Florey Department of Neuroscience and Mental Health - Research Publications

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 29
  • Item
    Thumbnail Image
    Advancing respiratory-cardiovascular physiology with the working heart-brainstem preparation over 25 years
    Paton, JFR ; Machado, BH ; Moraes, DJA ; Zoccal, DB ; Abdala, AP ; Smith, JC ; Antunes, VR ; Murphy, D ; Dutschmann, M ; Dhingra, RR ; McAllen, R ; Pickering, AE ; Wilson, RJA ; Day, TA ; Barioni, NO ; Allen, AM ; Menuet, C ; Donnelly, J ; Felippe, I ; St-John, WM (WILEY, 2022-04-07)
    Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.
  • Item
    Thumbnail Image
    Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia.
    Barioni, NO ; Derakhshan, F ; Tenorio Lopes, L ; Onimaru, H ; Roy, A ; McDonald, F ; Scheibli, E ; Baghdadwala, MI ; Heidari, N ; Bharadia, M ; Ikeda, K ; Yazawa, I ; Okada, Y ; Harris, MB ; Dutschmann, M ; Wilson, RJA (American Association for the Advancement of Science (AAAS), 2022-03-25)
    As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.
  • Item
    Thumbnail Image
    Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata
    Trevizan-Bau, P ; Dhingra, RR ; Furuya, WI ; Stanic, D ; Mazzone, SB ; Dutschmann, M (WILEY, 2021-01-05)
    Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-BötC, or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.
  • Item
    Thumbnail Image
    The role of glycinergic inhibition in respiratory pattern formation and cardio-respiratory coupling in rats.
    Furuya, WI ; Dhingra, RR ; Trevizan-Baú, P ; McAllen, RM ; Dutschmann, M (Elsevier BV, 2021)
    Cardio-respiratory coupling is reflected as respiratory sinus arrhythmia (RSA) and inspiratory-related bursting of sympathetic nerve activity. Inspiratory-related inhibitory and/or postinspiratory-related excitatory drive of cardiac vagal motoneurons (CVMs) can generate RSA. Since respiratory oscillations may depend on synaptic inhibition, we investigated the effects of blocking glycinergic neurotransmission (systemic and local application of the glycine receptor (GlyR) antagonist, strychnine) on the expression of the respiratory motor pattern, RSA and sympatho-respiratory coupling. We recorded heart-rate, phrenic, recurrent laryngeal and thoracic sympathetic nerve activities (PNA, RLNA, t-SNA) in a working-heart-brainstem preparation of rats, and show that systemic strychnine (50-200 ​nM) abolished RSA and triggered a shift of postinspiratory RLNA into inspiration, while t-SNA remained unchanged. Bilateral strychnine microinjection into the ventrolateral medullary area containing CVMs and laryngeal motoneurons (LMNs) of the nucleus ambiguus (NA/CVLM), the nucleus tractus solitarii, pre-Bötzinger Complex, Bötzinger Complex or Kölliker-Fuse nuclei revealed that only NA/CVLM strychnine microinjections mimicked the effects of systemic application. In all other target nuclei, except the Bötzinger Complex, GlyR-blockade attenuated the inspiratory-tachycardia of the RSA to a similar degree while evoking only a modest change in respiratory motor patterning, without changing the timing of postinspiratory-RLNA, or t-SNA. Thus, glycinergic inhibition at the motoneuronal level is involved in the generation of RSA and the separation of inspiratory and postinspiratory bursting of LMNs. Within the distributed ponto-medullary respiratory pre-motor network, local glycinergic inhibition contribute to the modulation of RSA tachycardia, respiratory frequency and phase duration but, surprisingly it had no major role in the mediation of respiratory-sympathetic coupling.
  • Item
    Thumbnail Image
    Laryngeal afferent modulation of swallowing interneurons in the dorsal medulla in perfused rats
    Fuse, S ; Sugiyama, Y ; Hashimoto, K ; Umezaki, T ; Oku, Y ; Dutschmann, M ; Hirano, S (WILEY, 2019-09-09)
    OBJECTIVES: The purpose of this study was to investigate the influence of laryngeal afferent inputs on brainstem circuits that mediate and transmit swallowing activity to the orofacial musculature. METHODS: Experiments were performed on 19 arterially perfused juvenile rats. The activities of swallowing interneurons in relation to their respective motor outputs in the hypoglossal and vagus nerves were assessed during fictive swallowing with or without concurrent laryngeal sensory stimulation at intensities of 20, 40, and 60 μA. RESULTS: The hypoglossal nerve activity was gradually enhanced with increasing intensity of the sensory stimulation, while the vagus nerve activity was not altered. The activities of various interneurons were modulated by the laryngeal stimulation, but more than 50% of the recorded neurons were inhibited by the stimulation. Some interneurons demonstrated no obvious change in their discharge rates with laryngeal sensory stimulation during fictive swallowing. CONCLUSION: Laryngeal afferent inputs partially modulated the swallowing motor activity via enhanced or suppressed activities of the swallowing interneurons, while the essential motor pattern underlying the pharyngeal stage of swallowing remained basically unchanged. Thus, the output patterns of the complex sequential movements of swallowing could be basically predetermined and further adjusted according to sensory information related to the properties of the ingested food by a swallowing central pattern generator. LEVEL OF EVIDENCE: NA Laryngoscope, 130: 1885-1893, 2020.
  • Item
    Thumbnail Image
    Activity of swallowing-related neurons in the medulla in the perfused brainstem preparation in rats
    Hashimoto, K ; Sugiyama, Y ; Fuse, S ; Umezaki, T ; Oku, Y ; Dutschmann, M ; Hirano, S (WILEY, 2019-02-01)
    OBJECTIVES/HYPOTHESIS: We aimed to investigate and validate the cellular activity patterns and the potential topographical organization of neurons of the medullary swallowing pattern generator (Sw-CPG). We used the perfused brainstem preparation as an innovative experimental model that allows for stable neuronal recording in the brainstem. STUDY DESIGN: Animal model. METHODS: Experiments were conducted in 14 juvenile Wistar rats. The activities of the phrenic, vagus, and hypoglossal nerves were recorded at baseline, and fictive swallowing was elicited by stimulation of the superior laryngeal nerve. Extracellular action potentials of 72 swallowing-related neurons were recorded in the Sw-CPG of the dorsal medulla oblongata. RESULTS: Neurons could be classified into three types: sensory relay, and neurons that were excited or inhibited during fictive swallowing. Approximately one-third of the neurons likely received monosynaptic input from the laryngeal afferents. One-third of neurons recorded showed respiratory-related activity, most of which exhibited inspiratory modulation. The neurons were widely distributed in the nucleus tractus solitarius and reticular formation. CONCLUSIONS: The perfused brainstem preparation of rat fully preserves the Sw-CPG. The recorded cellular activities and general topographical organization of swallowing neurons are in accordance with previous in vivo studies. Thus, the perfused brainstem preparation is an ideal experimental model to advance the understanding of neuronal mechanisms underlying swallowing. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E72-E79, 2019.
  • Item
    Thumbnail Image
    Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits
    Driessen, AK ; Farrell, MJ ; Dutschmann, M ; Stanic, D ; McGovern, AE ; Mazzone, SB (SPRINGER HEIDELBERG, 2018-12-01)
    Sensory neurons of the jugular vagal ganglia innervate the respiratory tract and project to the poorly studied medullary paratrigeminal nucleus. In the present study, we used neuroanatomical tracing, pharmacology and physiology in guinea pig to investigate the paratrigeminal neural circuits mediating jugular ganglia-evoked respiratory reflexes. Retrogradely traced laryngeal jugular ganglia neurons were largely (> 60%) unmyelinated and expressed the neuropeptide substance P and calcitonin gene-related peptide, although a population (~ 30%) of larger diameter myelinated jugular neurons was defined by the expression of vGlut1. Within the brainstem, vagal afferent terminals were confined to the caudal two-thirds of the paratrigeminal nucleus. Electrical stimulation of the laryngeal mucosa evoked a vagally mediated respiratory slowing that was mimicked by laryngeal capsaicin application. These laryngeal reflexes were modestly reduced by neuropeptide receptor antagonist microinjections into the paratrigeminal nucleus, but abolished by ionotropic glutamate receptor antagonists. D,L-Homocysteic acid microinjections into the paratrigeminal nucleus mimicked the laryngeal-evoked respiratory slowing, whereas capsaicin microinjections evoked a persistent tachypnoea that was insensitive to glutamatergic inhibition but abolished by neuropeptide receptor antagonists. Extensive projections from paratrigeminal neurons were anterogradely traced throughout the pontomedullary respiratory column. Dual retrograde tracing from pontine and ventrolateral medullary termination sites, as well as immunohistochemical staining for calbindin and neurokinin 1 receptors, supported the existence of different subpopulations of paratrigeminal neurons. Collectively, these data provide anatomical and functional evidence for at least two types of post-synaptic paratrigeminal neurons involved in respiratory reflexes, highlighting an unrecognised complexity in sensory processing in this region of the brainstem.
  • Item
    Thumbnail Image
    Decreased incidence, virus transmission capacity, and severity of COVID-19 at altitude on the American continent
    Arias-Reyes, C ; Carvajal-Rodriguez, F ; Poma-Machicao, L ; Aliaga-Raduan, F ; Marques, DA ; Zubieta-DeUrioste, N ; Accinelli, RA ; Schneider-Gasser, EM ; Zubieta-Calleja, G ; Dutschmann, M ; Soliz, J ; Verdonck, K (PUBLIC LIBRARY SCIENCE, 2021-03-29)
    The coronavirus disease 2019 (COVID-19) outbreak in North, Central, and South America has become the epicenter of the current pandemic. We have suggested previously that the infection rate of this virus might be lower in people living at high altitude (over 2,500 m) compared to that in the lowlands. Based on data from official sources, we performed a new epidemiological analysis of the development of the pandemic in 23 countries on the American continent as of May 23, 2020. Our results confirm our previous finding, further showing that the incidence of COVID-19 on the American continent decreases significantly starting at 1,000 m above sea level (masl). Moreover, epidemiological modeling indicates that the virus transmission rate is lower in the highlands (>1,000 masl) than in the lowlands (<1,000 masl). Finally, evaluating the differences in the recovery percentage of patients, the death-to-case ratio, and the theoretical fraction of undiagnosed cases, we found that the severity of COVID-19 is also decreased above 1,000 m. We conclude that the impact of the COVID-19 decreases significantly with altitude.
  • Item
    Thumbnail Image
    Volumetric mapping of the functional neuroanatomy of the respiratory network in the perfused brainstem preparation of rats
    Dhingra, RR ; Dick, TE ; Furuya, WI ; Galan, RF ; Dutschmann, M (WILEY, 2020-04-16)
    KEY POINTS: The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem-wide circuit are lacking. Here, we use silicon multi-electrode arrays to record respiratory local field potentials (rLFPs) from 196-364 electrode sites within 8-10 mm3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post-inspiration (PI) and late-expiration (E2). rLFPs peaked specifically at the three respiratory phase transitions, E2-I, I-PI and PI-E2. We show, for the first time, that only the I-PI transition engages a brainstem-wide network, and that rLFPs during the PI-E2 transition identify a hitherto unknown role for the dorsal respiratory group. Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease. ABSTRACT: While it is widely accepted that inspiratory rhythm generation depends on the pre-Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi-electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post-inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2-I, and PI-E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post-inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group-wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.
  • Item
    Thumbnail Image
    Modelling of synaptic interactions between two brainstem half-centre oscillators that coordinate breathing and swallowing
    Tolmachev, P ; Dhingra, RR ; Manton, JH ; Dutschmann, M ( 2021)
    Abstract Respiration and swallowing are vital orofacial motor behaviours that require the coordination of the activity of two brainstem central pattern generators (r-CPG, sw-CPG). Here, we use computational modelling to further elucidate the neural substrate for breathing-swallowing coordination. We progressively construct several computational models of the breathing-swallowing circuit, starting from two interacting half-centre oscillators for each CPG. The models are based exclusively on neuronal nodes with spike-frequency adaptation, having a parsimonious description of intrinsic properties. These basic models undergo a stepwise integration of synaptic connectivity between central sensory relay, sw- and r-CPG neuron populations to match experimental data obtained in a perfused brainstem preparation. In the model, stimulation of the superior laryngeal nerve (SLN, 10s) reliably triggers sequential swallowing with concomitant glottal closure and suppression of inspiratory activity, consistent with the motor pattern in experimental data. Short SLN stimulation (100ms) evokes single swallows and respiratory phase resetting yielding similar experimental and computational phase response curves. Subsequent phase space analysis of model dynamics provides further understanding of SLN-mediated respiratory phase resetting. Consistent with experiments, numerical circuit-busting simulations show that deletion of ponto-medullary synaptic interactions triggers apneusis and eliminates glottal closure during sequential swallowing. Additionally, systematic variations of the synaptic strengths of distinct network connections predict vulnerable network connections that can mediate clinically relevant breathing-swallowing disorders observed in the elderly and patients with neurodegenerative disease. Thus, the present model provides novel insights that can guide future experiments and the development of efficient treatments for prevalent breathing-swallowing disorders. Key points The coordination of breathing and swallowing depends on synaptic interactions between two functionally distinct central pattern generators (CPGs) in the dorsal and ventral brainstem. We model both CPGs as half-centre oscillators with spike-frequency adaptation to identify the minimal connectivity sufficient to mediate physiologic breathing-swallowing interactions. The resultant computational model(s) can generate sequential swallowing patterns including concomitant glottal closure during simulated 10s stimulation of the superior laryngeal nerve (SLN) consistent with experimental data. In silico, short (100 ms) SLN stimulation triggers a single swallow which modulates the respiratory cycle duration consistent with experimental recordings. By varying the synaptic connectivity strengths between the two CPGs and the sensory relay neurons, and by inhibiting specific nodes of the network, the model predicts vulnerable network connections that may mediate clinically relevant breathing-swallowing disorders.