Optogenetic Inhibition Of Striatal GABAergic Neuronal Activity Improves Outcomes After Ischemic Brain Injury

GABAergic neurons constitute the majority of neurons in the striatum and are known to regulate neurogenesis in adult mammalian brain.Studies showed that inhibition of striatal neuronal activity promoted neurogenesis and neurological function in the recovery phase after ischemic brain injury in mice.However,the specific effects of striatal GABAergic neuronal activity on neurological recovery in the sub-acute phase of ischemia and their underlying mechanisms remain unknown.Here,using optogenetics approach combined with two strains of transgenic mice,GAD2-Arch-GFP and GAD2-ChR2-tdTomato,to investigate the effects of GABAergic neuronal activities on the functional recovery and underline mechanisms.1.We used 530 nm or 473 nm laser stimulation to specifically inhibit or activate the striatal GABAergic neurons in GAD2-Arch-GFP and GAD2-ChR2-tdTomato transgenic mice.The laser was delivered via an implanted optical fiber to stimulate the opsins,Arch or ChR2,expressed on the membrane of GABAergic neurons.To confirm that lasers could change the activities of striatal GABAergic neurons,we administrated the light into brain and recorded their activities simultaneously.Results showed that both the local field potential and the firing rate were significantly reduced when green laser was applied.The power spectral density analysis(p<0.05)was also reduced when compared to the before and after the laser inhibition.473 nm blue pulse laser also induced neuronal activities.The firing rate was much higher than that before and after laser stimulation(p<0.05).A GAD staining was also performed to confirm that the neurons we stimulated were truly GABAergic neurons.We demonstrated that 530 or 473 nm laser could be used to inhibit or to activate the striatal GABAergic neuronal activities.2.Laser stimulation was performed from day 7 to day 13,twice a day,and fifteen minutes each session.A neurological severity score test was carried out before middle cerebral artery occlusion surgery and on day 1,day 3,day 7 and day 14 to assess the functional recovery following stroke.After a seven-day-stimulation,the results showed that inhibiting striatal GABAergic neuronal activity improved functional recovery and reduced infarct volume after ischemic stroke,while activating striatal GABAergic neurons aggravated the impairment.Open field test also showed that inhibiting striatal GABAergic neuronal activities promoted the functional recovery in mice.3.Using immunohistochemistry analysis,we examined brain atrophy volume,microvascular density and the changes of cellular morphology to investigate the mechanisms of inhibiting striatal GABAergic neuronal activities improved the functional recovery.We investigated the mechanism from two aspects,neurogenesis and neuroprotection.The results showed that there were no obvious signs of neurogenesis but found that the inhibition group has a higher microvascular density and less TUNEL~+cells in peri-infarct area,while the activation group has an opposite consequence.4.To further study the mechanism that inhibition of striatal GABAergic neuronal activities improved the microvascular density,we examined some factors that closely related to microvessels,including bFGF,VEGF,PDGF and BDNF.PCR results showed that only the bFGF was increased in the inhibition group and decreased in the activation group when compared to corresponding control group(p<0.05).Western blot and immunostaining also showed that the bFGF was increased in the inhibition group while decreased in the activation group(p<0.05).Besides,immunofluorescence co-localization analysis further demonstrated that the increased bFGF was mainly secreted from endothelial cells,indicating that inhibiting striatal GABAergic neuronal activities promotes the bFGF secretion from endothelial cells.5.To investigate the mechanisms that GABAergic neuronal activities changed the secretion of bFGF,we performed three different co-culture systems,including neurons/astrocytes/ECs,neurons/astrocytes and neurons/ECs.The results showed that 530 nm laser inhibition only increased the bFGF expression in neurons/astrocytes/ECs co-culture system.Similar results were not observed in neither neuron/astrocytes nor neurons/ECs system.Besides,the condition medium collected from laser stimulated three-cell co-culture system promoted another group cells’viability from oxygen-glucose deprivation treatment.Implying the improved outcomes after ischemic stroke may own to increased bFGF secretion induced by inhibiting striatal GABAergic neuronal activities.In conclusion,we studied the effects of striatal GABAergic neuronal activities on functional recovery after ischemic stroke,and further investigated its mechanisms.Results showed that inhibition of striatal GABAergic neuronal activity promoted functional recovery in mice and attenuated brain atrophy volume.These protection mechanisms may involve that inhibition of striatal GABAergic neuronal activities increased the bFGF expression from endothelial cells,depending on the presence of astrocytes,indicating that neurovascular unit played an important role in the signal regulation and neuronal circuits,providing a novel approach to promote neurological recovery after stroke.