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Significance N-methyl-D-aspartate receptors (NMDARs), which are critical in the brain, are increasingly being shown to signal without ion flux (i.e., “metabotropically”). What controls the metabotropic function of NMDARs is unknown. We discovered that a form of metabotropic signaling—glycine priming—is controlled by alternative splicing of the mRNA encoding one NMDAR subunit, GluN1. Our discovery was surprising because the spliced exon encodes a peptide cassette in the extracellular region of GluN1 far from the plasma membrane, and yet, metabotropic function requires signaling across the neuronal membrane. Moreover, we found that this metabotropic function of NMDARs is neuron cell–type specific: excitatory neurons show glycine priming, whereas inhibitory neurons do not. These findings have widespread implications for NMDARs in health and disease., N-methyl-D-aspartate (NMDA) receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. NMDARs can signal nonionotropically through binding of glycine alone to its cognate site on GluN1. A consequence of this signaling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit contains eight alternatively spliced isoforms produced by including or excluding the N1 and the C1, C2, or C2’ polypeptide cassettes. Whether GluN1 alternative splicing affects nonionotropic signaling by NMDARs is a major outstanding question. Here, we discovered that glycine priming of recombinant NMDARs critically depends on GluN1 isoforms lacking the N1 cassette; glycine priming is blocked in splice variants containing N1. On the other hand, the C-terminal cassettes—C1, C2, or C2’—each permit glycine signaling. In wild-type mice, we found glycine-induced nonionotropic signaling at synaptic NMDARs in CA1 hippocampal pyramidal neurons. This nonionotropic signaling by glycine to synaptic NMDARs was prevented in mice we engineered, such that GluN1 obligatorily contained N1. We discovered in wild-type mice that, in contrast to pyramidal neurons, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling nonionotropic signaling of NMDARs by activating the glycine site.

The Src family kinases (SFKs) are cytoplasmic non-receptor tyrosine kinases involved in multiple signalling pathways. In the central nervous system (CNS), SFKs are key regulators of N-methyl- d -aspartate receptor (NMDAR) function and major points of convergence for neuronal transduction pathways. Physiological upregulation of NMDAR activity by members of the SFKs, namely Src and Fyn, is crucial for induction of plasticity at Schaffer collateral-CA1 synapses of the hippocampus. Aberrant SFK regulation of NMDARs is implicated in several pathological conditions in the CNS including schizophrenia and pain hypersensitivity. Here, evidence is presented to highlight the current understanding of the intermolecular interactions of SFKs within the NMDAR macromolecular complex, the upstream regulators of SFK activity on NMDAR function and the role Src and Fyn have in synaptic plasticity and metaplasticity. The targeting of SFK protein-protein interactions is discussed as a potential therapeutic strategy to restore signalling activity underlying glutamatergic dysregulation in CNS disease pathophysiology.

Key points The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Botzinger complex (preBotC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBotC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. Abstract The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Botzinger Complex (preBotC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBotC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBotC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBotC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBotC inspiratory network, preBotC inspiratory neurons and cultured preBotC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBotC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

The profile of P2 receptor signaling in respiratory control has increased substantially since the first suggestions more than 15 years ago of roles in central chemoreception and modulating inspiratory motor outflow. Part of this reflects the paradigm shift that glia participate in information processing and that ATP is a major gliotransmitter. P2 receptors are a diverse family. Here, we review ATP signaling in respiratory control, highlighting G-protein coupled P2Y1 receptors that have been a focus of recent work. Despite strong evidence of a role for glia and P2 receptor signaling in the central chemosensitivity mediated by the retotrapezoid nucleus, P2Y1 receptors do not appear to be directly involved. Evidence that central P2 receptors and glia contribute to the hypoxic ventilatory response is compelling and P2Y1 receptors are the strongest candidate. However, functional significance in vivo, details of the signaling pathways and involvement of other receptor subtypes remain important questions.

Made available in DSpace on 2018-11-26T17:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-06-30 CIHR NSERC CFI WCHRI Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hydrogen Sulfide (H2S) is one of three gasotransmitters that modulate excitability in the CNS. Global application of H2S donors or inhibitors of H2S synthesis to the respiratory network has suggested that inspiratory rhythm is modulated by exogenous and endogenous H2S. However, effects have been variable, which may reflect that the RTN/pFRG (retrotrapezoid nucleus, parafacial respiratory group) and the preBotzinger Complex (preBotC, critical for inspiratory rhythm generation) are differentially modulated by exogenous H2S. Importantly, site-specific modulation of respiratory nuclei by H2S means that targeted, rather than global, manipulation of respiratory nuclei is required to understand the role of H2S signaling in respiratory control. Thus, our aim was to test whether endogenous H2S, which is produced by cystathionine-beta-synthase (CBS) in the CNS, acts specifically within the preBotC to modulate inspiratory activity under basal (in vitro/in vivo) and hypoxic conditions (in vivo). Inhibition of endogenous H2S production by bath application of the CBS inhibitor, aminooxyacetic acid (AOAA, 0.1-1.0mM) to rhythmic brainstem spinal cord (BSSC) and medullary slice preparations from newborn rats, or local application of AOAA into the preBotC (slices only) caused a dose-dependent decrease in burst frequency. Unilateral injection of AOAA into the preBotC of anesthetized, paralyzed adult rats decreased basal inspiratory burst frequency, amplitude and ventilatory output. AOAA in vivo did not affect the initial hypoxia-induced (10% O-2, 5 min) increase in ventilatory output, but enhanced the secondary hypoxic respiratory depression. These data suggest that the preBotC inspiratory network receives tonic excitatory modulation from the CBS-H2S system, and that endogenous H2S attenuates the secondary hypoxic respiratory depression. Univ Alberta, Women & Childrens Hlth Res Inst, Neurosci & Mental Hlth Inst, Dept Physiol,Fac Med & Dent, Edmonton, AB, Canada Sao Paulo State Univ, Dept Morphol & Anim Physiol, Jaboticabal, Brazil Univ Fed Piaui, Dept Biophys & Physiol, Teresina, Brazil Univ Sao Paulo, Fac Dent Ribeirao Preto, Dept Physiol, Ribeirao Preto, Brazil Hosp Sick Children, Peter Gilgan Ctr Res & Learning, Neurosci & Mental Hlth, Toronto, ON, Canada Sao Paulo State Univ, Dept Morphol & Anim Physiol, Jaboticabal, Brazil CIHR: 53085 CIHR: 130306 NSERC: 402532 NSERC: 434543 FAPESP: FAPESP-2012/02413-8 FAPESP: 2014/12951-2 : FAPESP 2013/17606-9

While once viewed as mere housekeepers, providing structural and metabolic support for neurons, it is now clear that neuroglia do much more. Phylogenetically, they have undergone enormous proliferation and diversification as central nervous systems grew in their complexity. In addition, they: i) are morphologically and functionally diverse; ii) play numerous, vital roles in maintaining CNS homeostasis; iii) are key players in brain development and responses to injury; and, iv) via gliotransmission, are likely participants in information processing. In this review, we discuss the diverse roles of neuroglia in maintaining homeostasis in the CNS, their evolutionary origins, the different types of neuroglia and their functional significance for respiratory control, and finally consider evidence that they contribute to the processing of chemosensory information in the respiratory network and the homeostatic control of blood gases.