Physiological and Morphological Characterization of Genetically Defined Classes of Interneurons in Respiratory Rhythm and Pattern Generation in Neonatal Mice

Breathing in mammals depends on an inspiratory-related rhythm that is generated by glutamatergic neurons in the preBotzinger complex (preBotC), a specialized site of the lower brainstem. Rhythm-generating preBotC neurons are derived from a single lineage that expresses the transcription factor (TF) Dbx1, but the cellular mechanisms of rhythmogenesis remain incompletely understood. to elucidate these mechanisms we comparatively analyzed Dbx1-expressing neurons (Dbx1 +) and Dbxl- neurons in the preBotC in knock-in transgenic mice. Whole-cell recordings in rhythmically active newborn mouse slice preparations showed that Dbx1 + neurons activate earlier in the respiratory cycle and discharge greater magnitude inspiratory bursts compared to Dbxl - neurons. Furthermore, Dbx1+ neurons required significantly less input current to discharge spikes (rheobase) in the context of network activity. The expression of intrinsic membrane properties indicative of A-current (IA) and hyperpolarization-activated current (Ih) was generally mutually exclusive in Dbx1 + neurons, which may indicate rhythmogenic function. In contrast, there was no such relationship in the expression of intrinsic currents I A and Ih in Dbxl- neurons. Confocal imaging and digital reconstruction of recorded neurons revealed dendritic spines on Dbxl- neurons, but Dbx1 + neurons were spineless. Dbx1 + neuron morphology was largely confined to the transverse plane whereas Dbxl- neurons projected dendrites to a greater extent in the parasagittal plane (rostrocaudally). A greater percentage of Dbx1 + neurons showed contralaterally projecting axons whereas Dbxl- neurons showed axons projecting in the rostral direction, which were severed by transverse cutting of the slice. Our data suggest that the rhythmogenic properties of Dbx1+ neurons include a higher level of intrinsic excitability that promotes burst generation in the context of network activity, which may be attributable to dendritic active properties that are recruited by excitatory synaptic transmission. Along with Dbxl, the TF Math1 has been shown to give rise to neurons that have important respiratory functions, including a potential role in coordinating the inspiratory and expiratory phases. to evaluate this role, we performed physiological and morphological characterizations of Math1+ neurons in transgenic mice and found that one out of six recorded Math1+ neurons showed expiratory activity. The expiratory Math1+ neuron appeared to be have a larger soma as well as a greater somatodendritic span in all axes (dorsal-ventral, medial-lateral and rostral-caudal) than the non-respiratory modulated Math1+ neurons. This suggests that respiratory modulated Math1+ neurons may be physiologically and morphologically specialized compared to non-rhythmic Mathl+ neurons. their larger morphological span and rhythmic expiratory modulation could be indicative of a function in coordinating phasic activity between inspiratory and expiratory oscillators. Although our findings are still preliminary, the data thus far are consistent with a hypothesized respiratory network model wherein the Math1+ neurons function in coordinating the pattern of inspiration and expiration. Identifying and characterizing hindbrain interneurons according to developmental genetic origins as well as physiological properties provides complementary information to help elucidate the cellular mechanisms underlying the generation and coordination of the respiratory rhythm

Computational modeling of spike generation in serotonergic neurons of the dorsal raphe nucleu

We consider here a single-compartment model of these neurons which is capable of describing many of the known features of spike generation, particularly the slow rhythmic pacemaking activity often observed in these cells in a variety of species. Included in the model are ten kinds of voltage dependent ion channels as well as calcium-dependent potassium current. Calcium dynamics includes buffering and pumping. In sections 3-9, each component is considered in detail and parameters estimated from voltage clamp data where possible. In the next two sections simplified versions of some components are employed to explore the effects of various parameters on spiking, using a systematic approach, ending up with the following eleven components: a fast sodium current $I_{Na}$, a delayed rectifier potassium current $I_{KDR}$, a transient potassium current $I_A$, a low-threshold calcium current $I_T$, two high threshold calcium currents $I_L$ and $I_N$, small and large conductance potassium currents $I_{SK}$ and $I_{BK}$, a hyperpolarization-activated cation current $I_H$, a leak current $I_{Leak}$ and intracellular calcium ion concentration $Ca_i$. Attention is focused on the properties usually associated with these neurons, particularly long duration of action potential, pacemaker-like spiking and the ramp-like return to threshold after a spike. In some cases the membrane potential trajectories display doublets or have kinks or notches as have been reported in some experimental studies. The computed time courses of $I_A$ and $I_T$ during the interspike interval support the generally held view of a competition between them in influencing the frequency of spiking. Spontaneous spiking could be obtained with small changes in a few parameters from their values with driven spiking.Comment: The abstract has been truncate

Rhythmogenic and Premotor Functions of Dbx1 Interneurons in the Pre-Bötzinger Complex and Reticular Formation: Modeling and Simulation Studies

Breathing in mammals depends on rhythms that originate from the preBötzinger complex (preBötC) of the ventral medulla and a network of brainstem and spinal premotor neurons. The rhythm-generating core of the preBötC, as well as some premotor circuits, consists of interneurons derived from Dbx1-expressing precursors but the structure and function of these networks remain incompletely understood. We previously developed a cell-specific detection and laser ablation system to interrogate respiratory network structure and function in a slice model of breathing that retains the preBötC, premotor circuits, and the respiratory related hypoglossal (XII) motor nucleus such that in spontaneously rhythmic slices, cumulative ablation of Dbx1 preBötC neurons decreased XII motor output by half after only a few cell deletions, and then decelerated and terminated rhythmic function altogether as the tally increased. In contrast, cumulatively deleting Dbx1 premotor neurons decreased XII motor output monotonically, but did not affect frequency nor stop functionality regardless of the ablation tally. This dissertation presents several network modeling and cellular modeling studies that would further our understanding of how respiratory rhythm is generated and transmitted to the XII motor nucleus. First, we propose that cumulative deletions of Dbx1 preBötC neurons preclude rhythm by diminishing the amount of excitatory inward current or disturbing the process of recurrent excitation rather than structurally breaking down the topological network. Second, we establish a feasible configuration for neural circuits including an Erdős-Rényi preBötC network and a small-world reticular premotor network with interconnections following an anti-preferential attachment rule, which is the only configuration that produces consistent outcomes with previous experimental benchmarks. Furthermore, since the performance of neuronal network simulations is, to some extent, affected by the nature of the cellular model, we aim to develop a more realistic cellular model based on the one we adopted in previous network studies, which would account for some recent experimental findings on rhythmogenic preBötC neurons

Mechanisms of rhythmic bursting involving Na+ and Ca2+ in excitatory networks of brainstem and spinal cord: a modeling study

The rhythmic, synchronized bursting of neurons in a network is an important phenomenon that underlies many rhythmic behaviors such as breathing, locomotion, feeding, etc. The basic mechanisms of rhythmic bursting in excitatory networks are currently under debate in some areas of the brainstem and spinal cord, and controversies exist regarding the role of network interactions and cellular properties in their generation. We focus on a specific controversy concerning the role of the membrane currents persistent sodium (INaP) and the calcium-activated nonspecific cation current (ICAN) in network rhythms existing in brainstem slices containing the preBotzinger complex (preBotC), an important preparation relevant for understanding respiratory rhythm generation. We also address another type of rhythm existing in some of these preparations which has been proposed as fictive sighing. Using ideas suggested by and data gathered from experiments in this field, we constructed a mathematical, physiologically realistic model, which includes a description of cellular properties such as INaP, ICAN, Ca2+ current (ICa), the Na+/K+ pump current (IPump), IP3-mediated intracellular Ca2+ release, inactivation of INaP and intracellular Ca2+ release, and network properties such as synaptic coupling and excitatory drive to the network. Using this model, we investigated how the behavior of an isolated neuron depends on the relative expression of these properties, and how the behavior of a network of coupled neurons depends on the distribution of these properties across the network. We show that the role of a particular current such as INaP or ICAN in a network rhythm may depend on this distribution, and on the conditions of excitatory drive to and the overall strength of synaptic interactions in the network, offering a possible resolution of the controversy regarding the role of these currents in respiratory rhythm generation. We also propose a model of fictive sighing. Although our model was designed to address a specific controversy in a preparation of the brainstem concerned with repiratory rhythmogenesis, we expect that some of our results may be applicable to understanding the basic mechanisms of rhythmic bursting in excitatory networks in other areas of the brainstem and spinal cord.Ph.D., Biomedical Engineering -- Drexel University, 201

A study of bursting in the preBotzinger Complex

The preBotzinger complex (PBC) of the mammalian brainstem is a heterogeneous neuronal network underlying the inspiration phase of the respiratory rhythm. Through excitatory synapses and a nontrivial network architecture, a synchronous, network-wide bursting rhythm emerges. On the other hand, during synaptic isolation, PBC neurons display three types of intrinsic dynamics: quiescence, bursting, or tonic activity. This work seeks to shed light on how the network rhythm emerges from the challenging architecture and heterogeneous population. Recent debate surrounding the role of intrinsically bursting neurons in the rhythmogenesis of the PBC inspires us to evaluate its role in a three-cell network. We found no advantage for intrinsically bursting neurons in forming synchronous network bursting; instead, intrinsically quiescent neurons were identified as a key mechanism. This analysis involved only studying the persistent sodium (NaP) current. Another important current for the PBC is the calcium-activated nonspecific cationic (CAN) current, which, when combined with a Na/K pump, was previously shown to be capable of producing bursts in coupled tonically active cells. In the second part of this study, we explore the interactions of the NaP and CAN currents, both currents are ubiquitous in the PBC. Using geometric singular perturbation theory and bifurcation analysis, we established the mechanisms through which reciprocally coupled pairs of neurons can generate various activity patterns. In particular, we highlighted how the NaP current could enhance the range of the strength of the CAN current for which bursts occur. We also were able to detail a novel bursting pattern seen in data, but not seen in previous models. With a foundation of understanding heterogeneity in the NaP and CAN currents, we again turned out attention to networks. For the third portion of the dissertation, we examine the effects that heterogeneity in the neuronal dynamics and coupling architecture can impose upon synchronous bursting of the entire network. We again found no significant advantage to including intrinsically bursting neurons in the network, and the best networks were characterized by an increased presence of quiescent neurons. We also described the way the NaP and CAN currents interact on the network scale to promote synchronous bursting

Multi-Scale Modeling of the Neural Control of Respiration

The generation of respiration in mammals begins in the lower brainstem where groups of neurons, that together comprise the respiratory central pattern generator (CPG), interact to produce a motor output that controls breathing. The pre-Bötzinger complex (pre-BötC) in the ventrolateral respiratory column (VRC) is believed to be a major contributor to rhythmic inspiratory activity that interacts with other neural compartments within the VRC as well as with other brainstem areas, including the pons. Though there has been a substantial push to understand the exact cellular and network mechanisms operating within the pre-BötC, as well as the way it is incorporated into the larger respiratory network, there is still much to be resolved. The overarching goal of the work presented in this dissertation is to contribute to our understanding of the neural control of respiration at several hierarchical levels. It is my hope that better insight into the complexities of these multiscale neural control mechanisms will provide a more complete framework for understanding various respiratory pathologies, and ultimately guide the development of novel therapies that will improve patient outcomes. I applied techniques from the fields of mathematics and computer science to develop computational models that reproduced results from electrophysiological recordings (done by our collaborators) and generated verifiable predictions. The scale of my modeling work encompasses the interaction of neurons in a single population, several interconnected populations of neurons that encompass the core of the mammalian respiratory network, and an integration of the respiratory network into a larger control system that includes afferent feedback loops. At each level I address specific, but related, topics that add to the general understanding of the neural control of respiration. The aims of my thesis address specific issues at each of the scales mentioned above. These issues may be summarized as follows: (i) the characteristic rhythmic bursting behavior observed in the pre-BötC, which was studied at the cellular levels with a particular interest in how this behavior impacts respiratory rhythmogenesis; (ii) a respiratory network connectome that defines interactions between several populations of neurons that together form the VRC, which produces an alternating pattern of inspiration, post-inspiration and expiration, and, how such a pattern may be affected by changes in chemical environment, e.g. elevated carbon dioxide or diminished oxygen concentrations; and (iii) the role of afferent feedback to the VRC, from the pons and lungs, which was studied in the context of respiratory phase switching mechanisms.Ph.D., Biomedical Engineering -- Drexel University, 201