Studies of Respiratory Rhythm Generation Maintained in Organotypic Slice Cultures

Breathing is an important rhythmic motor behavior whose underlying neural mechanisms can be studied in vitro. The study of breathing rhythms in vitro has depended upon reduced preparations of the brainstem that both retain respiratory-active neuronal populations and spontaneously generate respiratory-related motor output from cranial and spinal motor nerves. Brainstem-spinal cord en bloc preparations and transverse medullary slices of the brainstem have greatly improved the ability of researchers to experimentally access and thus characterize interneurons important in respiratory rhythmogenesis. These existing in vitro preparations are, however, not without their limitations. For example, the window of time within which experiments may be conducted is limited to several hours. Moreover, these preparations are poorly suited for studying subcellular ion channel distributions and synaptic integration in dendrites of rhythmically active respiratory interneurons because of tortuous tissue properties in slices and en bloc, which limits imaging approaches. Therefore, there is a need for an alternative experimental approach. Acute transverse slices of the medulla containing the preBötzinger complex (preBötC) have been exploited for the last 25 years as a model to study the neural basis of inspiratory rhythm generation. Here we transduce such preparations into a novel organotypic slice culture that retains bilaterally synchronized rhythmic activity for up to four weeks in vitro. Properties of this culture model of inspiratory rhythm are compared to analogous acute slice preparations and the rhythm is confirmed to be generated by neurons with similar electrophysiological and pharmacological properties. The improved optical environment of the cultured brain tissue permits detailed quantitative calcium imaging experiments, which are subsequently used to examine the subcellular distribution of a transient potassium current, IA, in rhythmically active preBötC interneurons. IA is found on the dendrites of these rhythmically active neurons, where it influences the electrotonic properties of dendrites and has the ability to counteract depolarizing inputs, such as post-synaptic excitatory potentials, that are temporally sparse in their occurrence (i.e., do not summate). These results suggest that excitatory input can be transiently inhibited by IA prior to its steady-state inactivation, which would occur as temporally and spatially summating synaptic inputs cause persistent depolarization. Thus, rhythmically active interneurons are equipped to appropriately integrate the activity state of the inspiratory network, inhibiting spurious inputs and yet yielding to synaptic inputs that summate, which thus coordinates the orderly recruitment of network constituents for rhythmic inspiratory bursts. In sum, the work presented here demonstrates the viability and potential usefulness of a new experimental model of respiratory rhythm generation, and further leverages its advantages to answer questions about dendritic synaptic integration that could not previously be addressed in the acute slice models of respiration. We argue that this new organotypic slice culture will have widespread applicability in studies of respiratory rhythm generation

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

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-Botzinger complex (preBotC) 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 preBotC 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 similar 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

Olfactory Inputs Modulate Respiration-Related Activity In The Prefrontal Cortex And Fear Behavior

Voluntary control of respiration, especially via rhythmic nasal breathing, alleviates negative feelings such as fear and is used clinically to manage certain types of panic attacks. However, the neural substrates that link nasal breathing to fear circuits remains unknown. Here we show that during conditioned fear-induced freezing behavior, mice breathe at a steady rate (~4 Hz) which is strongly correlated with a predominant 4 Hz oscillation observed in the olfactory bulb and the prelimbic prefrontal cortex (plPFC), a structure critical for the expression of conditioned fear behaviors. We demonstrate anatomical and functional connectivity between the olfactory pathway and plPFC via circuit tracing and optogenetic approaches. Disrupting olfactory inputs significantly reduces the 4 Hz oscillation in the plPFC suggesting that respiration-related signals from the olfactory system play a role in entraining this fear-related signal. Surprisingly, we find that without olfactory inputs, freezing times are significantly prolonged. Collectively, our results indicate that olfactory inputs modulate rhythmic activity in fear circuits and suggest a neural pathway that may underlie the behavioral benefits of respiration-entrained olfactory signals

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

Cellular And Molecular Insight Into Autonomic Function And Dysfunction

The autonomic nervous system (ANS) controls several vital functions of the body, especially the autonomic regulation of respiratory and cardiovascular systems. Dysfunction of either can be life-threatening. Some of cellular and molecular mechanisms underlying the respiratory and cardiovascular dysfunction is more critical and general. The demonstration of such general processes not only may help the understanding of etiology and pathophysiology of the diseases, but also suggests potential therapeutic modalities for the diseases. Severe breathing disorders including high apnea rate and breathing irregularity are found in Rett syndrome (RTT). In a novel rat model of RTT, we compared rat physical condition and behaviors with traditional mouse models of RTT. We found that the novel Mecp2−/Y rat model as an alternative RTT model recapitulated numerous RTT-like symptoms. To uncover the neuronal mechanisms underlying the RTT respiratory disorders, we performed in vivo recording from brainstem neurons in ventral respiratory column (VRC). Excessive activity of both inspiratory and expiratory neurons as well as ectopic discharge of phrenic nerve were detected in null rats. Such defects were likely caused by hyperexcitability of respiratory neurons due to inadequate synaptic inhibition necessary for phase switching. Then we took the GABAergic intervention to hyperexcitability of respiratory neurons, and successfully corrected the defects in neuronal firing patterns as well as the RTT breathing phenotypes. Similarly, change of cellular excitability was also observed in diabetic vascular complications. A critical player for the membrane excitability of vascular smooth muscle cells (VSMCs) is the KATP channel that is strongly suppressed by methylglyoxal (MGO) known to be overly produced with persistent hyperglycemia. The elevated level of microRNA (miR)-9a-3p contributed to the down-regulation of vascular KATP channels. miR-9a-3p inhibition using antisense oligonuecleotides corrected the dysfunction of KATP channels. Since VSMC membrane excitability plays an important role in vascular tone regulation, we generated a new strain of transgenic Tagln-ChR mouse model and demonstrate an alterative to manipulate VSMC membrane excitability and vascular tone using optogenetic approaches. Thus several molecular targets in cardiorespiratory system have been demonstrated underlying membrane excitability and the developments of several disease conditions in this thesis study

Autonomic dysfunction in epilepsy mouse models with implications for SUDEP research

Epilepsy has a high prevalence and can severely impair quality of life and increase the risk of premature death. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in drug-resistant epilepsy and most often results from respiratory and cardiac impairments due to brainstem dysfunction. Epileptic activity can spread widely, influencing neuronal activity in regions outside the epileptic network. The brainstem controls cardiorespiratory activity and arousal and reciprocally connects to cortical, diencephalic, and spinal cord areas. Epileptic activity can propagate trans-synaptically or via spreading depression (SD) to alter brainstem functions and cause cardiorespiratory dysfunction. The mechanisms by which seizures propagate to or otherwise impair brainstem function and trigger the cascading effects that cause SUDEP are poorly understood. We review insights from mouse models combined with new techniques to understand the pathophysiology of epilepsy and SUDEP. These techniques include in vivo, ex vivo, invasive and non-invasive methods in anesthetized and awake mice. Optogenetics combined with electrophysiological and optical manipulation and recording methods offer unique opportunities to study neuronal mechanisms under normal conditions, during and after non-fatal seizures, and in SUDEP. These combined approaches can advance our understanding of brainstem pathophysiology associated with seizures and SUDEP and may suggest strategies to prevent SUDEP

Suprapontine Structures Modulate Brainstem and Spinal Networks

Several spinal motor output and essential rhythmic behaviors are controlled by supraspinal structures, although their contribution to neuronal networks for respiration and locomotion at birth still requires better characterization. As preparations of isolated brainstem and spinal networks only focus on local circuitry, we introduced the in vitro central nervous system (CNS) from neonatal rodents to simultaneously record a stable respiratory rhythm from both cervical and lumbar ventral roots (VRs). Electrical pulses supplied to multiple sites of brainstem evoked distinct VR responses with staggered onset in the rostro-caudal direction. Stimulation of ventrolateral medulla (VLM) resulted in higher events from homolateral VRs. Stimulating a lumbar dorsal root (DR) elicited responses even from cervical VRs, albeit small and delayed, confirming functional ascending pathways. Oximetric assessments detected optimal oxygen levels on brainstem and cortical surfaces, and histological analysis of internal brain structures indicated preserved neuron viability without astrogliosis. Serial ablations showed precollicular decerebration reducing respiratory burst duration and frequency and diminishing the area of lumbar DR and VR potentials elicited by DR stimulation, while pontobulbar transection increased the frequency and duration of respiratory bursts. Keeping legs attached allows for expressing a respiratory rhythm during hindlimb stimulation. Trains of pulses evoked episodes of fictive locomotion (FL) when delivered to VLM or to a DR, the latter with a slightly better FL than in isolated cords. In summary, suprapontine centers regulate spontaneous respiratory rhythms, as well as electrically evoked reflexes and spinal network activity. The current approach contributes to clarifying modulatory brain influences on the brainstem and spinal microcircuits during development

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

Phenotypic properties and intrinsic currents of neurons involved in the neural generation of mammalian breathing

Breathing is essential for mammalian life. Although there is an emerging consensus that the inspiratory respiratory rhythm is generated in a lower brainstem region known as the preBotzinger Complex (preBotC), the mechanism of rhythmogenesis is still unclear. Additionally, the modulation of intrinsic currents within preBotC neurons has yet to be fully elucidated. This dissertation addresses both of these issues and relies on imaging, electrophysiological, and modeling techniques. The first chapter examines the size and composition of the preBotC. The chapter also decribes the means by which substance P (SP) excites the vast majority of preBotC neurons by illustrating the characteristics of the SP-activated current (/SP) in these neurons. In the subsequent chapter, we characterize a voltage-dependent potassium current that is involved in maintaining stable rhythms during normal fictive breathing. The third chapter presents a mathematical model of heterogeneous and rhythmogenic neurons that initiate network bursts. We show how this behavior relies on feedback synaptic connections within the network that reinforces activity, i.e., recurrent-excitation. We also compare model results to experimental data and make testable predictions. The final chapter elaborates on the discussion of /SP from the first chapter and presents evidence suggesting that a cyclic adenosine monophosphate (cAMP)-modulated non-specific cation channel may account for the depolarizing response in preBotC neurons from several neuromodulators. Altogether, this dissertation advances the field\u27s understanding on several fronts. We have distinguished possible functional roles of neurons from electrophysiological characteristics, estimated the number of neurons necessary for rhythmogenesis, characterized /SP , and clarified the distribution of SP-sensitive receptors among inspiratory neurons. We have identified and characterized a voltage-dependent potassium currrent important for inspiratory activity and analyzed its role. We have also described in detail how rhythmic bursts form from recurrent excitation and how this relates to experimental data. Finally, we have identified and begun characterizing a potentially important and novel mechanism for the modulation of membrane potentials in critical inspiratory neurons