0141 Ascending Projections From Parafacial Zone To The Medial Parabrachial Neurons. (12th April 2019) - De Luca, Roberto
Abstract: Introduction: The parafacial zone (PZ) is an important region for the generation of non-REM (NREM) sleep. We previously found that optogenetic-stimulation of PZ GABA projections inhibits lateral parabrachial neurons (lPB) that project to basal forebrain (BF) and we thought that through this circuit PZ neurons promoted NREM sleep. However some studies reported that glutamatergic neurons in mPB are necessary for spontaneous waking compared to those in lPB which promote arousal manily in response to hypercapnia or pain. We found that the PZ heavily innervates the mPB, suggesting that PZ might promote NREM sleep by inhibiting mPB. We used in vitro Channelrhodopsin2 (ChR2)-assisted circuit mapping (CRACM) to investigate the functional synaptic connectivity between the PZ and mPB neurons. We also studied in whole animal experiments the effects of activation of PZ GABAergic neurons using a chemogenetic approach. Methods: We recorded mPB neurons in brain slices from V gat-IRES-cre mice. To induce the expression of ChR2-mCherry in PZ GABA (PZ VGAT ) neurons, we injected an AAV-DIO-ChR2-mCherry unilaterally into the PZ of V gat-IRES-cre mice. We then recorded mPB neurons while photo-stimulating the PZ VGAT neurons axons and terminals in the PB. To induce the expression of hM3Dq-mCherry in PZ VGAT neurons, we injected bilaterally an AAV-DIO-hM3Dq-mCherry into the PZ of V gat-IRES-cre mice. We recorded EEG and EMG after intraperitoneal injections of saline (controls) or clozapine-N-oxide (CNO). Results: Optogenetic-stimulation of PZ VGAT ->mPB input evoked inhibitory postsynaptic currents (oIPSCs) in 21 out of 29 mPB neurons. We have previously found that activation of PZ VGAT ->lPB input evoked oIPSCs that were solely mediated by GABAA signaling. In mPB neurons, oIPSCs were abolished only when GABAA and glycine receptors antagonists were co-applied. In addition, we confirmed that injections of CNO were able to increase NREM sleep after activation of PZ VGAT neurons that expressed hM3Dq. Conclusion: Our results suggest that PZ neurons project to both lPB and mPB. They inhibit lPB neurons by GABA release and inhibit mPB neurons through GABA and glycine. We hypothetize that activation of PZ promotes NREM sleep by inhibiting mPB neurons. Support (If Any): NS091126 and NS092652