Volumetric mapping of the functional neuroanatomy of the brainstem respiratory network in the perfused brainstem preparation of rats

Abstract

One of the major goals of modern neuroscience is to understand the relationship between the functional neuroanatomy/connectivity of neural circuits and the behavior of an animal. The key challenge to addressing this goal with respect to control of breathing is the distributed nature of the respiratory network. While in cortical networks, short‐range connections (10–100 μm) out‐number long‐range connections (1+ mm), the opposite may be true of brainstem circuits. Specifically, anatomical tracing studies have identified many long‐range projections between brainstem respiratory nuclei that led to the concept that the brainstem respiratory network is compartmentally organized. Thus, measuring the functional neuroanatomy of the respiratory network—a task required to phenotype the functional role of neuronal populations identified in molecular mapping studies—requires monitoring the activity of respiratory neurons which may be spread over many millimeters.We hypothesized that the spatio‐temporal structure of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network in single preparations. To address our hypothesis, we developed an approach using silicon multi‐electrode arrays to record spontaneous LFPs from hundreds of electrode sites across the ponto‐medullary volume of the respiratory network while monitoring the respiratory motor pattern on phrenic and vagal nerves.Our results revealed the expression of rLFPs across the brainstem respiratory network. rLFPs were expressed selectively at the three transitions between respiratory phases: (1) from late‐expiration (E2) to inspiration (I), (2) from I to post‐inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions, rather than being equally distributed across the respiratory cycle. Spatially, the E2‐I (inspiratory on‐switch), and PI‐E2 transitions were localized to the ventral and dorsal respiratory groups, respectively, whereas the I‐PI (inspiratory off‐switch) transition was distributed across the ventral, dorsal and pontine respiratory groups. An independent component analysis (ICA) confirmed this spatio‐temporal organisation of rLFPs and identified a traveling wave of rLFPs that occurred at the I‐PI transition. Finally, a group‐wise ICA demonstrated that all preparations exhibited rLFPs with a similar temporal structure.Overall, under intact network conditions, our results confirm that inspiration is initiated by the pre‐Bötzinger complex, whereas post‐inspiration and late‐expiration depend on activity throughout the brainstem respiratory network. In conclusion, we have developed a general approach to volumetrically map spontaneous‐ or evoked‐respiratory network activity at the brainstem‐wide scale in single preparations to inform our understanding of the network mechanisms underlying the neural control of breathing.Support or Funding InformationThis work was supported by grants from the Australian Research Council (to MD), National Institutes of Health (to TED) and the Hartwell Foundation (to RFG).