Experimental Physiology

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1 Exp Physiol (214) pp Research Paper Research Paper Intrinsic chemosensitivity of rostral ventrolateral medullary sympathetic premotor neurons in the in situ arterially perfused preparation of rats Tadachika Koganezawa 1 and Julian F. R. Paton 2 1 Department of Physiology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki , Japan 2 School of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK Experimental Physiology New Findings What is the central question of this study? Brain hypoperfusion is a key factor triggering hypertension through activation of cardiovascular sympathetic vasomotor nerves. However, mechanisms of detecting brain hypoperfusion remain unclear. We hypothesized that the sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) can sense asphyxia and cause sympathoexcitation. What is the main finding and its importance? Functionally identified RVLM sympathetic premotor neurons were excited by hypoxia but less so by hypercapnia, before and after blockade of synaptic transmission. The RVLM sympathetic premotor neurons can act as an important oxygen sensor during brain hypoxia/hypoperfusion, which may be important in maintaining sympathetic nerve discharge to support blood pressure and hence maintain brain perfusion. Brainstem hypoperfusion is a major excitant of sympathetic activity triggering hypertension, but the exact mechanisms involved remain incompletely understood. A major source of excitatory drive to preganglionic sympathetic neurons originates from the ongoing activity of premotor neurons in the rostral ventrolateral medulla (RVLM sympathetic premotor neurons). The chemosensitivity profile of physiologically characterized RVLM sympathetic premotor neurons during hypoxia and hypercapnia remains unclear. We examined whether physiologically characterized RVLM sympathetic premotor neurons can sense brainstem ischaemia intrinsically. We addressed this issue in a unique in situ arterially perfused preparation before and after a complete blockade of fast excitatory and inhibitory synaptic transmission. During hypercapnic hypoxia, respiratory modulation of RVLM sympathetic premotor neurons was lost, but tonic firing of most RVLM sympathetic premotor neurons was elevated. After blockade of fast excitatory and inhibitory synaptic transmission, RVLM sympathetic premotor neurons continued to fire and exhibited an excitatory firing response to hypoxia but not hypercapnia. This study suggests that RVLM sympathetic premotor neurons can sustain high levels of neuronal discharge when oxygen is scarce. The intrinsic ability of RVLM sympathetic premotor neurons to maintain responsivity to brainstem hypoxia is an important mechanism ensuring adequate arterial pressure, essential for maintaining cerebral perfusion in the face of depressed ventilation and/or high cerebral vascular resistance. (Received 14 April 214; accepted after revision 4 July 214; first published online 11 July 214) Corresponding author T. Koganezawa: Department of Physiology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tennodai, Tsukuba, Ibaraki , Japan. t-kogane@md.tsukuba.ac.jp C 214 The Authors. Experimental Physiology C 214 The Physiological Society DOI: /expphysiol

2 1454 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp Introduction Previously, we found that reducing blood flow into the brainstem by acute bilateral occlusion of the vertebral arteries produced a prompt increase in arterial pressure and sympathetic nerve activity (Cates et al. 211). This response was originally described by Cushing (191), who increased cerebral vascular pressure by elevating intracranial pressure. As had been suggested previously (Cushing, 191; Rodbard & Stone, 1955; Dickinson & Thomson, 196), we emphasized the importance of an intracranial detection system that was sensitive to blood pressure and/or cerebral blood flow and oxygen delivery (Paton et al. 29) that would raise sympathetic activity and arterial pressure robustly to ensure adequate brain perfusion. Many premotor neurons for cardiovascular sympathetic preganglionic neurons are located in the rostral ventrolateral medulla (RVLM) and termed RVLM sympathetic premotor neurons (Dampney et al. 1979; Guyenet et al. 1989; Kumada et al. 199; Dampney, 1994). In numerous previous studies, the consensus of opinion is that RVLM sympathetic premotor neurons influencing vasomotor and cardiac sympathetic motor outflows have ongoing activity, which is inhibited by baroreceptors and excited by peripheral chemoreceptors (Kumada et al. 199) and distinct types of neighbouring respiratory neurons (Moraes et al. 214). Some studies have reported the possibility that RVLM sympathetic premotor neurons have chemosensitivity, especially to hypoxia, in vivo and in vitro (Sun et al. 1992; Sun & Reis, 1994a,b; Mazza et al. 2; D Agostino et al. 29). However, in the in vivo studies, synaptic inputs into RVLM sympathetic premotor neurons were either intact or only partly blocked (Sun et al. 1992; Sun & Reis, 1994b). Therefore, the effects of synaptic inputs from other neurons to RVLM sympathetic premotor neurons could not be ignored. In contrast, in the in vitro studies, dissociated neurons were intermingled with non-cardiovascular neurons from the RVLM (Sun & Reis, 1994a; Mazza et al. 2; D Agostino et al. 29). Therefore, it remains unclear whether the RVLM sympathetic premotor neurons themselves can directly sense hypoxia and/or hypercapnia. In the present study, we hypothesized that a locus for the detection system of brainstem hypoperfusion is the rostral ventrolateral medulla and that RVLM sympathetic premotor neurons will be intrinsically sensitive to ischaemia. Our aim, therefore, was to assess changes of the firing of single RVLM sympathetic premotor neurons during hypercapnic hypoxia and hypoxia alone both before and after blocking fast excitatory and inhibitory synaptic transmission. This is not possible in vivo because global blockade of excitatory and inhibitory synaptic transmission would be lethal. Thus, we have employed the in situ arterially perfused rat preparation (Paton, 1996), which allowed long-term extracellular recording, repeated and reversible bouts of severe hypercapnic hypoxia and extreme manipulation of the extracellular milieu without detrimental effects on brainstem viability. We report that RVLM sympathetic premotor neuronal firing is elevated in response to ischaemia in the presence of fast excitatory and inhibitory synaptic transmission. This unique characteristic makes the RVLM a prime locus for maintaining sympathetic discharge in conditions of cerebral hypoperfusion. Methods All procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and were approved by the Animal Experiment Committees of the University of Bristol and the University of Tsukuba. In situ arterially perfused preparations Seventy-four experiments were performed in decerebrate, unanaesthetized and in situ arterially perfused preparations of Wistar rats (male, 6 9 g; University of Bristol colony or Japan SLC, Hamamatsu, Japan). General methods were based on those described previously (Paton, 1996). Briefly, a rat was anaesthetized deeply by inhalation of 5% halothane until there was no sign of withdrawal reflexes after pinching the tail or a forepaw. The rat was bisected subdiaphragmatically and its upper body immersed in ice-chilled mock cerebrospinal fluid. Rats were decerebrated at the precollicular level, and the fourth ventricle was exposed by cerebellectomy. The lungs were removed. After transferral to a recording chamber, a double-lumen perfusion catheter (4 french; Braintree Scientific, Braintree, MA, USA) was introduced into the left ventricle and advanced into the ascending aorta. Perfusion solution (see below for constituents) was maintatined at constant flow using a roller pump (14 16 ml min 1 ; 55LA; Watson Marlow, Wilmington, MA, USA). Perfusion pressure was recorded from the second lumen of the catheter. Preparations were paralysed with vecuronium bromide (4 mg l 1 ; Organon, Roseland, NJ, USA). [Arg8]-Vasopressin acetate salt (.8 nm; Sigma, St. Louis, MO, USA) was added into the perfusate to increase peripheral vascular resistance. The composition of the perfusate was as follows (mm): NaCl, 125 (Fisher Scientific, Loughborough, UK); NaHCO 3, 24 (Fisher Scientific, Loughborough, UK); KCl, 5 (The British Drug House, Poole, UK); CaCl 2, 2.5 (Sigma, St. Louis, MO, USA); MgSO 4, 1.25 (The British Drug House, Poole, UK); KH 2 PO 4, 1.25 (Sigma, St. Louis, MO, USA); and dextrose, 1(TheBritishDrugHouse,Poole,UK).Thecomposition of the perfusate containing a low concentration of Ca 2+ C 214 The Authors. Experimental Physiology C 214 The Physiological Society

3 Exp Physiol (214) pp Chemosensitivity of the sympathetic premotor neurons 1455 and high concentration of Mg 2+ was as follows (mm): NaCl, 117; NaHCO 3, 24; KCl, 5; CaCl 2,.2; MgSO 4, 1.25; KH 2 PO 4, 1.25; MgCl 2, 4; and dextrose, 1 (The British Drug House, Poole, UK). The perfusate volume was 2 ml and contained 1.25% Ficoll R 7 (Sigma, St Louis, NJ, USA), which is a synthetic polymer of sucrose used to increase osmotic pressure, and prewarmed to 31 C. In control conditions, the perfusate was equilibrated with 95% oxygen and 5% carbon dioxide. Electrocardiogram and peripheral nerve recording The electrocardiogram was recorded and the R wave discriminated and used to generate Transistor-Transistor- Logic (TTL) pulses, from which heart rate (HR) was derived. The left phrenic nerve (PN) was isolated at the level of thorax, cut close to the diaphragm, and its activity was recorded via a glass suction electrode. The left central vagus nerve (VN) at the cervical level and the left thoracic sympathetic chain (SC) at T8 T9 were isolated and recorded using glass suction electrodes. Simultaneous activities of the PN, VN and SC were amplified ( 1,), filtered (1 5 Hz) and integrated (. time constant). The inspiratory motor pattern consisted of an incrementing discharge indicative of a eupnoeic-like pattern (St-John & Paton, 23), which was used to gauge the viability of the preparation. The SC displayed both respiratory- and non-respiratory-modulated activity, as described before (Pickering et al. 23). Recording the activity of single RVLM sympathetic premotor neurons Single-unit activity of neurons located in the left RVLM was recorded by micropipettes filled with.5 M sodium acetate and 2% Pontamine Sky Blue for marking recording sites. Micropipettes were held in a three-dimensional micromanipulator and driven into the RVLM in 2 μm steps using a custom-built stepper motor. The tip resistance of the microelectrodes was between 8 and 12 M. Single-unit activity was amplified (Axoprobe-1A; Axon Instruments, Sunnyvale, CA, USA) with reference to an earth electrode attached to a neck muscle; signals were filtered (1 5 Hz; Neurolog, Digitimer, Letchworth Garden City, UK). The site of unit recording was marked by iontophoretic deposition of dye ( 1V, 1 min). Recordingprocedureswereasfollows.Onencountering a spontaneously active neuron in the RVLM, we tested baroreceptor sensitivity. Aortic baroreceptors were stimulated by distension of the aortic arch using a balloon-tipped catheter (2 french; Edwards Lifesciences, Irvine, CA, USA), which was advanced towards the arch via the descending aorta. The final position of the balloon was optimized to produce a reflex bradycardia when distended. If the neuron was inhibited by baroreceptor stimulation, we proceeded to test its reaction to peripheral chemoreceptor stimulation. Peripheral chemoreceptors were activated by an intra-aortic injection of NaCN (.3%, 5 μl; The British Drug House, Poole, UK) via a side-port of the aortic perfusion cannula. This produced reflex bradycardia and sympathoexcitation recorded in the SC. If the unit was excited by this stimulus, it was deemed an RVLM sympathetic premotor unit. Finally, we tested for its bulbospinal projections by using conventional antidromic activation from the spinal cord. Following a cervical laminectomy, bipolar cashew-coated tungsten wire electrodes (custom made; tip diameter, <1 μm) were placed into the dorsolateral funiculi under visual control at the C7 segmental level. The stimulus site was determined by producing powerful excitation of the SC activity evoked using three-pulses stimulation (.2 ms width, 1 Hz, 1 V). For antidromic activation of medullary neurons, single-pulse stimulation and an intensity of 1.5 times threshold for SC activation was employed (typically <1 V; range, 3 6 V). The conduction velocity of the descending axon was estimated by the onset latency for the antidromic spike and distance between the stimulating and the recording electrodes. Identification of antidromic spikes was assured using the collision test (Lipski, 1981) in all cases. Exposure to hypercapnic hypoxia, hypercapnia and ischaemia AfterthecontrolactivityofSCorRVLMsympathetic premotor neurons had been recorded for several minutes, the control perfusate equilibrated with 95% oxygen and 5% carbon dioxide (ph 7.4) was then switched to one pre-equilibrated with 5% oxygen, 8% carbon dioxide and 87% nitrogen in order to provide exposure to hypercapnic hypoxia (ph 7.1). The preparation was exposed to this perfusate until gasping appeared (12 36 s), which followed both the excitatory and depressant phases of the hypoxic respiratory response. ing was determined by a decrementing pattern of inspiratory discharge and loss of postinspiratory activity in the VN (Paton et al. 26); this occurred approximately 2 s ( s) after the induction of hypercapnic hypoxia. The appearance of gasping was a sure sign of brainstem hypoxia. After gasping was established, a mixture of 95% oxygen and 5% carbon dioxide was reintroduced into the perfusate, and eupnoea was re-established. There was minimal effect on perfusion pressure. Note that these stimuli often caused tonic activity in the PN, which persisted after blockade of synaptic transmission (Fig. 4A and B), implying a direct effect at the spinal motoneuron level. In order to expose the preparation to hypercapnia, the control perfusate equilibrated with 95% oxygen and 5% carbon dioxide was switched to one pre-equilibrated with a mixture of 92% oxygen and 8% carbon dioxide (ph 7.). In peripheral C 214 The Authors. Experimental Physiology C 214 The Physiological Society

4 1456 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp nerve recording studies only, ischaemia was introduced by arresting perfusion for approximately 6 s; this test was not used while recording single units due to the mechanical instability of the brainstem when perfusion is arrested. After gasping was observed, perfusion was restarted and eupnoea resumed. Hypercapnic hypoxia, hypercapnia and ischaemia were repeatable and reversible. Administration of drugs Antagonists of receptors mediating fast excitatory and inhibitory neurotransmission were added directly into the perfusate as before (St-John et al. 29). For blockade of excitatory synaptic transmission (NMDA, non-nmda and kainate receptors),kynurenic acid (3 1 mm; Sigma) was added. Given that both disinhibition and release from inhibition can produce action potentials, we blocked fast chloride-mediated inhibition using either bicuculline methiodide (1 μm; Sigma, St. Louis, MO, USA) or bicuculline free-base (1 μm; Tocris, Bristol, UK) to antagonize GABA A receptors. In addition, strychnine (1 μm; Sigma, St. Louis, MO, USA) was added to block synaptic inhibition by glycine receptors. To block the persistent sodium current, riluzole (5 1 mm; Tocris, Bristol, UK) was added, which has been shown to block this current in medullary respiratory neurons using voltage clamp in vitro (Paton et al. 26). Sodium cyanide (.3%, 5 4 μl) was applied by an intra-aortic injection via a side-port of the aortic perfusion cannula. Histological examination At the end of each experiment, the brain was removed and immersed in.1 M PBS containing 1% formalin for 3 days. The medulla was sectioned transversely (5 μm) and stained with Neutral Red so that the location of recording sites could be assessed microscopically and documented. Data analysis Discharges of reticulospinal neurons and nerves, perfusion pressure, ECG and stimulus marks were recorded directly onto a computer hard drive through an AD converter (141 plus; Cambridge Electronic Design, Cambridge, UK) and analysed using the data capture and analysis software (Spike2; Cambridge Electronic Design, Cambridge, UK). The response magnitude of the activity of nerves or neurons to stimulations was expressed as the percentage change from the prestimulus level. Thus, the percentage change was expressed as follows: (response control) 1/control (%). The firing rate of a neuron during eupnoea, hypercapnic hypoxia and hypercapnia was expressed as both the peak firing rate and the mean firing rate over epochs. In addition, spike-triggered averaging was performed to determine RVLM sympathetic premotor neuronal activity and its relationship with SC activity. We also performed phrenic-triggered averaging to determine respiratory modulation of SC and RVLM neuron activities. Numerical data were expressed as means ± SEM. To compare two different groups statistically, we used Student s paired t test. To compare more than three groups, we used one-way ANOVA. The level of significance was taken as P <.5. Results Sympathetic chain and respiratory activity during eupnoea and gasping; role of persistent sodium current We initially determined the response of the sympathetic outflow during hypercapnic hypoxia. was defined as described previously (St-John & Paton, 23; Paton et al. 26), consisting of a ramp inspiratory motor pattern in PN, postinspiratory discharge in VN and pre-inspiratory and late-inspiratory/early postinspiratory discharges (relative to the PN activity) within SC. Ischaemia (n = 4) or hypercapnic hypoxia (n = 4) both induced gasping (Fig. 1A and C). During gasping, defined by synchronized inspiratory discharges of PN and VN and loss of postinspiratory discharge in VN (Paton et al. 26), SC activity exhibited an inspiratory-related discharge superimposed on a significantly raised level of tonic discharge. Tonic sympathetic activity increased by ± 43.7% (P =.7) during ischaemia-induced gasping and 17.4 ± 43.2% (P =.44) in hypercapnic hypoxia-induced gasping compared with eupnoeic levels. GiventhatC1RVLMneuronalfiring(Kangrga&Loewy, 1995) has been shown to be dependent on the persistent sodium currentin vitro, we next assessed the involvement of this current in the generation of the SC activity response during hypercapnic hypoxia. We administered riluzole, an antagonist of the persistent sodium current, to the perfusate at concentrations that were shown previously to block gasping in thein situ preparation and the persistent sodium current in voltage-clamped respiratory neurons in vitro (Paton et al. 26). During eupnoea, riluzole did not affect PN, VN or SC discharges but abolished gasping when evoked by either arresting perfusion or hypercapnic hypoxia (Fig. 1B and D). In contrast, the evoked increase in tonic SC activity appeared unaffected and not different from the activity levels reached predrug and during the time when gasping was expected to occur (perfusion arrested, ± 42.5%, P =.194; and hypercapnic hypoxia, 83.6 ± 32.9%, P =.194). Thus, the mechanisms for generation of SC activity during eupnoea and asphyxia are not dependent upon the persistent sodium current, whereas gasping is. It was next important to assess whether RVLM sympathetic premotor neurons could maintain their discharge during hypoxic hypercapnia. C 214 The Authors. Experimental Physiology C 214 The Physiological Society

5 Exp Physiol (214) pp Chemosensitivity of the sympathetic premotor neurons 1457 Characteristics of RVLM sympathetic premotor neurons Sixty-six RVLM sympathetic premotor neurons were recorded and confirmed by post hoc histology to be located in the RVLM (Fig. 2A). At the time of their recording, RVLM sympathetic premotor neurons were characterized further by the following: (i) their spontaneous activity (firing rate, 9.5 ±.8 pulses s 1 ); (ii) inhibition by stimulation of baroreceptors as induced by distension of the aortic arch (which also depressed SC activity: neuron, 94.1 ± 1.5%; and nerve, 96.6 ±.7%; Fig. 2B); (iii) activation by stimulation of peripheral chemoreceptors, which was induced by an arterial injection of sodium cyanide (neuron, ± 55.1%; and nerve, ± 15.7%; Fig. 1C); and (iv) synchronization to SC activity as revealed by spike-triggered averaging in many cases (Fig. 2D). Of the 66 RVLM sympathetic premotor neurons, 4 had activity related to the thoracic SC. The interval between the discharge of the neuron and the peak of the SC slow wave was 98.3 ± 13.1 ms. The positive correlation at relatively short latency to SC activity is consistent with these RVLM neurons having relatively direct spinal projections; (vi) of the 66 RVLM sympathetic premotor neurons, we confirmed existence of a descending projection in 11 using the antidromic collision test (Fig. 2E). The estimated conductionvelocity was 1.36 ±.3 m s 1 (range, m s 1 ). GiventhatithasbeenshownthatRVLMsympathetic premotor neurons receive respiratory modulation, we also analysed respiratory modulation of the neurons (McAllen, 1987; McAllen et al. 21; Ootsuka et al. 22). From phrenic-triggered averaging, 52 of the 66 neurons were A B Riluzole No gasp No gasp HR (beats 4 HR 4 (beats min -1 ) min ) PP 16 PP 16 (mmhg) (mmhg) Ischaemia Ischaemia C D Riluzole No gasp No gasp HR 4 HR 4 (beats (beats min -1 ) min -1 ) PP 16 PP 16 (mmhg) (mmhg) Hypercapnic Hypercapnic hypoxia hypoxia Figure 1. Sympathetic chain activity during ischaemia and hypercapnic hypoxia is insensitive to blockade of persistent sodium current A and B, responses to ischaemia in the absence (A) and presence of 7 μm riluzole (B), a blocker of the persistent sodium current. C and D, reponses to hypercapnic hypoxia in the absence (C) and presence of 7 μm riluzole (D). In each left panel, from top to bottom, the traces are as follows: integrated activities of the phrenic nerve ( PN), the central vagus nerve ( VN), the thoracic sympathetic chain ( SC), heart rate (HR) and perfusion pressure (PP). Horizontal bars show the periods of ischaemia and hypercapnic hypoxia. To the right of panels A D, an expanded time scale of recordings at the time points indicated by arrows in each left panel is depicted. C 214 The Authors. Experimental Physiology C 214 The Physiological Society

6 1458 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp respiratory modulated in the following phases: inspiration (and pre-inspiration; n = 21; Fig. 2F); inspiration and postinspiration (n = 13; Fig. 2G); postinspiration (n = 11) and pre-inspiration (Fig. 2H); and postinspiration (and inspiration; n = 7; Fig. 2I). Effects of hypercapnic hypoxia on RVLM sympathetic premotor neuronal firing We tested the effect of hypercapnic hypoxia to the point of gasping on the firing in 51 of the 66 RVLM sympathetic A B C D Sp5 RVLM non-rvlm Amb IO py E o 5 4 HR (beats min -1 ) x F Distension G 5 4 H NaCN I o ms Figure 2. Identification of baro- and peripheral chemoreceptor-sensitive sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) A, recording sites of 66 RVLM sympathetic premotor neurons (filled circles) and three baroreceptor- and peripheral chemoreceptor-insensitive neurons (non-rvlm sympathetic premotor neurons; open circles). Abbreviations: Amb, nucleus ambiguus; IO, nucleus olivaris; py, tractus pyramidalis; and Sp5, spinal trigeminal nucleus. B and C, responses of an RVLM sympathetic premotor neuron to stimulation of aortic baroreceptors and peripheral chemoreceptors. From top to bottom, traces are as follows: integrated activities of the phrenic nerve ( PN), thoracic sympathetic chain ( SC), raw unitary discharge () with its firing rate (bin size, 5 ms) and heart rate (HR). The timing of baroreceptor stimulation is indicated by the horizontal bar in B. Stimulation of peripheral chemoreceptors with sodium cyanide (NaCN; 5 μl of.1%) is indicated by the arrow in C. D, neuronal spike-triggered average of the SC activity (based on 16,432 spikes). Arrow indicates the time of the trigger by the neuronal spike. Integrated SC activity preceded (left of time ) and followed (right of time ) relative to the action potential (at time ) of the RVLM sympathetic premotor neuron. E, antidromic collision test (five superimposed traces). Stimuli (arrows) were applied 16 ms (top traces) and 15 ms (bottom traces) after spontaneously occurring spikes (open circles) to the dorsolateral funiculus of the seventh segment of the cervical spinal cord. Antidromic spikes (cross) were evoked only after a critical delay after a spontaneously occurring spike. F I, phrenic-triggered average of PN and SC and phrenic-triggered firing rate of an RVLM sympathetic premotor neuron (based on 32, 26, 32 and 59 trials in F I, respectively). Arrow indicates the time of the trigger. The neurons in F I had modulation in the pre-inspiratory and inspiratory phases, the inspiratory and postinspiratory phases, the postinspiratory phase and the pre-inspiratory and postinspiratory phases, respectively. C 214 The Authors. Experimental Physiology C 214 The Physiological Society

7 Exp Physiol (214) pp Chemosensitivity of the sympathetic premotor neurons 1459 premotor neurons described above. Of these, firing persistedin42rvlmsympatheticpremotorneurons (Fig. 3A) during the stimulus, but in the remaining nine cells it was either depressed severely or abolished (Fig. 3B). In the hypercapnic hypoxia-activated neurons (i.e. 42 of 66 RVLM sympathetic premotor neurons), a peak tonic firing response of 25.3 ± 2.4 pulses s 1 (control, 9.9 ± 1.1 pulses s 1 ; i.e ± 11.6% increase) occurred 47 ± 4 s after the start of the hypercapnic hypoxic stimulation and, hence, before gasping (Fig. 3A). With prolonged exposure to the stimulus to induce gasping, the tonic firing rate became somewhat reduced (i.e. steady state, 17.1 ± 1.9 pulses s 1, P = ; Fig.3A); the latter value was measured immediately prior to the onset of gasping (i.e. 143 ± 9 s). There was A B 15 1 Hypercapnic hypoxia Hypercapnic hypoxia 3 s 3 s Figure 3. Response patterns of RVLM sympathetic premotor neurons to hypercapnic hypoxic stimulation The RVLM sympathetic premotor neuron in A was sensitive to hypercapnic hypoxia and became excited. This was representative of 82% of cells recorded, whereas the activity of 18% of cells was depressed (B). Each left panel shows the response of an RVLM sympathetic premotor neuron to hypercapnic hypoxia. From top to bottom, traces are as follows: integrated activities of the phrenic nerve ( PN), the thoracic sympathetic nerve ( SC) and firing frequency (bin size, ) of the RVLM sympathetic premotor neuron (). Horizontal bars show the period of stimulation. Each right panel shows a record on an expanded time scale during eupnoea and gasping from the time points indicated by arrows in the left panels; raw neuronal firing records of the RVLM sympathetic premotor neurons are shown instead of rate histogram plots. no significant difference in the firing responses among groups categorized by respiratory-related modulation (Table 1). A post-stimulus depression of firing was typical, but all neurons eventually returned to their control firing levels some minutes after the hypercapnic hypoxic stimulus was withdrawn and carbogen gas reinstated. For neurons depressed by hypercapnic hypoxia, a firing level of 7.1 ± 1.5 pulses s 1 before stimulation was reduced significantly to.7 ±.5 pulses s 1 (P =.2) as measured during gasping; again, this was not confined to types of respiratory-modulated neurons. Effect of blockade of fast excitatory and inhibitory synaptic transmission on RVLM sympathetic premotor neuronal activity In 26 of the 42 hypercapnic hypoxia-activated RVLM sympathetic premotor neurons (Fig. 4A), we next assessed the effect of blocking fast excitatory and inhibitory synaptic transmission (see Methods for cocktail of antagonists) on both ongoing firing and the hypercapnic hypoxia-induced excitatory firing response. As previously published, the cocktail of antagonists used in the present study blocked fast synaptic transmission effectively in the in situ preparation after 2 min exposure (Paton, 1997; Paton & St-John, 25; Paton et al. 26; St-John et al. 29). As observed previously, effective blockade in this study was further assured based on the following characteristics (see Fig. 4B): (i) abolition of PN activity; (ii) significant attenuation of SC activity to levels comparable to those previously seen after hexamethonium treatment to block ganglionic transmission in situ (Chizh et al. 1998); and (iii) abolition of the pronounced peripheral chemoreceptor reflex-mediated changes in PN, SC and heart rate. In all 26 RVLM sympathetic premotor neurons tested, their steady-state activity persisted unabatedly during blockade of fast synaptic transmission (before blockade, 11.4 ± 1.4 pulses s 1 ; and after blockade, 9.9 ± 1.4 pulses s 1 ; P =.31). We also tested the effect of blockade of synaptic transmission by perfusate containing a low concentration of Ca 2+ and high concentration of Mg 2+ (n = 5). Firing of these neurons remained during blockade of synaptic transmission (before blockade, 7.8 ± 1.9 pulses s 1 ;and after blockade, 11. ± 1.7 pulses s 1 ; P =.33). These data suggest that RVLM sympathetic premotor neurons have the ability to generate tonic discharge independently of both fast excitatory and inhibitory synaptic transmission. Rostral ventrolateral medullary sympathetic premotor neuronal response to hypercapnic hypoxia with fast synaptic transmission blocked In six of the 26 RVLM sympathetic premotor neurons isolated synaptically by the cocktail of C 214 The Authors. Experimental Physiology C 214 The Physiological Society

8 146 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp Table 1. Peak tonic firing and steady-state firing responses of rostral ventrolateral medullary sympathetic premotor neurons to hypercapnic hypoxia Respiratory modulation Peak tonic firing response (%) Steady-state firing response (%) Inspiration (n = 1) ± ± 27.3 Inspiration and postinspiration (n = 9) ± ± 25.5 Postinspiration (n = 8) ± ± 3.1 Pre-inspiration and postinspiration (n = 4) ± ± 36.8 No modulation (n = 11) ± ± Values are means ± SEM. The percentage changes were expressed as (response control) 1/control (%). These present the changes in firing rate of those neurons that increased their discharge to the hypercapnic hypoxia, i.e. those that decreased their activity, are not included (nine neurons). antagonists (see the previous paragraph), we successfully maintained recordings during repeated hypercapnic hypoxia challenges. As observed before blockade of synaptic transmission, hypercapnic hypoxia continued to excite all neurons, giving a peak firing response of 21.2 ± 4.3 pulses s 1 (Fig. 4B). This represented a ± 78.9% increase in firing above basal levels, which occurred 57± after the hypercapnic hypoxic stimulus. The hypercapnic hypoxia steady-state-evoked activity remained significantly above control levels throughout the stimulus (control, 9.7 ± 3. pulses s 1 versus hypercapnic hypoxia, 13.5 ± 3.2 pulses s 1, P =.1). Note that the timings of these measurements are comparable to those made with synaptic transmission intact, because they were made at the expected time to produce gasping based on the control response (i.e. 137 ± 23 s after the starting point of hypercapnic hypoxia). This steady-state firing rate was somewhat lower (8.4 ± 2.7 pulses s 1 )than that seen during hypercapnic hypoxia before blockade of synaptic transmission (P =.13) but, unlike control conditions, firing rate did not recede, suggesting that in the intact system there is active hyperpolarization coincident with the time of gasping. After reintroduction of the control perfusate (carbogen gassed), two RVLM sympathetic premotor neurons fell silent transiently ( 2 s), but all neurons returned to their control firing rate within 5 s. It was also notable that the residual tonic SC activity (recorded simultaneously) was maintained during hypercapnic hypoxia, reflecting that activity in some postganglionic neurons was maintained. We also tested the effect of hypercapnic hypoxia (65 8 s) on five functionally identified RVLM sympathetic premotor neurons in perfusate containing a low concentration of Ca 2+ and high concentration of Mg 2+ (Fig. 4C). All of these RVLM sympathetic premotor neurons showed an excitatory response to hypercapnic hypoxia by ± 15.7% (control, 11. ± 1.7 pulses s 1 versus peak firing, 29. ± 5.8 pulses s 1 ). This peak firing occurred 53 ± 13 s after the hypercapnic hypoxic stimulus. After reintroduction of the control perfusate, one RVLM sympathetic premotor neuron fell silent transiently ( 2 s), but all neurons returned to their control firing rate within 5 s. In sum, our data support the notion that RVLM sympathetic premotor neurons have an intrinsic ability to respond to hypercapnic hypoxia in the absence of fast excitatory and inhibitory synaptic transmission. Rostral ventrolateral medullary sympathetic premotor neuronal response to hypercapnia and NaCN with fast synaptic transmission blocked In order to examine the effect of hypercapnia and hypoxia separately, 1 functionally identified RVLM sympathetic premotor neurons were exposed to hypercapnia and sodium cyanide during blockade of fast synaptic transmission by the cocktail of antagonists. During hypercapnia and blockade of fast synaptic transmission, firing of seven RVLM sympathetic premotor neurons was depressed by 86.2 ± 9.2% (Fig. 5A; control, 7. ± 1.5 pulses s 1 ; and peak firing, 1.2 ±.8 pulses s 1 ; P =.2). On the contrary, three neurons were slightly activated during hypercapnia by 83.6 ± 4.2% (control, 1.5±.4 pulses s 1 ; and peak firing, 2.4 ±.5 pulses s 1 ; P =.55). Stimulation of peripheral chemoreceptors using low doses of sodium cyanide (15 μg) was no longer effective in exciting these neurons (before blockade, 84.4 ± 15.3%; and after blockade, 14.8 ± 4.2%; P <.1), but they were activated by an intra-aortic injection of high doses of sodium cyanide in a dose-dependent manner (Fig. 5B and C). In order to examine whether a post-stimulus depression of RVLM sympathetic premotor neurons in response to hypercapnic hypoxia is caused by hypoxia, in five of 1 RVLM sympathetic premotor neurons, we also tested the effect of hypercapnic hypoxia during blockade of fast synaptic transmission by drugs. During hypercapnic hypoxia, all RVLM sympathetic premotor neurons showed an excitatory response by ± 25.% (control, 4.1 ± 1.5 pulses s 1 versus hypercapnic hypoxia, 9. ± 3.2 pulses s 1 ; P =.22). This peak firing occurred 65 ± 15 s after the hypercapnic hypoxic C 214 The Authors. Experimental Physiology C 214 The Physiological Society

9 Exp Physiol (214) pp Chemosensitivity of the sympathetic premotor neurons 1461 A B C 5 Blockade of synaptic transmission 5 Hypercapnic hypoxia Hypercapnic hypoxia Blockade of synaptic transmission 25 Time of gasp Hypercapnic hypoxia Hypercapnic hypoxia 3 s 3 s 3 s Blockade of synaptic transmission Blockade of synaptic transmission Time of gasp Hypercapnic hypoxia Figure 4. Excitatory response of RVLM sympathetic premotor neurons induced by hypercapnic hypoxia persists after blockade of fast synaptic transmission A and B, before(a) and after blockade of synaptic transmission by the cocktail of antagonists (B). Each left panel shows the response of an RVLM sympathetic premotor neuron to hypercapnic hypoxia. From top to bottom, traces are as follows: integrated activities of phrenic nerve ( PN), the thoracic sympathetic nerve ( SC) and firing frequency (bin size, ) of an RVLM sympathetic premotor neuron (). Horizontal bars show the period of hypercapnic hypoxic stimulation. Each right panel shows a recording on an expanded time scale during eupnoea and gasping taken from time points indicated by arrows in the left panels. Raw discharges of RVLM sympathetic premotor neurons are shown. This RVLM sympathetic premotor neuron is different from the one depicted in Fig. 3A. Note that before blockade of synaptic inputs, all RVLM sympathetic premotor neurons tested showed the same excitatory pattern of response to hypercapnic hypoxia. Typically, neurons showed post-stimulus depression in firing rate but always recovered to control firing rate with time. C, after blockade of synaptic transmission by the perfusate containing a low concentration of Ca 2+ and a high concentration of Mg 2+. This RVLM sympathetic premotor neuron is different from the one depicted in Figs 3A and 4A and B. stimulus. Two RVLM sympathetic premotor neurons showed a post-stimulus depression of firing, and these were the neurons which were depressed by the hypercapnic stimulus (Fig. 5D). In two RVLM sympathetic premotor neurons, we also tested the effect of a high dose of sodium cyanide during the hypercapnia-induced depression of firing (Fig. 5E). During the depression of firing in response to the hypercapnic stimulus, high doses of sodium cyanide could activate RVLM sympathetic premotor neurons. We also observed that RVLM sympathetic premotor neurons (n = 2) exposed to a low concentration of Ca 2+ and a high concentration of Mg 2+ in the perfusate were also activated by the high concentration of sodium cyanide. These data suggest that the intra-aortic injection of a high concentration of sodium cyanide can directly affect RVLM sympathetic premotor neurons without synaptic transmission. Non-rostral ventrolateral medullary sympathetic premotor neuronal responses to hypercapnic hypoxia with fast synaptic transmission blocked In order to determine whether the persistent firing response of RVLM sympathetic premotor neurons during hypercapnic hypoxia before and after blockade of fast synaptic transmission was either unique to this group of cardiovascular neurons or a general characteristic of neurons in this region of the medulla oblongata, we recorded from an additional three neurons within the RVLM area (Fig. 1A). In all cases, these exhibited ongoing firing (1.9 pulses s 1, expiratory modulated; 9.6 pulses s 1, late expiratory modulated; and 12.7 pulses s 1, no respiratory modulation) but were insensitive to baroreceptor and peripheral chemoreceptor stimulation and could not be activated antidromically from the spinal cord. During control trials of hypercapnic hypoxia-induced gasping, neuronal firing was depressed or ceased. After blockade of fast synaptic transmission by the cocktail of antagonists, neuronal activity levels became very low, with occasional spiking only. On subsequent exposure to hypercapnic hypoxia, all neurons fell silent. Discussion Hypertension produced in response to brain hypoperfusion is an important mechanism to maintain brain perfusion (Cushing, 191; Rodbard & Stone, 1955; Dickinson & Thomson, 196; Paton et al. 29). However, the mechanism for detecting brain hypoperfusion remains incompletely understood. Using the in situ arterially perfused preparation, this study reports on the intrinsic response of functionally identified sympathetic premotor neurons in the RVLM to hypoxia and hypercapnia. We C 214 The Authors. Experimental Physiology C 214 The Physiological Society

10 1462 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp observed that functionally identified RVLM sympathetic premotor neurons responded to hypoxia even after blockade of fast synaptic transmission. This may indicate that sympathetic premotor neurons in the RVLM have intrinsic chemosensitivity and can respond to brain hypoperfusion to ensure brain perfusion or that they are activated by signals emanating from local blood vessels and/or astrocytes. We also made a number of other novel observations, as follows. First, the tonic firing of the majority (82%) of RVLM sympathetic premotor neurons was elevated during hypercapnic hypoxia at a time when gasping indicated severe brainstem ischaemia. Our data imply some heterogeneity within the RVLM population, because 18% of neurons were depressed by hypercapnic hypoxia. Whether these sympathetic premotor neurons have distinct projections and a functional role (i.e. non-cardiovascular) remains an open question and is discussed later. Second, respiratory modulation of RVLM sympathetic premotor neurons provides a major synaptic input at rest, but in hypercapnic hypoxia this drive is lost yet tonic firing persists. Third, RVLM sympathetic premotor neurons continued to fire after blockade of fast excitatory and inhibitory synaptic transmission, A B C 1 %Change of Hypercapnia 15 μg 15 μg 3 μg 6 μg 9 μg 12 μg Blockade of synaptic transmission 3 s D s 5 s 5 s 15 μg NaCN 15 μg NaCN 9 μg NaCN Blockade of synaptic transmission Hypercapnic hypoxia 6 s E μg NaCN Hypercapnia 6 s Figure 5. Excitatory response of RVLM sympathetic premotor neurons induced by NaCN after blockade of fast synaptic transmission A, response of an RVLM sympathetic premotor neuron to hypercapnia after blockade of synaptic transmission by the cocktail of antagonists. From top to bottom, traces are as follows: integrated activities of phrenic nerve ( PN), the thoracic sympathetic nerve ( SC) and firing frequency (bin size, ) of an RVLM sympathetic premotor neuron (). Horizontal bars show the period of hypercapnic stimulation. B, responses of an RVLM sympathetic premotor neuron before (left panel) and after application of NaCN (15 or 9 μg, middle and right panel, respectively) into the perfusate as indicated by the arrows. After blockade of synaptic transmission, the RVLM sympathetic premotor neuronal activity did not change with a low dose of NaCN, which can excite peripheral chemoreceptors, but the high dose of NaCN could activate the RVLM sympathetic premotor neuron directly. C, changes of RVLM sympathetic premotor neuronal activity in response to application of NaCN (from 15 to 12 μg) before (open bar) and after blockade of synaptic transmission (filled bars). D and E, responses of an RVLM sympathetic premotor neuron to hypercapnic hypoxia, hypercapnia and a high dose of NaCN after blockade of synaptic transmission. Horizontal bars show the periods of hypercacnic hypoxic and hypercapnic stimulation. Application of NaCN (9 μg) into the perfusate is indicated by an arrow. C 214 The Authors. Experimental Physiology C 214 The Physiological Society

11 Exp Physiol (214) pp Chemosensitivity of the sympathetic premotor neurons 1463 confirming auto-active properties described previously in vitro(guyenet et al. 1989). Fourth, SC activity (basal and in the presence of hypercapnic hypoxia) is riluzole insensitive and presumably independent of the persistent sodium current. Fifth, synaptically isolated RVLM sympathetic premotor neurons typically showed reduced firing in response to hypercapnia, making them distinct from central chemoreceptive retrofacial neurons (Mulkey et al. 24; Guyenet, 26). We acknowledge that there was a surprising paucity of neurons (11 of 66) that we could confirm as spinally projecting by the antidromic collision test. Such negative data are difficult to interpret. We believe that many neurons may have had a spinal projection, and our inability to demonstrate this electrophysiologically most probably relates to a technical issue, because all other major criteria used to characterize these neurons were fulfilled. Moreover, the finding that the discharge of more than half of the neurons studied correlated positively and at relatively short latency to SC activity is consistent with these RVLM neurons having relatively direct spinal projections. It should be emphasized that we did not expect all RVLM neurons to show a positive correlation, because our SC recording was from limited segmental levels. Based on this and the expected responses to baro- and chemoreceptor stimulation and respiratory modulation together with their location in the RVLM, we believe that the majority of neurons analysed in this study were RVLM sympathetic premotor neurons. Some may have had supramedullary projections. These could be involved in the generation of sympathetic circuits via indirect pathways, such as via the hypothalamus (Haselton & Guyenet, 199; Verberne et al. 1999). We found that after blockade of fast synaptic transmission, hypercapnic hypoxia and hypoxic stimulation alone (induced by a high dose of sodium cyanide) continued to excite RVLM sympathetic premotor neurons. This raises the possibility that these neurons are central oxygen sensors. Previous reports have already suggested that RVLM neurons have chemosensitivity (Sun et al. 1992; Sun & Reis, 1994a,b; Mazza et al. 2; D Agostino et al. 29). However, these reports failed to exclude effects of synaptic transmission in the RVLM (Sun et al. 1992; Sun & Reis, 1994b) or to identify whether they are genuinely sympathetic premotor neurons or not (Sun & Reis, 1994a; Mazza et al. 2; D Agostino et al. 29). This is important because RVLM sympathetic premotor neurons are intermixed with ventral column respiratory neurons, amongst others. Indeed, any discussion about the chemosensitivity of physiologically uncharacterized RVLM neurons cannot ascribe their functional role (Mazza et al. 2; D Agostino et al. 29). In the present study, we identified neurons in the RVLM functionally as sympathetic premotor neurons. Our data clearly indicate that RVLM sympathetic premotor neurons can sense and respond to hypoxia. This mechanism may contribute to the Cushing response (Dickinson, 199), in which sympathetic activity is generated to cause hypertension to increase blood flow to the brain in conditions of poor perfusion or oxygen debt. Future studies need to assess the RVLM tissue oxygen threshold that triggers increases in RVLM sympathetic premotor neuronal activity to appreciate the importance of its physiological and/or pathophysiological role fully. In this study, we also recorded non-barosensitive and non-chemosensitive neurons in the RVLM and tested the effect of hypercapnic hypoxia. The activity of these neurons was always depressed by hypercapnic hypoxia before and after blockade of fast synaptic transmission; therefore, excitatory responsiveness to hypoxia appears to be a relatively unique characteristic of RVLM sympathetic premotor neurons, at least in the RVLM. In our study, we blocked fast synaptic transmission with a cocktail of antagonists, i.e. bicuculline, strychnine and kynurenic acid. We fully recognize that this treatment will not block all synaptic inputs to RVLM sympathetic premotor neurons, such as metabotropic receptors. However, we did observe a similar excitatory response of RVLM sympathetic premotor neurons to both hypercapnic hypoxia and hypoxic stimuli when using a perfusate containing a low concentration of Ca 2+ and a high concentration of Mg 2+. Thus, these data are consistent with the viewpoint that the response of these neurons was intrinsic and not dependent upon neuronal synaptic transmission. We found a substantial fraction of RVLM sympathetic premotor neurons (18%) that were either inhibited or their activity abolished by hypercapnic hypoxia. Such findings have previously been reported in anaesthetized cats, rats and rabbits also (McAllen, 1992; Koshiya et al. 1993; Koganezawa & Terui, 27). Interestingly, the activities of different sympathetic postganglionic nerves change in a non-uniform way during hypoxia (Iriki et al. 1971). For example, hypoxia activated visceral and muscle vasoconstrictors but inhibited cutaneous vasoconstrictors and cardiac outflows (Iriki & Kozawa, 1975; Gregor & Jänig, 1977). It has been suggested that these differences are caused by the distinct responses of sympathetic premotor neurons (Ootsuka et al. 22; Cao et al. 24; Koganezawa & Terui, 25). Our findings of hypercapnic hypoxia-sensitive and -insensitive RVLM sympathetic premotor neurons could provide a substrate for these differential sympathetic postganglionic nerve responses. During eupnoea, the SC exhibited a respiratory-related discharge, which could originate from the RVLM because 82% of neurons received respiratory modulation confined mostly to the inspiratory phase, which is consistent with previous reports (McAllen, 1987; McAllen et al. 21; Ootsuka et al. 22). Thus, during eupnoea and the early C 214 The Authors. Experimental Physiology C 214 The Physiological Society

12 1464 T. Koganezawa and J. F. R. Paton Exp Physiol (214) pp stages of a hypercapnic hypoxic stimulus, a substantial source of excitatory drive to RVLM sympathetic premotor neurons appears to be of a synaptic nature, originating, in part, from the respiratory oscillator, as recently described (Moraes et al. 214). In contrast, in conditions of prolonged hypercapnic hypoxia, RVLM sympathetic premotor neurons exhibit greater amounts of tonic activity as the respiratory oscillator fails. In these conditions, respiratory drives cannot be relied upon as a source of excitatory drive due to hypoxia-related respiratory depression (Schmidt et al. 1995). Theintrinsicmechanism we report here comes into play for maintaining tonic excitatory drive. But what provides this tonic drive, whether in normoxia or in prolonged hypercapnic hypoxia? The network versus pacemaker debate for RVLM sympathetic premotor neurons is well voiced (Dampney et al. 2; Lipski et al. 22; Guyenet, 26). For the pacemaker theory, spontaneous discharge of RVLM sympathetic premotor neurons was generated by an intrinsic pacemaker potential, producing ramp-like depolarizations and tonic spiking in RVLM sympathetic premotor neurons recorded intracellularly in vitro (Sun et al. 1988a), but this property was restricted to non-c1 neurons (Sun et al. 1988b). The latter finding appears controversial itself, in that C1 neurons were shown to generate pacemaker potentials by others and are mediated by the persistent sodium current in vitro (Kangrga & Loewy, 1995). Our studies do not allow comment on RVLM sympathetic premotor neuronal activity and its dependence on the persistent sodium current because this was not tested directly. However, we did not find any effect of blocking this membrane conductance with riluzole on the tonic discharge of the SC during either control conditions or prolonged exposure to hypercapnic hypoxia, even though gasping was blocked (as shown previously; Paton et al. 26), confirming drug penetration and viability. This may indicate that the majority of sympathoexcitation generated by the brainstem, including RVLM sympathetic premotor neurons, is dependent upon other mechanisms for pacemaking (i.e. independent of persistent sodium current) that are gained up during hypercapnic hypoxia in the in situ preparation. This may be voltage dependent as we have shown for some inspiratory burster neurones in situ (St-John et al. 29) and as reviewed recently (Smith et al. 213; Richter & Smith, 214). For the network theory, spiking of RVLM sympathetic premotor neurons depends on fast excitatory synaptic drive. Lipski et al. (1996) reported that in anaesthetized in vivo rats generating eupnoea, and in which tissues were well oxygenated, intracellularly recorded RVLM sympathetic premotor neurons did not show evidence of pacemaker-like discharges but had spontaneous irregular discharges that were triggered by excitatory postsynaptic potentials. These authors concluded that the spontaneous discharge of RVLM sympathetic premotor neurons recorded in vivo was generated by synaptic inputs in their experimental conditions. WeproposethatRVLMsympatheticpremotorneurons can switch from being synaptically driven to becoming intrinsically auto-active when exposed to hypercapnic hypoxia and that this transition is both seamless and reversible. Previous studies have clearly shown the importance of intrinsic membrane potassium and calcium currents for the auto-activity of RVLM sympathetic premotor neurons exposed to hypoxia in vitro (Sun & Reis, 1994a; Golanov & Reis, 1999; Wang et al. 21). Moreover, hypercapnia and hypoxia can activate intrinsic membrane conductances to induce tonic firing, which include oxygen-sensitive potassium (Archer et al. 26), calcium-dependent potassium (Kemp et al. 26) and TASK channels (Berg et al. 24). Note that both neurons and glia within hypoxic tissue normally leak potassium ions that can further boost membrane depolarization (Ballanyi, 24), which can enhance pacemaking. The contribution of these mechanisms for generating RVLM sympathetic premotor neuronal activity in ischaemia in vivo awaits further experimental investigation. In conclusion, some RVLM sympathetic premotor neurons may be intrinsically sensitive to low oxygen levels and capable of generating sustained tonic activity automatically. This may be critical for ensuring high levels of sympathetic activity to maintain arterial pressure and adequate perfusion of vital organs when oxygen levels are reduced severely. 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