No abstract

To regulate the internal electrochemical environment of the body, the respiratory control system, including the brain, must detect ionic alterations in the intracellular and extracellular environment. At birth, most neonatal animals are capable of detecting changes in breathing‐related stimuli. However, the early development of this respiratory‐related chemosensitivity, as well as its role in maintaining developmental rhythms, is still unclear. Thus, we aim to describe the early embryonic development and maturation of pH chemosensitivity in the developing medulla oblongata of the altricial zebra finch. The zebra finch model allows breathing related rhythmic spontaneous neural activity (rSNA) to be recorded continuously from embryonic day 4 (E4), when it is undifferentiated, through hatching on E14, when air breathing has been established. The objective of this study is to test whether breathing related rhythms, observed during the embryonic period (E4–E10), and functional breathing rhythms, observed during the perinatal period (E11–E14), are responsive to alterations in extracellular pH (pHe). We hypothesized that changes in pHe will alter rSNA and breathing‐related frequency in an age‐dependent manner. To test our hypotheses, we employed electrophysiological recordings from the isolated medulla. We specifically targeted cranial nerve XI (accessory nerve), which innervates muscles in the neck, and is thought to expand and stiffen the airway during inspiration movements in birds. After baseline recordings were collected, the medullary tissue was exposed to low or high pHe via the bath superfusate. Following the low or high pHe treatment periods, we cleared the pHe solution with control aCSF to restore baseline conditions. We hypothesize that low pHe exposure will decrease respiratory‐related rhythmogenesis in animals younger than E11 and increase rhythmic activity in embryos older than E11. We also anticipate that high pHe exposure will have the opposite effect on the excitability of rhythmic activity. Interestingly, past studies have shown that neural circuits seem to transition at E11 and several breathing‐related phenotypes emerge at this age. Specifically, this transitional period is coincident with continuous air breathing in the developing zebra finch. Results show that young embryos (E4–E7) were unresponsive to low pHe exposure, while animals between E8 – E10 exhibited a decrease in rhythmic activity after exposure to low pHe. In contrast, animals older than E11 increased rhythmic activity in response to low pHe. When exposed to high pHe, all embryos between E4–10 exhibited an increase in rhythmic activity and older animals (E12–E14) responded by decreasing rhythmic activity. These data are consistent with our hypotheses and suggests that specific, specialized aspects of central breathing control gradually evolve over the incubation period. Future studies will address the mechanisms behind the observed changes in pHe chemosensitivity.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence (INBRE) Program Grant # P20 GM103408 (National Institute of General Medical Sciences) to JRW and CWL, and the National Institute of Neurological Disorders and Stroke via the Health Academic Research Enhancement Award (AREA) Program Grant # 1R15NS087521‐01A1 to JQP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The execution of rhythmic behaviors such as breathing requires proper construction of neural circuits during embryogenesis and throughout the perinatal period. A unique feature of developing circuits that may support neuronal circuit organization is the appearance and maintenance of rhythmic spontaneous neural activity (rSNA). Although rSNA has been studied extensively using a variety of in vitro preparations, rarely has rSNA been observed continuously from its onset until it contributes to mature behaviors. Little is known about how rSNA is maintained throughout the embryonic and perinatal periods due to difficulties accessing mammalian embryos and the delicate nature of embryonic tissue. To better understand these details, we used the zebra finch embryo brainstem preparation, and recorded rSNA and subsequent breathing rhythms continuously from embryonic day (E) 4 through hatching on E14. Our objective was to test whether breathing‐related rhythms during the aqueous embryonic period and aerial breathing rhythms during the perinatal period are sensitive to changes in CO2 (hypercapnia or hypocapnia). We hypothesized that hypercapnic and hypocapnic stimuli would alter rSNA frequency according to embryonic age. We predicted prenatal embryos (<E11) would show changes in rSNA that corresponded with generalized dampening or strengthening of neural activity based on whether pH was more acidic or alkaline, respectively. In non‐respiratory neurons, acidic stimuli typically lower activity and alkaline stimuli raise it. In contrast, we predicted the most mature, and continuously air breathing, embryos (>E11) would show respiratory‐related directional alterations in breathing‐related rhythm frequency similar to neonatal mammals exposed to hypercapnia or hypocapnia. To test our hypotheses, we superfused zebra finch brainstems with artificial cerebrospinal fluid equilibrated with control (90% O2, 5% CO2), hypercapnic (90% O2, 8% CO2), or hypocapnic (90% O2, 2% CO2) gases. During the hour‐long treatments, rSNA (<E11) or breathing‐related rhythms (>E11) were recorded from the spinal accessory nerve. Results suggest that CO2 modifies rSNA in younger embryos as well as breathing rhythms in older embryos, however the polarity of the response reversed near E11. Specifically, hypercapnia decreased rSNA frequency up to approximately 66 ± 14% of control frequency in prenatal embryos (<E11) and increased respiratory‐related neural activity frequency to approximately 112 ± 10% of control in perinatal embryos (>E11). Moreover, preliminary data from experiments where [NaHCO3] was raised such that pH was 7.7 show that hypocapnia increased the frequency of rSNA in E4–E11 embryos and leads to slow, irregular breathing‐related rhythms in E11–E14 embryos. These data indicate that the undifferentiated future respiratory circuits of the developing embryo are capable of sensing changes in CO2 and that this signal may serve to regulate rhythmic oscillations prior to air breathing. As predicted, near E11, when air breathing begins, the response to CO2 switches from the embryonic condition to the mature condition—consistent with CO2 effects in perinatal and newborn rodents and humans.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence (INBRE) Program Grant # P20 GM103408 (National Institute of General Medical Sciences) to JRW and CWL, and the National Institute of Neurological Disorders and Stroke via the Health Academic Research Enhancement Award (AREA) Program Grant # 1R15NS087521‐01A1 to JQP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.