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

Abstract

Key pointsr The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem-wide circuit are lacking.r Here, we use silicon multi-electrode arrays to record respiratory local field potentials (rLFPs) from 196-364 electrode sites within 8-10 mm 3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post-inspiration (PI) and late-expiration (E2). r rLFPs peaked specifically at the three respiratory phase transitions, E2-I, I-PI and PI-E2. r We show, for the first time, that only the I-PI transition engages a brainstem-wide network, and that rLFPs during the PI-E2 transition identify a hitherto unknown role for the dorsal respiratory group.r Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease.Abstract While it is widely accepted that inspiratory rhythm generation depends on the pre-Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization 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. We developed an approach using silicon multi-electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the Rishi R. Dhingra is currently a Senior Research Officer in the Florey Institute of Neuroscience & Mental Health at the University of Melbourne. He received his PhD in Neurosciences in 2014 from Case Western Reserve University, Cleveland, USA. His research interest is to develop and apply innovative approaches to monitor and model the activity of brainstem respiratory and cardiovascular circuits at the systems scale in vertebrates. J Physiol 598.11 respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically 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. Spatially, the E2-I, and PI-E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post-inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group-wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thu...

The post‐inspiratory complex (PiCo), what is the evidence?

“…In summary, the data in the paper of Dhingra et al (2020) are in contradiction to the concept of the PiCo being a central rhythm generator for respiratory activity and to the (triple) oscillator hypothesis. I hope that this letter can be a crystallization nucleus for a broader and open-minded discussion about the generation of PI activity, the PiCo and the triple oscillator hypothesis.…”

“…Now, in a very elegant study, recently published in The Journal of Physiology , Dhingra and colleagues showed how neuronal activity of the respiratory network is spatially distributed during the different phases of the respiratory rhythm (Dhingra et al . 2020). Based on respiratory local field potentials (rLFP) recoded with multi‐electrode arrays in the pons and medulla they provide, for the first time, a four‐dimensional reconstruction of the functional neuroanatomy of the entire respiratory network.…”

“…7 from Dhingra et al . (2020). Red circles: Overlay of the region of the putative post‐inspiratory complex (PiCo).…”

Clarity of the rhythmic brainstem

“…The study by Dhingra et al . (2020) has addressed this shortfall by demonstrating the dynamics of activity/inactivity of neuronal populations from the complete ponto‐medullary respiratory network of single rats in real time . Using the in situ arterially perfused preparation of rat, which generates a three‐phase respiratory motor pattern of inspiration, post‐inspiration (first stage of expiration) and stage two expiration, the authors used state‐of‐the‐art silicon‐based ultrafine multi‐electrode array technology to record macroscopic local field potentials from 196–364 sites per rat with known anatomic coordinates in three‐dimensional space.…”

“…The study is analogous to the Clarity procedure where brain volumes are made transparent to allow visualisation of a defined neuronal network. However, unlike Clarity, Dhingra et al .’s (2020) study also adds the electrical activity to the network and hence provides Clarity of the rhythmic brainstem.…”