Albert J. Berger - US grants

The overall objective of our research is to explain the behavior of the respiratory control system based on an understanding of the basic properties of and interactions among individual respiratory neurons. Thus we believe that the behavior of the respiratory system can be synthesized based on an understanding of individual neuronal elements and their interconnections. This proposal focuses on in vitro studies of an important class of respiratory motoneuron, the hypoglossal motoneuron. A specific goal of the research is to understand basic mechanisms responsible for the behavior of the motoneurons. The first specific aim is to continue our studies of membrane currents present in these motoneurons. By using voltage-clamping of hypoglossal motoneurons, with both conventional and patch-type microelectrodes in thick-and thin-medullary slices of rat, we will investigate three classes of membrane currents that are activated or de-inactivated by membrane hyperpolarization. These three include Ca2+ currents and channels, transient K+ currents and the mixed cationic inward rectifier current, Ih. These currents will be isolated to determine, in detail, their ionic bases, pharmacology, voltage-dependency and kinetics. The second specific aim is to investigate the direct effects on hypoglossal motoneurons of two neurotransmitters, serotonin (5-HT) and thyrotropin- releasing hormone (TRH). Specific membrane ionic and intracellular second- messenger mechanisms by which these transmitters act will be determined. The third specific aim is to use the rhythmically active in vitro neonatal brainstem spinal cord preparation, with voltage-clamp recording, to test the hypothesis that respiratory-related rhythmic activation of hypoglossal motoneurons is partly determined by voltage-and-time-dependent conductances. The results of these studies will provide fundamental information about the normal functioning of hypoglossal motoneurons. In addition, since hypoglossal motoneurons regulate the contraction of the tongue and this muscle determines in part patency of the upper airway, a comprehensive understanding of these motoneurons may be essential to our understanding of certain pathological states, such as obstructive sleep apnea and obstructive apnea of prematurity.

It is hypothesized that postnatal changes occur in intrinsic membrane currents and in the direct responses to neurotransmitters in neonatal rat hypoglossal motoneurons. These changes may be responsible for differences in both the sub- and supra-threshold behaviors of these motoneurons as well as differences in the responses to various neurotransmitters. It is further hypothesized that the changes in the neurotransmitter responses may be, in part, correlated with changes in neurotransmitter receptor gene expression in these neonatal motoneurons. The first two specific aims investigate two inward voltage- and time- dependent ionic currents that are important in the sub- and supra- threshold firing behaviors of hypoglossal motoneurons. The first specific aim is to investigate the postnatal development of properties of the mixed cationic inward rectifier current activated on hyperpolarization, Ih. Single electrode voltage-clamping of hypoglossal motoneurons in conventional brainstem slices will be utilized to study the properties of this current. The second specific aim is to investigate the postnatal development of properties of the low-voltage-activated Ca2+ current. Whole-cell currents measured with patch-type electrodes in brainstem slices will be utilized to study the properties of this current. Specific aims three and four investigate two neurotransmitters that have direct excitatory effects on hypoglossal motoneurons; their release may be state-dependent (sleep vs. awake). The third specific aim is to investigate the postnatal development of the response to thyrotropin- releasing hormone (TRH). It will also be determined whether these changes correlate with TRH receptor mRNA expression in hypoglossal motoneurons. The fourth specific aim is to investigate the postnatal development of the response to norepinephrine (NE). It will also be determined whether these changes correlate with alpha1-adrenergic receptor mRNA receptor mRNA expression in hypoglossal motoneurons. This proposal focuses on in vitro studies of hypoglossal motoneurons because these motoneurons may have an important function in regulating upper airway patency particularly during sleep states. Results from these studies will provide new information concerning the normal and postnatal development of these important motoneurons. The upper airway has been suggested as a site for airway obstruction; therefore a comprehensive understanding of these motoneurons may provide new insights into certain pathologic states, including apnea of prematurity and Sudden Infant Death Syndrome.

DESCRIPTION (Applicant's abstract): Our long-term objective is to understand how respiratory-related neurons synaptically interact. This application focuses on glycine and gamma-aminobutyric acid (GABA) ligand-gated receptors (glycine-Rs and GABAA-Rs, respectively). We hypothesize that they are important in brainstem and spinal cord physiology related to the neurobiology of breathing. The upper airway is a site of airway obstruction: thus understanding of synaptic transmission to hypoglossal motoneurons (HMs) may provide new insights into airway pathologies such as obstructive sleep apnea. We continue to focus our research on HMs because the tongue has a critical position in the upper airway, thereby affecting upper airway resistance and patency. Also, HMs are important in other behaviors including mastication, swallowing (deglutition), sucking, licking, and vocalization. The experiments proposed employ two different in vitro brainstem slice preparations to study glycinergic and GABAergic synaptic transmission to HMs. In both, rat HMs will be visualized using infra-red differential interference optics and we will use whole-cell and outside-out patch recordings from these neurons. HMs will be studied in the absence (standard slice preparation) and in the presence (rhythmic slice preparation) of ongoing rhythmic respiratory activity. Specific aim 1 proposes to characterize GABAA -R-mediated synaptic transmission to HMs. We hypothesize that GABAA-Rs have differential pharmacological and kinetic properties as compared to glycine-Rs. Specific Aim 2 proposes to investigate glycine and GABA cotransmission at individual synaptic terminals apposed to HMs. We hypothesize that glycine and GABA are co-released and that following co-release they activate co-localized glycine and GABAA receptors at individual synaptic terminals. Specific Aim 3 proposes to investigate whether glycine and/or GABAA receptor mediated synaptic events are important for the respiratory-related behavior of HMs recorded in the rhythmic medullary slice preparation. We hypothesize that glycine and/or GABA mediated synaptic events shape the inspiratory phase behavior of HMs, mostly via shunting inhibition of excitatory respiratory drive. The fourth Specific Aim proposes to investigate in HMs postsynaptic mechanisms whereby ethanol modulates glycine-Rs. We hypothesize that ethanol increases the affinity of glycine-Rs for glycine and thereby potentiates inhibitory synaptic events. This may be an important mechanism whereby alcohol consumption exacerbates obstructive sleep apnea.