To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states.DOI: http://dx.doi.org/10.7554/eLife.03427.001

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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 ...

Cerebral amyloid angiopathy (CAA), the deposition of β-amyloid (Aβ) in cerebrovascular walls, is the most common cause of lobar hemorrhagic stroke. Previous studies show that cerebrovascular Aβ induces expression and activation of matrix metalloproteinase 9 (MMP-9) in cerebral vessels of amyloid precursor protein (APP) transgenic mice. Here we extended these findings and evaluated MMP-9 expression in postmortem brain tissues of human CAA cases. MMP-9 co-localized with CAA, correlated with the severity of the vascular pathology, and was detected in proximity to microbleeds. We characterized a novel assay using longitudinal multi-photon microscopy and a novel tracer to visualize and quantify the magnitude and kinetics of hemorrhages in 3-dimensions in living mouse brains. We demonstrated that topical application of recombinant MMP-9 resulted in a time- and dose-dependent cerebral hemorrhage. APP mice with significant CAA developed more extensive hemorrhages which also appeared sooner after exposure to MMP-9. Our data suggest an important role for MMP-9 in development of hemorrhages in the setting of CAA. Inhibition of MMP-9 may present a preventive strategy for CAA-associated hemorrhage.