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Breathing responses produced by optogenetic stimulation of adrenergic C1 neurons are dependent on the connection with preBtzinger complex in rats

Malheiros-Lima, MR;Totola, LT;Lana, MVG;Strauss, BE;Takakura, AC;Moreira, TS;

Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBtzinger complex (preBtC) is a group of neurons located in the intermediate aspect of the ventrolateral medulla, critical for respiratory rhythmogenesis, and is modulated by glutamate and catecholamines. Our hypothesis is that selective activation of C1 neurons leads to breathing responses by excitatory connections with the preBtC neurons. Anatomical connection between C1 cells and preBtC was evaluated using retrograde (Cholera Toxin b; preBtC) and anterograde (LVV-PRSx8-ChR2-eYFP; C1 region) tracers. LVV-PRSx8-ChR2-eYFP (viral vector that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter (PRSx8) was also injected into the C1 region of male Wistar rats for the functional experiments. Anatomical results demonstrated that preBtC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBtC was evaluated by photostimulation of ChR2-transduced C1 neurons before and after unilateral injection of the ionotropic glutamate antagonist, kynurenic acid (kyn), or cocktail of adrenergic antagonists in the preBtC. Kyn injection into preBtC blocked the increase in DiaEMG frequency without changing the MAP increase elicited by photostimulation of C1 neurons, while the injection of adrenergic antagonists into the preBtC did not change DiaEMG frequency and MAP increase induced by photostimulation of C1 cells. Our results suggest that the increase in breathing produced by photostimulation of C1 neurons can be caused by a direct glutamatergic activation of preBtC neurons.