 in the hippocampus.