Mapping Circuit Connections of Brainstem Respiratory Regions with Retrogradely Transporting Viral Vectors in Adult Transgenic Rats

In mammals, breathing is a constantly regulated key motor behavior that is generated by a rhythmic neural process in the brainstem. The preBötzinger complex (preBötC), a medullary region critical for generating the breathing rhythm, has been proposed to have circuit connections ipsilaterally and contralaterally to functionally distinct neighboring medullary regions, such as the respiratory Bötzinger complex (BötC), the rostral and caudal Ventral Respiratory Groups (rVRG/cVRG), and retrotrapezoid nucleus (RTN) for coordination of the rhythmic activity of multiple respiratory neuronal populations. Because breathing is integrated with other volitional and physiological behaviors, it is proposed that brainstem respiratory circuits, especially within the preBötC, act as critical nodes for control of breathing, and may have connections to neurons in other regions throughout the brain involved in motor control and behavioral integration such as motor cortex for volitional control of breathing or other aspects of motor integration. However, the axonal projections of preBötC neurons throughout the brain, and the input connections from other brain regions have not been mapped. In the present study, we therefore performed brain‐wide mapping employing adult transgenic rats expressing Cre recombinase under control of the glutamic acid decarboxylase 1 (GAD1) promoter to specifically target GABAergic inhibitory neurons. To map input connections to the preBötC, we used combinations of Cre‐dependent and Cre‐independent retrogradely transporting viral vectors (rAAV2‐retro) driving expression of different fluorescent proteins to allow connections of GABAergic neurons to be differentiated from connections by other neuron types. This approach allows for targeted mapping of local circuit neurons and their axonal projections as well as retrograde labeling of neurons with long‐range input connections. With this approach, we have obtained evidence of retrograde GABAergic and non‐GABAergic neuronal labeling at multiple levels of the brainstem (including BötC, rVRG, RTN, and pontine Kölliker‐Fuse/Parabrachial nuclei) and numerous suprabrainstem regions including cortical and subcortical regions. Our results indicate that various constructs of rAAV2‐retro in combination with specific Cre transgenic lines are effective tools for brain‐wide mapping of input connections to circuits in functionally defined respiratory regions, including providing genetic access to specific populations of projection neurons. Support or Funding Information IRP of NIH, NINDS