Development of respiratory centers in the bullfrog tadpole brainstem

Thesis (Ph.D.) University of Alaska Fairbanks, 2017Among vertebrates, rhythmic motor behaviors such as breathing, swallowing, and sucking are controlled by rhythm generators or neural oscillators located at various sites in the medulla of the brainstem. That all vertebrates exhibit these behaviors, leads investigators to hypothesize common ancestry for the cellular networks responsible for homeostatic rhythm generation in the brainstem. While the locations and functions of rhythm generating sites controlling some of these behaviors have been well investigated, details regarding the development of these sites remain largely unknown. Recent work has suggested that neural oscillators in the rostral and caudal medulla, which contribute to ventilation in amphibians, may be homologous with those controlling breathing in mammals. I first investigated the developmental contributions of these regions to CO₂ sensitivity and rhythm generation in bullfrog tadpoles at different stages of metamorphosis. I then characterized the function and structure of a neural oscillator essential for lung rhythmogenesis in the tadpoles and compared it to similar oscillators in mammals. To investigate functional aspects of brainstem, I used a combination of single-unit and whole-nerve electrophysiology in the presence of pharmacological agents (neuronal receptor agonists and antagonists) or following removal of portions of the isolated brainstem of bullfrog tadpoles at different stages of metamorphosis. Structural studies were accomplished using immunohistochemistry, staining for phenotypic markers common to mammalian rhythmogenic sites, and assessing the difference between early and late metamorphic bullfrog tadpoles. Taken together, my results suggest that amphibians may indeed have a rhythmogenic site in the rostral medulla that is homologous to a mammalian rhythmogenic site; it is both structurally and functionally similar to the mammalian parafacial respiratory group/retrotrapezoid nucleus complex. This region undergoes structural and functional changes as tadpoles develop through metamorphosis. Understanding the development of respiration in amphibians may provide clues into the evolution and development of breathing in mammals

Lung breathing in the bullfrog: generating respiratory rhythm and pattern

Thesis (M.S.) University of Alaska Fairbanks, 2008This research investigated location of the lung respiratory rhythm generator (RRG) in the bullfrog brainstem using neurokinin-1 (NK1R) and [mu]opioid ([mu]OR) receptor colocalization and characterized the role of these receptors in breathing pattern formation. colocalization was distinct near the facial nucleus in juvenile bullfrogs but not in tadpoles. NK1R intensity exhibited no developmental change, while [mu]OR intensity increased from late-stage tadpoles to juvenile frogs. Substance P (NK1R agonist; bath applied) increased lung burst frequency, lung burst cycle frequency (BCF), episode frequency, lung burst amplitude and area, but decreased number of lung bursts per episode and lung burst duration. Antagonist D decreased lung burst frequency and BCF, episode frequency, and the number of lung bursts per episode, and increased lung burst duration and area. DAMGO ([mu]OR agonist; bath applied) decreased lung burst frequency and BCF, episode frequency, and number of lung bursts per episode, but increased all lung burst parameters. Naloxone ([mu]OR antagonist) increased lung burst frequency and BCF, episode frequency, lung bursts per episode but decreased all lung burst parameters. Together these results indicate that NK1R and [mu]OR colocalization represents the lung RRG, and that episode formation is intrinsic to the respiratory control network but may or may not originate in the RRG

A phylogenetic hypothesis for the origin of hiccough

Summary The occurrence of hiccoughs (hiccups) is very widespread and yet their neuronal origin and physiological significance are still unresolved. Several hypotheses have been proposed. Here we consider a phylogenetic perspective, starting from the concept that the ventilatory central pattern generator of lower vertebrates provides the base upon which central pattern generators of higher vertebrates develop. Hiccoughs are characterized by glottal closure during inspiration and by early development in relation to lung ventilation. They are inhibited when the concentration of inhaled CO 2 is increased and they can be abolished by the drug baclofen (an agonist of the GABA B receptor). These properties are shared by ventilatory motor patterns of lower vertebrates, leading to the hypothesis that hiccough is the expression of archaic motor patterns and particularly the motor pattern of gill ventilation in bimodal breathers such as most frogs. A circuit that can generate hiccoughs may persist in mammals because it has permitted the development of pattern generators for other useful functions of the pharynx and chest wall muscles, such as suckling or eupneic breathing

Astroglial Control of Respiratory Rhythm Generating Circuits

Astrocytes, the most numerous glial cells of the central nervous system, are well known to provide neuronal circuits with essential structural and metabolic support. There is also evidence that astrocytes may modulate the activities of neuronal circuits controlling motor rhythms including those of the brainstem’s preBötzinger complex (preBötC) that generates the rhythm of breathing in mammals. However, the extent and mechanisms of active astroglial control of the respiratory rhythm-generating circuits remain unknown. The morphological features of astrocytes in this critical brainstem region are also unknown. In this dissertation, viral gene transfer approaches designed to block or activate astroglial signaling pathways were used to determine the role of preBötC astrocytes in the control of breathing using in vitro and in vivo experimental models. Computer-aided morphometric analyses were used to investigate the structural features of brainstem astrocytes potentially contributing to their functional role. The results from these complementary, multi-faceted experiments show that (i) morphologically, preBötC astrocytes are larger, have more branches, and longer processes when compared to astrocytes residing in other regions of the brainstem; (ii) in conscious adult rats, blockade of vesicular release mechanisms or ATP-mediated signaling in preBötC astrocytes by virally-induced bilateral expression of either the light chain of tetanus toxin (TeLC), the dominant-negative SNARE proteins (dnSNARE), or a potent ectonucleotidase – transmembrane prostatic acid phosphatase – results in a significant reduction of resting respiratory frequency and frequency of sighs, augmented breaths that engage preBötC circuits to increase inspiratory effort; (iii) hypoxic- and CO2-induced ventilatory responses are significantly reduced when vesicular release mechanisms in preBötC astrocytes are blocked; (iv) activation of preBötC astrocytes expressing Gq-coupled Designer Receptor Exclusively Activated by Designer Drug is associated with higher frequency of both normal inspirations and sighs; (v) blockade of vesicular release mechanisms (expression of TeLC or dnSNARE) in preBötC astrocytes is associated with a dramatic reduction of exercise capacity. These data suggest that astroglial mechanisms involving exocytotic vesicular release of signaling molecules (gliotransmitters), provides tonic excitatory drive to the inspiratory rhythm-generating circuits of the preBötC and contributes to the generation of sighs. The role of preBötC astrocytes in central nervous mechanisms controlling breathing becomes especially important in conditions of metabolic stress requiring homeostatic adjustments of breathing such as systemic hypoxia, hypercapnia, and exercise, when enhanced respiratory efforts are critical to support physiological and behavioral demands of the body

Impact des minéralocorticoïdes, glucocorticoïdes et de l'hormone thyroïdienne sur le développement respiratoire de Lithobates catesbeianus

L’émergence de la respiration aérienne au cours du développement de l’amphibien demande d’importants changements au niveau du circuit cérébral qui génère et régule le rythme respiratoire. La métamorphose étant influencée par différentes hormones métamorphiques, nous avons testé l’hypothèse proposant qu’exposer le cerveau à ces hormones augmenterait le rythme respiratoire aérien fictif chez les têtards de Lithobates catesbeianus. Nous utilisons une technique d’isolation du tronc cérébrale qui est ensuite exposé à différentes hormones, soit l’aldostérone, la corticostérone et l’hormone T3. En comparant les préparations exposées aux hormones sur 24h avec les contrôles, nous démontrons que l’exposition aux hormones augmente de manière générale la respiration aérienne. La ventilation fictive des préparations exposées aux hormones présentait des similarités avec celles provenant d’adultes. Nous concluons que, grâce à leurs effets à long terme, les hormones régulant la métamorphose peuvent déclencher la maturation des circuits neuronaux qui génèrent et régulent la respiration de cette espèce.The emergence of air breathing during amphibian development requires significant changes to the brainstem circuits that generate and regulate breathing. Because this metamorphosis is regulated by metamorphic hormones, we tested the hypothesis that exposing the brainstem to these hormones augments the fictive air breathing frequency in Lithobates catesbeianus tadpoles. Brainstems were isolated from pre-metamorphic tadpoles and a hormone exposure was made with the following hormones: aldosterone, corticosterone, T3. By comparison with preparations subjected to sham treatment, hormone exposure generally increased fictive air breathing frequency. Brainstem preparations showed a response to a change in the aCSF pH only before the 24h incubation, suggesting a chemosensitivity lost following the incubation. However, the patterns of the exposed brainstem presented an activity that is reminiscent of those observed in adult frogs. We conclude that hormones play an important role in the maturation of the neural circuits that generate and regulate breathing in this species

A common role for astrocytes in rhythmic behaviours?

Authors acknowledge the Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791) and the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M021793/1) for their funding and support.Astrocytes are a functionally diverse form of glial cell involved in various aspects of nervous system infrastructure, from the metabolic and structural support of neurons to direct neuromodulation of synaptic activity. Investigating how astrocytes behave in functionally related circuits may help us understand whether there is any conserved logic to the role of astrocytes within neuronal networks. Astrocytes are implicated as key neuromodulatory cells within neural circuits that control a number of rhythmic behaviours such as breathing, locomotion and circadian sleep-wake cycles. In this review, we examine the evidence that astrocytes are directly involved in the regulation of the neural circuits underlying six different rhythmic behaviours: locomotion, breathing, chewing, gastrointestinal motility, circadian sleep-wake cycles and oscillatory feeding behaviour. We discuss how astrocytes are integrated into the neuronal networks that regulate these behaviours, and identify the potential gliotransmission signalling mechanisms involved. From reviewing the evidence of astrocytic involvement in a range of rhythmic behaviours, we reveal a heterogenous array of gliotransmission mechanisms, which help to regulate neuronal networks. However, we also observe an intriguing thread of commonality, in the form of purinergic gliotransmission, which is frequently utilised to facilitate feedback inhibition within rhythmic networks to constrain a given behaviour within its operational range.PostprintPeer reviewe