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

Nicotine exposure in the developing bullfrog: influences on neuroventilatory responses to CO₂

Thesis (M.S.) University of Alaska Fairbanks, 2008Developmental exposure to the neuroteratogen nicotine may affect ventilatory responses to hypercapnia. Developmental changes in normocapnic and hypercapnic neuroventilation of the isolated bullfrog brainstem preparation have been previously characterized. I investigated the effect of 3- and 10-wk chronic nicotine (30 [mu]g/L) exposure on lung burst frequency exhibited by early and late metamorphic bullfrog tadpoles during normocapnia (1.5 % CO₂) and hypercapnia (5.0 % CO₂). Chronic nicotine exposure impairs the hypercapnic neuroventilatory response of early metamorphic tadpoles following both 3- and 10-wk exposure. Late metamorphic tadpoles demonstrated an impaired hypercapnic neuroventilatory response only after 10-wk exposure. Chronic nicotine exposure had no effect on normocapnic neuroventilation. Brainstem preparations from early and late metamorphic tadpoles and juvenile bullfrogs were exposed acutely to 18 [mu]g/L nicotine. Acute nicotine had no effect on normocapnic or hypercapnic neuroventilation of early metamorphic tadpoles. Late metamorphic tadpoles and juvenile bullfrogs demonstrated depressed normocapnic neuroventilation in response to acute nicotine exposure, while late metamorphic tadpole brainstems responded significantly to hypercapnia during acute exposure. This suggests that bullfrogs have a differential response to acute nicotine exposure that increases with development. Collectively these data suggest that the consequences of developmental nicotine exposure differ between acute and chronic exposure and throughout bullfrog development.1. Timing and duration of developmental nicotine exposure contribute to attenuation of the tadpole hypercapnic response -- 2. Nicotine affects the normocapnic and hypercapnic neuroventilation of bullfrogs in a developmental stage dependent manner -- General conclusions -- Literature cited

Central CO2 chemosensitivity in tadpoles: impairment and the role of serotonin

Thesis (M.S.) University of Alaska Fairbanks, 2012Nicotine and ethanol are known neuroteratogens and prenatal exposure correlates with Sudden Infant Death Syndrome (SIDS), which is thought to result from failure to maintain pH homeostasis through respiratory adjustments. This failed homeostatic control is believed to be serotonergic in origin. In previous studies nicotine or ethanol exposure ablated the robust hypercapnic response of early-stage tadpoles. These findings lead us to question if the ablation occurred through a serotonindependent mechanism. This study investigated the role of serotonin (5- HT) in the nicotine- or ethanol-induced abolishment of the hypercapnic response. We found that toxin-exposed animals were insensitive to hypercapnia and also failed to respond to concomitant exposure to hypercapnia and 8-OH-DPAT, supporting our hypothesis that toxininduced abolishment of the hypercapnic response is mediated by 5-HTia receptors. Immunofluorescence data from brainstem slices of ethanolexposed animals showed a decrease in 5-HTia receptors and the serotonin-synthesizing enzyme tryptophan hydroxylase. In contrast, 3- wk nicotine-exposed animals displayed no significant difference in immunofluorescence for either protein. Taken together the electrophysiological and immunofluorescence data suggest the effects of ethanol or nicotine exposure, which impair the hypercapnic response, include a failure of serotonergic signaling and that this failure is not simply the reflection of a global reduction in serotonin levels

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

Neuroplasticity And Neurotoxicology: Central Breathing Control Following Developmental Nicotine Or Ethanol Exposure

Thesis (Ph.D.) University of Alaska Fairbanks, 2010Nicotine or ethanol exposure early in development are both risk factors for Sudden Infant Death Syndrome (SIDS). I tested the hypothesis that both nicotine and ethanol may be linked to SIDS by impairing central breathing control responses to low oxygen (hypoxia) and high carbon dioxide (hypercapnia) stressors. Experiments were conducted in bullfrog tadpoles, a model system for respiratory neurotoxicology research. I addressed three specific aims: to characterize the effect of chronic ethanol on central responses to hypercapnia and hypoxia, to characterize the effect of chronic nicotine on central hypoxic responses, and to determine the persistence of hypercapnic impairments following 10-wk exposure to either nicotine or ethanol. 10-wk nicotine exposure resulted in neuroplastic changes that eliminated the central hypoxic responses of early but not late metamorphic tadpoles. Thus, central responses to both hypoxia and hypercapnia were impaired following nicotine exposure. The attenuated central hypercapnic response of nicotine-exposed tadpoles persisted for 1 - 3 wk. Following 10-wk chronic ethanol exposure central responses to hypercapnia and hypoxia were lost regardless of the developmental timing of exposure. Impairments in central hypercapnic responses persisted for 3 - 6 wk after ethanol exposure ended. The recovery of central hypercapnic responses in nicotine- and ethanol-exposed tadpoles may be an example of recuperative neuroplasticity resulting in either a reinstatement of network components and functions or an accommodation to deleterious nicotine- and ethanol-evoked neuroplastic changes. Collectively these data suggest that both nicotine and ethanol may target adaptive and compensatory mechanisms in central breathing control. The teratogen-induced impairments were developmentally dependent in the case of nicotine, and they persisted longer following ethanol exposure. The overall result of exposure to either neuroteratogen was an inability to respond to central breathing stressors, supporting the possible link to SIDS

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