Elsevier

NeuroImage

Volume 98, September 2014, Pages 460-474
NeuroImage

The preBötzinger complex as a hub for network activity along the ventral respiratory column in the neonate rat

https://doi.org/10.1016/j.neuroimage.2014.04.073Get rights and content

Highlights

  • High-speed optical recordings of respiratory networks were made in rat hindbrain.

  • Coupling relations were estimated using the normalized transfer entropy metric.

  • Deterministic neuronal interactions at multiple time scales were detected.

  • A functional hub with dense recurrent coupling to the larger network was found.

  • Structured coupling in networks and macroscopic directed flow were detected.

Abstract

In vertebrates, respiratory control is ascribed to heterogeneous respiration-modulated neurons along the Ventral Respiratory Column (VRC) in medulla, which includes the preBötzinger Complex (preBötC), the putative respiratory rhythm generator. Here, the functional anatomy of the VRC was characterized via optical recordings in the sagittaly sectioned neonate rat hindbrain, at sampling rates permitting coupling estimation between neuron pairs, so that each neuron was described using unitary, neuron-system, and coupling attributes. Structured coupling relations in local networks, significantly oriented coupling in the peri-inspiratory interval detected in pooled data, and significant correlations between firing rate and expiratory duration in subsets of neurons revealed network regulation at multiple timescales. Spatially averaged neuronal attributes, including coupling vectors, revealed a sharp boundary at the rostral margin of the preBötC, as well as other functional anatomical features congruent with identified structures, including the parafacial respiratory group and the nucleus ambiguus. Cluster analysis of attributes identified two spatially compact, homogenous groups: the first overlapped with the preBötC, and was characterized by strong respiratory modulation and dense bidirectional coupling with itself and other groups, consistent with a central role for the preBötC in respiratory control; the second lay between preBötC and the facial nucleus, and was characterized by weak respiratory modulation and weak coupling with other respiratory neurons, which is congruent with cardiovascular regulatory networks that are found in this region. Other groups identified using cluster analysis suggested that networks along VRC regulated expiratory duration, and the transition to and from inspiration, but these groups were heterogeneous and anatomically dispersed. Thus, by recording local networks in parallel, this study found evidence for respiratory regulation at multiple timescales along the VRC, as well as a role for the preBötC in the integration of functionally disparate respiratory neurons.

Introduction

Over 100 years ago, it was postulated that a “noeud vital” was responsible for breathing (Flourens, 1824). More recently, the persistence of stable respiratory motor output in the neonate rodent brainstem-spinal cord preparation, and then in still more reduced slice and island preparations supported the conjecture that highly reduced networks located in the pre-Bötzinger Complex (preBötC) in ventrolateral medulla were sufficient to generate respiratory rhythm (Johnson et al., 2001, Smith and Feldman, 1987, Smith et al., 1991a). More recently, targeted bilateral lesions to the preBötC established the necessity of this structure for normal respiratory rhythm generation in otherwise intact animals (Gray et al., 1999, Tan et al., 2008). These studies support the consensus that respiratory rhythm generation arises out of the activity of functionally specialized, anatomically compact networks within the preBötC. The VRC is comprised of respiration-modulated neurons with a wide range of firing patterns. These networks mediate expiratory-inspiratory (E-I) and inspiratory-expiratory (I-E) transitions (Cohen, 1979); relay motor drive (Haxhiu et al., 2005); couple respiratory rhythm with cardiac rhythm (Garcia et al., 2013) as well as other rhythmic orofacial behaviors (Moore et al., 2013); and integrate afferent feedback from chemo- and mechanoreceptors (Feldman et al., 2003). Interactions between the preBötC and these functionally heterogeneous networks along the VRC are poorly understood.

The goal of this study was to gain a better understanding of the function of VRC networks by developing a macroscopic description of coupling relations along the ventral respiratory column (VRC), which in neonate rats extends over 1500 μm caudal to the facial nucleus (VIIn). To this end, serial optical recordings were made along the VRC using the sagittaly sectioned rat hindbrain preparation (Mellen and Funk, 2013), which exposes the column along its surface. Datasets were aligned using anatomical landmarks, and each neuron was described using unitary, neuron-system, and coupling attributes. These attributes were analyzed to infer anatomical and functional properties of VRC networks. On one hand, spatial averaging of individual attributes revealed functional parcellation consistent with identified anatomical structures. On the other hand, attributes were combined using cluster analysis to group neurons with similar attribute profiles, and these disparate groups overlapped anatomically. Some of the resulting groups were broadly distributed along the VRC and heterogeneous, and are interpreted to reflect the functional organization of the VRC. Other groups were homogeneous and anatomically compact, and thus could be interpreted as functional-anatomical structures. At the level of local networks, structured coupling between neuron pairs that spanned moderate and slow time scales was detected, and averaging of coupling relations revealed transient directed coupling at a macroscopic scale in the peri-inspiratory interval.

Section snippets

Brainstem preparation

In accordance with methods approved by the Institutional Animal Care and Use Committee, neonate Sprague-Dawley rat pups (P0-P3) were anesthetized with isoflurane. The neuraxis was then transected at the level of the cerebellar peduncles. Hindbrain and spinal cord were rapidly isolated in chilled aCSF made up of (in mM) 128.0 NaCl, 3.0 KCl, 1.2 CaCl2, 1.0 MgSO4, 21.0 NaHCO3, 0.5 NaH2PO4, and 30.0 glucose, equilibrated with 95% O2 5% CO2, to a pH = 7.4. The preparation was mounted on an attachment

Results

The sagittaly sectioned rat hindbrain preparation (SSRH) isolated from neonate (P0-P2) Sprague-Dawley rat pups exposes the VRC along its major axis (Fig. 1A). In this preparation respiratory motor output was recorded from ventral root C1 (gray trace Fig. 2B), together with optical recordings of neuronal activity (black traces, Fig. 2B), from which spike times were extracted (Fig. 1B). In 39 experiments, 946 neurons (neurons per experiment = 24.3 ± 9.8) were recorded over a region extending 400 μm

Discussion

Here, analysis of somatic Ca2+ transients obtained from a large and heterogeneous sample of neurons along the VRC was exploited to generate inferences about its functional organization. On one hand spatial averages of the central tendency of individual attributes revealed functional parcellation that corroborated known functional anatomical features along the VRC. On the other hand, cluster analysis identified six groups of neurons based on their shared and functionally interpretable attribute

Conflict of interest

Dr. Mellen is negotiating a licensing agreement with Explora Nova to disseminate the image acquisition and semi-automated ROI detection software used in this study.

Acknowledgements

Thanks to Drs. Muriel Thoby-Brisson, David Magnusson, and Alona Ben-Tal for their careful reading of drafts of this manuscript.

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