Projects | Funding | Techniques

Projects

Our laboratory is interested in the cellular and molecular mechanisms by which cells sense changes in O2, CO2, and pH, and make appropriate physiological responses. For example, we study specialized receptor cells and neurons that respond to low O2 (hypoxia) by activating a signalling cascade leading to regulation of plasma membrane K+ channels, and release of neurotransmitters or neuromodulators (e.g. catecholamines, ATP, ACh, 5-HT, GABA, nitric oxide). We combine primary cell culture, patch clamp electrophysiology, ratiometric calcium imaging, carbon fibre amperometry, confocal immunofluorescence, (Q)RT-PCR, chemiluminescence methods, Western blotting techniques, and microarrays on cell lines to characterize key components in the signalling pathway.

Mechanisms of chemotransduction by carotid bodies and sensory transmission to afferent neurons

Peripheral chemoreceptors (type I cells) located in the carotid bodies (CBs) act as polymodal sensors that relay sensory information to the brainstem via afferent nerve fibers. There is a consensus that CBs sense blood PO2 and PCO2/pH, however, the mechanisms of sensory processing are only slowly being unraveled. Our long-term objectives are to understand at the cellular and molecular level the transduction mechanisms by which peripheral chemoreceptors sense and integrate diverse chemostimuli, and the role of neurotransmitters/neuromodulators in sensory processing.

Anatomical and functional characterization of aortic bodies

In addition to the CBs, which mainly elicit respiratory reflexes in response to chemoexcitation, another group of peripheral chemoreceptors, the aortic bodies (ABs), are primarily involved in regulating cardiovascular reflexes (e.g. hypertension). In general, less is known about the anatomy, physiology and function of ABs as compared to CBs. In our lab, we are interested in locating and characterizing AB chemoreceptor cells, identifying their adequate stimuli and neurotransmitter output, as well as functionally identifying their sensory afferents.

Chemosensing mechanisms in adrenal chromaffin cells

During vaginal delivery the newborn is subjected to episodes of asphyxia, i.e. low PO2, high PCO2, low pH and low glucose. These asphyxial episodes are important physiologically, since they trigger catecholamine release from the adrenal gland that is vital for survival of the neonate. Impairment of this stress response increases neonatal morbidity and mortality. In our lab, we are investigating how the hormone-producing chromaffin cells in the adrenal medulla sense blood O2, CO2, pH and glucose, leading to hormone secretion.

Developmental regulation of chemosensing in adrenal chromaffin cells

Despite their chemosensitivity at birth, adrenal chromaffin cells lose their ability to respond directly to asphyxial stimuli during postnatal development. Interestingly, the time course of this developmental change parallels innervation of the adrenal gland by the splanchnic nerve, and it has been suggested that substances released by the nerve trigger changes in ion channel expression, for example, leading to suppression of the ‘direct’ stress response. Our lab is interested in understanding the mechanisms by which adrenal chromaffin cells lose their chemosensitivity, particularly the role of chemicals released by the splanchnic nerve.  For example, we are examining how certain chemicals (e.g. nicotine, opiates), which mimic the effects of natural chemical transmitters released from the splanchnic nerve, alter chromaffin cell physiology. Furthermore, these chemicals are health risk factors and are linked to respiratory distress in the newborn (i.e. Sudden Infant Death Syndrome, SIDS).

Regulatory effects of hypoxia-inducible (transcription) factors (HIFs): Mechanisms underlying chemoreceptor plasticity

In addition to examining the responses of chemoreceptor cells to acute (i.e. seconds to minutes) hypoxia, other research in our lab focuses on the effects of chronic (i.e. hours to days) hypoxia. Under these conditions, adaptation can occur via activation of a family of transcription factors known as the hypoxia-inducible factors (HIFs). Microarray analyses performed in our laboratory on an immortalizedO2-sensitive chromaffin cell line (MAH cells) reveal that chronic hypoxia induces changes in the expression of many genes that alter the physiology of these cells, and that some of these changes are HIF-2α-dependent. With the aid of a MAH cell line deficient in HIF-2α, we are investigating the role of HIF-2α in gene regulation during chronic hypoxia.

Chemosensory mechanisms in gills of water-breathing vertebrates

We are interested in the evolution of O2- and CO2/H+- sensing in vertebrates and have carried out pioneering studies on isolated putative O2-receptors, i.e. 5-HT -containing neuroepithelial cells or NECs, located in the gills of zebrafish. In electrophysiological studies, we demonstrated that zebrafish NECs express O2 sensing properties that resemble those in mammalian CB and adrenal chromaffin cells.Using a comparative approach we are extending these studies to other water-breathing vertebrates including larval amphibians (Xenopus), as well as testing for the potential of NECs to act as CO2/H+ sensors. These electrophysiological studies will be complemented with other functional assays developed in the lab, including ratiometric Ca2+ and pH imaging and non-invasive monitoring of aminesecretion by carbon fiber amperometry. Additionally, confocal immunofluorescence techniques are used to study the localization of gill NECs and their innervation pattern in situ.

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Funding

  1. Canadian Institutes of Health Research (2015-2020)
  2. Natural Sciences and Engineering Research Council, Discovery (2015-2020)
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Techniques

  • Cell culture

    Short- and long-term culture of primary chemoreceptor and neuronal cells, immortalized cell lines.

  • Patch clamp electrophysiology

    Single- and double-patch clamp recordings

    Voltage clamp and current clamp recordings allow the measurement of membrane currents and membrane potential, respectively, as well as changes in these parameters during application of (chemo)stimuli

  • Carbon fibre amperometry

    Measures amine (e.g. catecholamine) secretion from individual cells in real time, and provides information regarding vesicle content.

  • Ratiometric Ca2+ imaging

    The fluorescent probe, Fura-2 is used to measure changes in intracellular free Ca2+ concentration.

  • Immunofluorescence and confocal microscopy

    Fluorescently-labelled antibodies allow the (sub)cellular localization of specific proteins.

  • Chemiluminescence (Luminometry)

    Measures ATP release and reactive oxygen species production in living cells and tissues.

  • (Q)RT-PCR, Western blot analysis

    Allows quantification of mRNA and protein levels in cells and tissues exposed to different treatments.

  • Microarrays

    Tool that aids in the identification of genes whose expression patterns (i.e. mRNA levels) are altered under different experimental conditions.