Chloride Modulates Central pH Sensitivity and Plasticity of Brainstem Breathing-Related Biorhythms in Zebra Finch Embryos.

All terrestrial vertebrate life must transition from aquatic gas exchange in the embryonic environment to aerial or pulmonary respiration at birth. In addition to being able to breathe air, neonates must possess functional sensory feedback systems for maintaining acid-base balance. Respiratory neurons in the brainstem act as pH sensors that can adjust breathing to regulate systemic pH. The central pH sensitivity of breathing-related motor output develops over the embryonic period in the zebra finch (Taeniopygia guttata). Due to the key role of chloride ions in electrochemical stability and developmental plasticity, we tested chloride's role in the development of central pH sensitivity. We blocked gamma-aminobutyric acid-A receptors and cation-chloride cotransport that subtly modulated the low-pH effects on early breathing biorhythms. Further, chloride-free artificial cerebrospinal fluid altered the pattern and timing of breathing biorhythms and blocked the stimulating effect of acidosis in E12-14 brainstems. Early and middle stage embryos exhibited rebound plasticity in brainstem motor outputs during low-pH treatment, which was eliminated by chloride-free solution. Results show that chloride modulates low-pH sensitivity and rebound plasticity in the zebra finch embryonic brainstem, but work is needed to determine the cellular and circuit mechanisms that control functional chloride balance during acid-base disturbances.

Inhibitory synaptic transmission is impaired in the Kölliker-Fuse of male, but not female, Rett syndrome mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that mainly affects females due to silencing mutations in the X-linked MECP2 gene. One of the most troubling symptoms of RTT is breathing irregularity, including apneas, breath-holds, and hyperventilation. Mice with silencing mutations in Mecp2 exhibit breathing abnormalities similar to human patients and serve as useful models for studying mechanisms underlying breathing problems in RTT. Previous work implicated the pontine, respiratory-controlling Kölliker-Fuse (KF) in the breathing problems in RTT. The goal of this study was to test the hypothesis that inhibitory synaptic transmission is deficient in KF neurons from symptomatic male and female RTT mice. We performed whole cell voltage-clamp recordings from KF neurons in acute brain slices to examine spontaneous and electrically evoked inhibitory post-synaptic currents (IPSCs) in RTT mice and age- and sex-matched wild-type mice. The frequency of spontaneous IPSCs was reduced in KF neurons from male RTT mice but surprisingly not in female RTT mice. In addition, electrically evoked IPSCs were less reliable in KF neurons from male, but not female, RTT mice, which was positively correlated with paired-pulse facilitation, indicating decreased probability of release. KF neurons from male RTT mice were also more excitable and exhibited shorter-duration action potentials. Increased excitability of KF neurons from male mice was not explained by changes in axon initial segment length. These findings indicate impaired inhibitory neurotransmission and increased excitability of KF neurons in male but not female RTT mice and suggest that sex-dependent mechanisms contribute to breathing problems in RTT.NEW & NOTEWORTHY Kölliker-Fuse (KF) neurons in acute brain slices from male Rett syndrome (RTT) mice receive reduced inhibitory synaptic inputs compared with wild-type littermates. In female RTT mice, inhibitory transmission was not different in KF neurons compared with controls. The results from this study show that sex-specific alterations in synaptic transmission occur in the KF of RTT mice.

Glucagon-Like Peptide-1 Receptors in the Gustatory Cortex Influence Food Intake.

The gustatory cortex (GC) region of the insular cortex processes taste information in manners important for taste-guided behaviors, including food intake itself. In addition to oral gustatory stimuli, GC activity is also influenced by physiological states including hunger. The specific cell types and molecular mechanisms that provide the GC with such abilities are unclear. Glucagon-like peptide 1 (GLP-1) is produced by neurons in the brain, where it can act on GLP-1 receptor-expressing (GLP-1R+) neurons found in several brain regions. In these brain regions, GLP-1R agonism suppresses homeostatic food intake and dampens the hedonic value of food. Here, we report in mice of both sexes that cells within the GC express Glp1r mRNA and further, by ex vivo brain slice recordings, that GC GLP-1R+ neurons are depolarized by the selective GLP-1R agonist, exendin-4. Next we found that chemogenetic stimulation of GLP-1R+ neurons, and also pharmacological stimulation of GC-GLP-1Rs themselves, both reduced homeostatic food intake. When mice were chronically maintained on diets with specific fat contents and then later offered foods with new fat contents, we also found that GLP-1R agonism reduced food intake toward foods with differing fat contents, indicating that GC GLP-1R influences may depend on palatability of the food. Together, these results provide evidence for a specific cell population in the GC that may hold roles in both homeostatic and hedonic food intake.SIGNIFICANCE STATEMENT The present study demonstrates that a population of neurons in the GC region of the insular cortex expresses receptors for GLP-1Rs, these neurons are depolarized by agonism of GLP-1Rs, and GC GLP-1Rs can influence food intake on their activation, including in manners depending on food palatability. This work is significant by adding to our understanding of the brain systems that mediate ingestive behavior, which holds implications for metabolic diseases.

Activation of orexin-2 receptors in the KÓ§lliker-Fuse nucleus of anesthetized mice leads to transient slowing of respiratory rate.

Orexins are neuropeptides originating from the hypothalamus that serve broad physiological roles, including the regulation of autonomic function, sleep-wake states, arousal and breathing. Lack of orexins may lead to narcolepsy and sleep disordered breathing. Orexinergic hypothalamic neurons send fibers to KÓ§lliker-Fuse (KF) neurons that directly project to the rostroventral respiratory group, and phrenic and hypoglossal motor neurons. These connections indicate a potential role of orexin-modulated KF neurons in functionally linking the control of wakefulness/arousal and respiration. In a reduced preparation of juvenile rats Orexin B microinjected into the KF led to a transient increase in respiratory rate and hypoglossal output, however Orexin B modulation of the KF in intact preparations has not been explored. Here, we performed microinjections of the Orexin B mouse peptide and the synthetic Orexin 2 receptor agonist, MDK 5220, in the KF of spontaneously breathing, isoflurane anesthetized wild type mice. Microinjection of Orexin-2 receptor agonists into the KF led to transient slowing of respiratory rate, which was more exaggerated in response to Orexin-B than MDK 5220 injections. Our data suggest that Orexin B signaling in the KF may contribute to arousal-mediated respiratory responses.

Development and regulation of breathing rhythms in embryonic and hatchling birds.

For many, if not all, air-breathing vertebrates, breathing-like movements begin while the embryo is still ensconced in an aqueous environment. This is because primordial regions of the CNS become spontaneously active during early gestation and then must functionally transform and specialize once air breathing commences. The degree to which the embryonic ventilatory control system is established and competent at birth is variable, however, even between different components of the respiratory system. Moreover, the embryological experiences of an individual can also affect the outcomes and responsiveness of ventilation to respiratory stimuli and these details have major clinical implications. The broad field of respiratory neurobiology still has much to learn about the ontogeny of breathing control systems, and the oviparity of birds provides a unique model to examine how early rhythms transform day-to-day as they become functional. This hybrid review and research article will highlight the contributions of birds to the study of breathing control during early development. We will detail what is currently known about the onset and maturation of respiratory rhythm generation and also provide novel data about the development of central chemosensitivity. Finally, we will review data regarding the development of peripheral afferent inputs during early development and discuss whole-animal reflex responsiveness to common respiratory stimuli, both chronic and acute, during the incubation period and following hatching.

Classroom-Based Research Experiences to Support Underserved STEM Student Success: From Introductory Inquiry to Optogenetics in the Embryonic Chicken.

In order to help overcome barriers to success for undergraduate STEM students from disadvantaged backgrounds, we developed two classroom-based research experiences (REs), Connecting Life (CL) and the Summer Research Institute (SRI). These REs were implemented over a two-year period (2014-2015) for regional community college students as part of the Southern Illinois Bridges to the Baccalaureate (SI Bridges) program. CL and SRI, broadly centered in biomedical sciences research, are designed to be offered in tandem. CL utilizes a guided inquiry approach with microscopy work-stations in experimental cell biology to experientially introduce research while building skills and confidence. CL serves as the gateway experience for the SRI, an intensive summer RE in which scholars engage in authentic research using modern technologies including optogenetics. We piloted the REs in year 1 (9 scholars) and made refinements in year 2 (10 scholars). Participants ("Bridges scholars") were enrolled full-time at one of two regional, rural community colleges, and came on-site to Southern Illinois University at Carbondale (SIUC) for the paid REs. Here we report the development, design and implementation of CL and the SRI, and report improved STEM research-related attitudes and aptitudes as a result of these experiences. Our findings suggest that guided inquiry with increasingly technical authentic research projects in a classroom-based and supportive learning community-style setting is a positive model for the transformation of underserved community college students into confident, motivated scientists with research-ready skills, and is likely translatable to other research novices.