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Aging is associated with cognitive decline and memory loss in humans. In rats, aging-associated neuronal excitability changes and impairments in learning have been extensively studied in the hippocampus. Here, we investigated the roles of L-type calcium channels (LTCCs) in the rat piriform cortex (PC), in comparison with those of the hippocampus. We employed spatial and olfactory tasks that involve the hippocampus and PC. LTCC blocker nimodipine administration impaired spontaneous location recognition in adult rats (6–9 months). However, the same blocker rescued the spatial learning deficiency in aged rats (19–23 months). In an odor-associative learning task, infusions of nimodipine into either the PC or dorsal CA1 impaired the ability of adult rats to learn a positive odor association. Again, in contrast, nimodipine rescued odor associative learning in aged rats. Aged CA1 neurons had higher somatic expression of LTCC Cav1.2 subunits, exhibited larger afterhyperpolarization (AHP) and lower excitability compared with adult neurons. In contrast, PC neurons from aged rats showed higher excitability and no difference in AHP. Cav1.2 expression was similar in adult and aged PC somata, but relatively higher in PSD95− puncta in aged dendrites. Our data suggest unique features of aging-associated changes in LTCCs in the PC and hippocampus.

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1–2 weeks), young adult (2–3 months) and aged (20–25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.

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.

SummaryN-methyl-D-aspartate 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. Gating of the NMDARs requires binding of four co-agonist molecules, but the receptors can signal non-ionotropically through binding of glycine, alone, to its cognate site on GluN1. A consequence of this signalling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit is not a singular molecular species in the CNS, rather there are 8 alternatively spliced isoforms of this subunit produced by including or excluding the N1 and the C1, C2 or C2’ polypeptide cassettes. Whether alternative splicing affects glycine priming signalling is unknown. Here, using recombinant NMDARs expressed heterologously we discovered that glycine priming of NMDARs critically depends on alternative splicing: the four splice isoforms lacking the N1 cassette, encoded in exon 5, are primed by glycine whereas glycine priming is blocked in the four splice variants containing the N1 cassette. On the other hand, the C-terminal cassettes – C1, C2 or C2’ – had no effect on glycine priming signalling. Nor was glycine priming affected by the GluN2 subunit in the receptor. In wild-type mice we found that glycine primed internalization of synaptic NMDARs in CA1 hippocampal pyramidal neurons. With mice we engineered such that GluN1 obligatorily contained the N1 cassette, glycine did not prime synaptic NMDARs in pyramidal neurons. In contrast to pyramidal neurons, we discovered that in wild-type mice, 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 non-ionotropic signalling of NMDAR by glycine and the consequential cell surface dynamics of the receptors.

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

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‐Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y(1) receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y(1) receptor‐mediated release of Ca(2+) 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‐Bötzinger Complex (preBötC) inspiratory rhythm‐generating network acts via P2Y(1) receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC 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 preBötC contributes to the hypoxia‐induced increases in ventilation. Unilateral blockade of P2Y(1) receptors in the preBötC 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 P2Y(1) receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y(1) receptor‐mediated increase in fictive inspiratory frequency involves Ca(2+) recruitment from intracellular stores leading to increases in intracellular Ca(2+) ([Ca(2+)](i)) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y(1) receptors on inspiratory neurons (and/or glia) to evoke Ca(2+) release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

ATP signalling in the CNS is mediated by a three-part system comprising the actions of ATP (and ADP) at P2 receptors (P2Rs), adenosine (ADO) at P1 receptors (P1Rs), and ectonucleotidases that degrade ATP into ADO. ATP excites preBotzinger complex (preBotC) inspiratory rhythm-generating networks where its release attenuates the hypoxic depression of breathing. Its metabolite, ADO, inhibits breathing through unknown mechanisms that may involve the preBotC. Our objective is to understand the dynamics of this signalling system and its influence on preBotC networks. We show that the preBotC of mouse and rat is sensitive to P2Y(1) purinoceptor (P2Y(1)R) activation, responding with a >2-fold increase in frequency. Remarkably, the mouse preBotC is insensitive to ATP. Only after block of A(1) ADORs is the ATP-evoked, P2Y(1)R-mediated frequency increase observed. This demonstrates that ATP is rapidly degraded to ADO, which activates inhibitory A(1)Rs, counteracting the P2Y(1)R-mediated excitation. ADO sensitivity of mouse preBotC was confirmed by a frequency decrease that was absent in rat. Differential ectonucleotidase activities are likely to contribute to the negligible ATP sensitivity of mouse preBotC. Real-time PCR analysis of ectonucleotidase isoforms in preBotC punches revealed TNAP (degrades ATP to ADO) or ENTPDase2 (favours production of excitatory ADP) as the primary constituent in mouse and rat, respectively. These data further establish the sensitivity of this vital network to P2Y(1)R-mediated excitation, emphasizing that individual components of the three-part signalling system dramatically alter network responses to ATP. Data also suggest therapeutic potential may derive from methods that alter the ATP-ADO balance to favour the excitatory actions of ATP.

The diffusion of receptors within the two-dimensional environment of the plasma membrane is a complex process. Although certain components diffuse according to a random walk model (Brownian diffusion), an overwhelming body of work has found that membrane diffusion is nonideal (anomalous diffusion). One of the most powerful methods for studying membrane diffusion is single particle tracking (SPT), which records the trajectory of a label attached to a membrane component of interest. One of the outstanding problems in SPT is the analysis of data to identify the presence of heterogeneity. We have adapted a first-passage time (FPT) algorithm, originally developed for the interpretation of animal movement, for the analysis of SPT data. We discuss the general application of the FPT analysis to molecular diffusion, and use simulations to test the method against data containing known regions of confinement. We conclude that FPT can be used to identify the presence and size of confinement within trajectories of the receptor LFA-1, and these results are consistent with previous reports on the size of LFA-1 clusters. The analysis of trajectory data for cell surface receptors by FPT provides a robust method to determine the presence and size of confined regions of diffusion.

The hypoxic ventilatory response (HVR) comprises an initial increase in ventilation followed by a secondary depression. We tested the hypothesis that the exocytotic release of ATP by astrocytes in ...

To examine the amount of exhaled carbon dioxide (ECO2) with different breathing patterns in spontaneously breathing preterm infants after birth.Preterm infants had a facemask attached to a combined carbon dioxide/flow sensor placed over their mouth and nose to record ECO2 and gas flow. A breath-by-breath analysis of the first 5 minutes of the recording was performed.Thirty spontaneously breathing preterm infants, gestational age (mean ± SD) 30 ± 2 weeks and birth weight 1635 ± 499 g were studied. ECO2 from normal breaths and slow expirations was significantly larger than with other breathing patterns (P.001). ECO2 per breath also increased with gestational age P.001. The expiratory hold pattern was the most prevalent breathing pattern both during the first minute of recording and overall. Breathing pattern proportions also varied by gestational age. Finally, ECO2 from the fifth minute of recording was significantly greater than that produced during the first 4 minutes of recording (P ≤ .029).ECO2 varies with different breathing patterns and increases with gestational age and over time. ECO2 may be an indicator of lung aeration and that postnatal ECO2 monitoring may be useful in preterm infants in the delivery room.

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.