Developmental, Physiological, and Transcriptomic Analyses of Neurons involved in the Generation of Mammalian Breathing

Breathing is a rhythmic motor behavior with obvious physiological importance: breathing movements are essential for respiration, which sustains homeostasis and life itself in a wide array of animals including humans and all mammals. The breathing rhythm is produced by interneurons of the brainstem preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1. However, the cellular and synaptic neural mechanisms underlying respiratory rhythmogenesis remain unclear. The first chapter of this dissertation examines a Dbx1 transgenic mouse line often exploited to study the neural control of breathing. It emphasizes the cellular fate of progenitors that express Dbx1 at different times during development. I couple tamoxifen-inducible Dbx1 Cre-driver mice with Cre-dependent reporters, then show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Further, I quantify the temporal assemblage of Dbx1 neurons and glia in the preBötC and provide practical guidance on breeding and tamoxifen administration strategies to bias reporter protein expression toward neurons (or glia), which can aid researchers in targeting studies to unravel their functions in respiratory neurobiology. The second chapter of this dissertation exploits the mouse model characterized in the first chapter and then focuses on mechanisms of respiratory rhythmogenesis. The breathing cycle consists of inspiratory and expiratory phases. Inspiratory burst-initiation and burst-sustaining mechanisms have been investigated by many groups. Here, I specifically investigate the role of short-term synaptic depression in burst termination and the inspiratory-expiratory phase transition using rhythmically active medullary slice preparations from Dbx1 Cre-driver mice coupled with channelrhodopsin reporters. I demonstrate the existence of a post- inspiratory refractory period that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1-derived preBötC neurons. I show that postsynaptic factors cannot account for the refractory period, and that presynaptic vesicle depletion most likely underlies the refractory period. The third chapter of this dissertation focuses on transcriptomic analysis of Dbx1 preBötC neurons, and differences in gene expression between Dbx1-derived and non- Dbx1-derived preBötC neurons. I analyze and quantify the expression of over 20,000 genes, and make the raw data publicly available for further analysis. I argue that this full transcriptome approach will enable our research group (and others) to devise physiological studies that target specific subunits and isoforms of ion channels and integral membrane proteins to examine the role(s) of Dxb1- derived neurons and glia at the molecular level of breathing behavior. In addition to predictable gene candidates (such as ion channels, etc) this transcriptome analysis delivers unanticipated novel gene candidates that can be investigated in future respiratory physiology experiments. Knowing the site (preBötC) and cell class (Dbx1) at the point of origin of respiration, this dissertation provides tools and specific investigations that advance understanding of the neural mechanisms of breathing

Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBotzinger Complex in Neonatal Mice

The brainstem preBotzinger complex (preBotC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx1-expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory-expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that afferent axonal projections to Dbx1 preBotC neurons undergo activity-dependent depression and we identify a refractory period (similar to 2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 preBotC neurons. We demonstrate that the duration of the refractory period-but neither the cycle period nor the magnitude of endogenous inspiratory bursts-is sensitive to changes in extracellular Ca2+. Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca2+. Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx1 preBotC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis

Fate mapping neurons and glia derived from Dbx1-expressing progenitors in mouse preBotzinger complex

The brainstem preBotzinger complex (preBotC) generates the inspiratory breathing rhythm, and its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors. To study the neural bases of breathing, tamoxifen-inducible Cre-driver mice and Cre-dependent reporters are used to identify, record, and perturb Dbx1 preBotC neurons. However, the relationship between tamoxifen administration and reporter protein expression in preBotC neurons and glia has not been quantified. To address this problem, we crossed mice that express tamoxifen-inducible Cre recombinase under the control of the Dbx1 gene (Dbx1(CreERT2)) with Cre-dependent fluorescent reporter mice (Rosa26(tdTomato)), administered tamoxifen at different times during development, and analyzed tdTomato expression in the preBotC of their offspring. We also crossed Rosa26(tdTomato) reporters with mice that constitutively express Cre driven by Dbx1 (Dbx1(Cre)) and analyzed tdTomato expression in the preBotC of their offspring for comparison. We show that Dbx1-expressing progenitors give rise to preBotC neurons and glia. Peak neuronal tdTomato expression occurs when tamoxifen is administered at embryonic day 9.5 (E9.5), whereas tdTomato expression in glia shows no clear relationship with tamoxifen timing. These results can be used to bias reporter protein expression in neurons (or glia). Tamoxifen administration at E9.5 labels 91% of Dbx1-derived neurons in the preBotC, yet only 48% of Dbx1-derived glia. By fate mapping Dbx1-expressing progenitors, this study illustrates the developmental assemblage of Dbx1-derived cells in preBotC, which can be used to design intersectional Cre/lox experiments that interrogate its cellular composition, structure, and function

Identification of the Pre-Botzinger Complex Inspiratory Center in Calibrated “Sandwich” Slices from Newborn Mice with Fluorescent Dbx1 Interneurons

Inspiratory active pre‐Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow‐up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1‐derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0–4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging‐based studies, as well as the application of other Cre‐dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory‐related oscillations of preBötC Dbx1‐derived neurons and thus breathing

Transcriptome of Neonatal PreBotzinger Complex Neurones in Dbx1 Reporter Mice

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBotzinger Complex (preBotC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBotC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBotC neurons express kappa-opioid receptors in addition to mu-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBotC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O-2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBotC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBotC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development

Using historical accounts of harpsichord touch to empirically investigate the production and perception of dynamics on the 1788 Taskin.

This article investigates the extent of production and perception of dynamic differences on a French historical harpsichord, extensively revised in 1788 by Pascal Taskin. A historical review reports on the descriptions of two different types of touch found in treatises of the 18th century. These two touches (loud/struck and soft/pressed) were used to perform single tones on the lower, upper, peau de buffle (PDB) registers (the last of which Taskin is credited with having invented) and the coupled 8-foot registers to investigate differences in dynamics. Acoustic measurements show varied differences of up to 11 dB for the two types of touch over different pitches in each register. The strongest difference is measured in the first harmonic of note F2 on the PDB. A listening experiment was conducted to test whether these differences are perceivable. Participants performed a discrimination task using pairs of single tones. Participants were able to perform significantly better than chance in correctly identifying whether pairs of single tones were same or different with respect to loudness [t(24) = 12.01, p < 0.001]. Accuracies were influenced by pitch and register, the PDB providing the strongest accuracies over the four registers tested

The role of glutamate in transmission of the respiratory rhythm onto cranial motor neurons in the American bullfrog (lithobates catesbeinana)

Bibliography: p. 65-73Includes copy of animal protocol approval. Original copy with original Partial Copyright Licence

A History of the Harpsichord – Insert

Insert from the book "A History of the Harpsichord"