Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

Abstract

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Bötzinger Complex (preBötC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBötC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 ± 45 neurons stopped spontaneous respiratory rhythm, and our data suggest ≈82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBötC can sustain insults that destroy no more than ≈18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.central pattern generator | calcium imaging | brainstem C entral pattern generator (CPG) networks produce coordinated motor patterns that underlie behaviors such as breathing, locomotion, and chewing (1, 2). A key issue is not just which cell types generate these patterns of activity, but how many? This question pertains to whether and how cumulative cell loss will impair and, possibly preclude, network function.Breathing consists of inspiratory and expiratory phases. The preBötzinger Complex (preBötC) of the medulla putatively contains the inspiratory CPG (3, 4). Over several days in vivo, saporinmediated destruction of preBötC neurons that express neurokinin-1 receptors (NK1Rs) causes sleep-disordered breathing and fatal respiratory pathology (5, 6). Similarly, acute cell-silencing of a subset of the same NK1R-expressing population of preBötC neurons stops spontaneous breathing in awake adult rats (7). These studies helped to determine the cellular composition of the preBötC and confirmed that it was essential for breathing in an otherwise intact animal, but could not measure the resiliency of the preBötC when faced with silencing or deleting its core piecewise.We examined this issue by using slice preparations that capture the preBötC and spontaneously generate fictive breathing-related activity in vitro. We developed a computer-automated system that detects rhythmically active interneurons via two-photon calcium imaging, stores their physical locations in memory, and then laser ablates the targets sequentially, while electrophysiologically monitoring the motor output of the circuit. We applied this system to the preBötC to test the hypothesis that piecewise destruction of the CPG core would cause graded loss of motor activity up to rhythm cessation. We surmised that this effect ...

Robust network oscillations during mammalian respiratory rhythm generation driven by synaptic dynamics

“…Normalizing the number of lesioned neurons to the total required for complete rhythm cessation resulted in exponential increases in the CPs and variability (Fig. 2Ac), comparable to those obtained in previous physiological in vitro experiments (20,25). The proportion of lesioned neurons required to stop bursting is nevertheless higher than observed experimentally in vitro (Discussion and SI Appendix, Fig.…”

“…These electrophysiological data match the predicted model dynamics of I syn in Fig. 1E and would suggest that, together with the rest of the favorable modeling tests performed here recapitulating experimental results (20,26,28,34), the modeling approach we exercised is applicable to the respiratory rhythm-generating network in vitro.…”

“…This study provides a generally applicable mechanism for other central pattern generator systems that are less well understood. connected neurons with dynamic synapses, we simulated the membrane potential (V) in 400 neurons (Materials and Methods), comparable to the number of preBötC rhythm-generating neurons (20). For each neuron, the key active properties are the RP and RRP for synaptic vesicles (Fig.…”

KCC2-mediated regulation of respiration-related rhythmic activity during postnatal development in mouse medulla oblongata

“…First, the brainstem-spinal cord preparation includes many respiratory-related regions (Alheid and McCrimmon, 2008;Carroll and Agarwal, 2010;Smith et al, 2007), whereas, the medullary slice preparation is more suitable for the elucidation of the role of each nucleus involved in respiration in the medulla, specifically (Hayes et al, 2012;Janczewski et al, 2013;Koizumi et al, 2008;Sebe and Berger, 2008). Second, even though the blocking action of DIOA appears to be more specific to KCC2 than that of furosemide, it is unlikely that DIOA would completely block the activity of all KCC2 proteins in the slice preparations.…”

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice

“…The destruction of ∼120 rhythmic neurons irreversibly stopped respiratory rhythmogenesis (Hayes et al, 2012). However, inspiratory-modulated preBötC neurons may be excitatory or inhibitory.…”