Breathing responses produced by optogenetic stimulation of adrenergic C1 neurons are dependent on the connection with preBötzinger complex in rats
Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBötzinger complex (preBötC) 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 preBötC neurons. Anatomical connection between C1 cells and preBötC was evaluated using retrograde (Cholera Toxin b; preBötC) 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 preBötC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBötC 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 preBötC. Kyn injection into preBötC 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 preBötC 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 preBötC neurons.
KeywordsCatecholamines Glutamate Hypoxia Optogenetics Rostroventrolateral medulla Ventilation
We would like to acknowledge Dr. Eugenia Constanzi-Strauss for providing the infrastructure that allowed us to perform the virus injection in the brain.
MRML, ACT, and TSM designed research; MRML and LTT performed electrophysiology and anatomy research; MRML and MVGL produced the lentivirus; BES provided material and technical assistance to lentivirus production; MRML and TSM analyzed data; MRML, ACT, and TSM wrote the paper. MRML, LTT, MVGL, BES, ACT, and TSM performed critical review of the manuscript. All authors approved the final version.
This work was supported by the São Paulo Research Foundation (FAPESP; grants 2016/23281-3 to ACT; 2015/23376-1 and 2016/22069-0 to TSM) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant 471283/2012-6 to TSM). FAPESP fellowship (2014/07698-6 to MRML) and CNPq fellowship (301219/2016-8 to ACT and 301904/2015-4 to TSM).
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Conflict of interest
The authors declare that they have no conflicts of interest.
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