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W4312331490 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Regulation of systemic PCO2 is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO2 chemosensory nuclei mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pFL). Here, we use GCaMP6 expression and head-mounted mini-microscopes to image Ca2+ activity in these nuclei in awake adult mice during hypercapnia. Activity in the pFL supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO2, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours. Editor's evaluation This paper presents a novel application of endoscopic imaging with miniscopes in awake-behaving mice to address the important problem of analyzing multicellular activity by calcium activity imaging within circumscribed regions of the medulla oblongata that are proposed to have chemosensory functions for the homeostatic regulation of breathing in response to elevated systemic CO2 (hypercapnia). The authors importantly demonstrate chemosensory responses of neurons in the retrotrapezoid nucleus-RTN, Raphe magnus and pallidus nuclei, and lateral parafacial region- pFL, and they describe regional heterogeneity of cellular responses to hypercapnia in these regions with important functional implications. Analyzing chemosensory properties of these medullary regions has been the focus of numerous studies, but the problem of analyzing regional multicellular chemosensory responses in the awake freely behaving rodent has not been previously addressed, so this paper represents an advance for the field. The authors present a novel catalog of neuronal responses, and they illustrate the feasibility of this imaging approach, paving the way for further development/application of this approach, while indicating its limitations. https://doi.org/10.7554/eLife.70671.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The precise control of breathing is fundamental to the survival of all terrestrial vertebrates. Breathing fulfils two essential functions: provision of oxygen to support metabolism; and removal of the metabolic by-product, CO2. Rapid and regulated removal of CO2 is essential because its over-accumulation in blood will result in death from the consequent drop in pH. Understanding of the complexity of the brain microcircuit regulating CO2 and pH remains incomplete. Whilst the primacy of the ventral medulla surface (VMS) in central chemoreception was described almost 60 years ago, this region contains multiple chemosensory nuclei that are candidates to mediate adaptive control of breathing (Nattie and Li, 1994; Richerson, 1995). This has led to the idea that central chemosensitivity is mediated by a network of chemosensory nuclei distributed throughout the brainstem and extending as far as the limbic system (Huckstepp and Dale, 2011b). Furthermore, there are multiple phenotypes of neurons within single chemosensory nuclei (Huckstepp et al., 2018; Iceman et al., 2014; Iceman and Harris, 2014; Johansen et al., 2015), and even within previously well-defined subpopulations of neurons (Shi et al., 2017; Stornetta et al., 2009). Recently, brain imaging techniques have been developed to allow recording of activity of defined cell populations in awake, freely moving animals even in deep brain structures such as the hypothalamus (Ziv and Ghosh, 2015). These methods require: the expression of genetically encoded Ca2+ indicators such as GCaMP6 in the relevant neurons; the implantation of gradient refractive index (GRIN) lenses at the correct stereotaxic position; and a head-mounted mini-epifluorescence microscope to enable image acquisition during the free behaviour of the mouse. We have now adapted these methods to enable recording of defined neuronal populations in the rostral medulla oblongata of mice to analyze the chemosensory control of breathing. In this paper, we study the activity of neurons in the retrotrapezoid nucleus (RTN) (Mulkey et al., 2004; Nattie and Li, 1994; Ramanantsoa et al., 2011) and the rostral medullary raphe (Bradley et al., 2002; Ray et al., 2011; Richerson, 1995). Evidence strongly favours their involvement in the central chemosensory response, but thorough understanding as to how these nuclei contribute to central chemosensitivity has been hampered by the inability to record the activity of their constituent cells in awake freely behaving adult rodents. Instead, neuronal recordings have been made from young (<14 d) or adult rodents under anaesthesia. Both of these methods have significant drawbacks -the chemosensory control of breathing matures postnatally; and anaesthesia is known to depress the activity of respiratory neurons and reconfigure the circuit. We also assess the contribution of the lateral parafacial region (pFL) to an often-overlooked aspect of the hypercapnic ventilatory response, active expiration. During resting eupneic breathing, there is little active expiration instead the expiratory step involves elastic rebound of the respiratory muscles to push air out of the lungs. However, when the intensity of breathing is increased for example during exercise or hypercapnia, active expiration (recruitment of abdominal muscles to push air out of the lungs) occurs. Mounting evidence suggests the pFL may contain the expiratory oscillator and that neurons in this nucleus are recruited to evoke active expiration (Huckstepp et al., 2015; Huckstepp et al., 2016; Pagliardini et al., 2011). Nevertheless, the key step of recording activity of these neurons in awake behaving animals and linking it to active expiration has not been achieved. Our data show the pFL did not undergo sustained activation during hypercapnia, but instead contributed to acute transient high amplitude expiratory events. We found that neurons in the RTN and Raphe exhibited a range of responses to hypercapnia. Many RTN neurons exhibited fast adapting responses. This response type was less common in the Raphe, and many Raphe neurons exhibited a slower graded response. Our data are consistent with a tiered chemosensory network, with the RTN and Raphe responsible for detecting different aspects of the hypercapnic stimulus. These novel findings illuminate the fundamental functional components of chemosensory nuclei and show that neuronal responses to hypercapnia are considerably more complex than anticipated. Results Optical characteristics of the recording system The GRIN lens has a diameter of 600 µm with a focal plane ~300 µm below the lens (Jennings et al., 2015; Resendez et al., 2016). However, the focal plane varies depending upon the distance between the camera and GRIN lens, which can be altered via a manual turret adjustment to optimise the focus on fluorescent cells. To document the range of focal plane depth in our system, we imaged fluorescent beads entrapped in agarose under two conditions: the turret at its lowest (i.e. camera is against the end of the GRIN lens, and the focal plane is at its closest to the lens) and its maximum setting (i.e. the camera is as far from the end of the GRIN lens as it can be, and the focal plane is at its deepest relative to the lens) (Figure 1—figure supplement 1). The focal depth (in which the beads were sharply focussed) was 100 µm at any single setting and the full focal plane ranged from 150 to 450 µm below the end of the lens as the turret moved from its lowest to highest setting. The turret setting is given in all figure legends. Inclusion/exclusion criteria for cells in the study Following assessment for inclusion/exclusion criteria, data from 18 mice comprising recordings from 194 cells were included for analysis (Figure 1A). As the mice were unrestrained and able to move freely during the recordings, to visualize physical movements during Ca2+ analysis high-definition videos of mice were synchronized to the simultaneous plethysmograph and Inscopix Ca2+ imaging recordings in Inscopix Data Processing Software and Spike2. Our recordings are performed at a single wavelength (Figure 1B). Therefore, it was essential to verify that any signals result from changes in Ca2+ rather than from movement artefacts. Figure 1 with 9 supplements see all Download asset Open asset Experimental approach and movement artefact. (A) A CONSORT flow diagram for inclusion/exclusion of experiments and cells from the study. (B) Representation of GRIN lens (microendoscope), baseplate, and mini epifluorescence camera placement for recording of brainstem nuclei. (C–D) GFP control. RTN neurons were transduced with AAV-Syn-GFP (not Ca2+ sensitive) and GRIN lens implanted. Fluorescence was recorded going from 0 to 6% inspired CO2 (gray bar). WBP: whole body plethysmography trace showing breathing movements. No signals are seen that resemble the Ca2+ transients observed with GCaMP6. (E) Comparison of Ca2+ fluorescence signals observed with GCaMP6 versus movement from WBP trace (WBP sonogram) and head movement as observed from video recording (Mov sonogram). In expanded traces- a,b, Examples of lack of fluorescence signal during movement of head/body; c,f, examples of fluorescence signal in the absence of movement; d,e, examples of fluorescence signals during movement. (Scalebar- 20 μm) (F) Seizures caused increase in activity of RTN neurons that was corelated with changes in breathing in anaesthetized mouse. Changes in mouse WBP before and after induction of non-behavioural seizures with intraperitoneal injection of kainic acid time matched with the activity of RTN neurons (red). The dotted square is changes in activity of neurons (ROIs) time matched with WBP and expanded in panel G. (G) Increase in the activity of RTN neurons correlated with changes in breathing. The start of the GCaMP6 transient is shown by the dotted line. Ca2+ transients with a fast rise and exponential fall (pink traces); and slower sustained changes (turquoise traces). We examined the origins of the changes in fluorescence in three ways. Firstly, we transduced RTN neurons of three mice with AAV-syn-GFP and performed imaging during a hypercapnic challenge (Figure 1C–D, Figure 1—video 1). This allowed measurement of comparable levels of fluorescence to GCaMP6 to determine whether movement by itself, in the absence of any Ca2+ reporter activity, could generate confounding fluorescent signals. Analysis of changes in fluorescence showed only small amplitude (often negative-going) fluctuations. In no case did we observe large increases in fluorescence of the type we routinely observed when GCaMP6 was expressed (e.g. Figure 1E), suggesting that movement per se cannot give fluorescence signals that resemble Ca2+ signals. Secondly, we analyzed the movement of the mouse simultaneously with the Ca2+ recordings (Figure 1E) via two methods. The first was to analyze the headmount movements in the video recordings of the mouse in the plethysmography chamber using AnyMaze tracking software and convert these movements into a colour coded sonogram. The brighter colours in the sonogram indicate headmount movement. The second method analyzed the large excursions on the whole body plethysmography (WBP) trace. These arise artefactually from movement. Comparison of the sonogram based on the WBP trace (WBP Sonogram) and that based on head movement (Mov Sonogram) showed that the two measures correlated well and that large excursions of the WBP trace often coincided with head movement (compare sonograms in Figure 1E). When we examined the GCaMP6 fluorescence in conjunction with the sonograms of head and body movement, we observed that: (i) large movements of the head/body did not evoke noticeable changes in GCaMP6 fluorescence (Figure 1Ea,b); (ii) GCaMP6 Ca2+ signals characterized by a fast rise and exponential decay occurred in the absence of any movement (Figure 1Ec,f); and (iii) similar shaped Ca2+ signals occurred during movement (Figure 1Ed,e). Together this suggests that movement of the head or body of the animal does not prevent the recording of genuine changes in GCaMP6 fluorescence related to changes in intracellular Ca2+. Thirdly, we used the same paradigm of stimulation for all experiments as can be seen from the records in Figure 1—figure supplements 2–4. This enabled averaging of the responses aligned either to the onset of hypercapnia, or features of the WBP trace. This averaging brought out consistent features of the presumed Ca2+ signals that were temporally related to either the onset of hypercapnia or alterations of breathing. While for the most part we could not record from the same neurons in different recording sessions from the same mouse, we did observe consistent patterns of neuronal firing between recording sessions (Figure 1—figure supplement 5). As movement that could give rise to artefact would not be expected to be similar from recording session to recording session, or from mouse to mouse, the consistency of the patterns of the signals between mice and recording sessions strongly suggests a true biological rather than artefactual origin of the recorded signals. In some cases, it was possible to identify the same neuron from recording sessions on different days in the same mouse. This was only the case in a minority of sessions perhaps because the brainstem is extracranial and more mobile and thus it is harder to reproduce the same exact focal plane between different recording sessions. When we were able to identify the same neurons their activity patterns were remarkably similar between separate recording sessions (Figure 1—figure supplement 6). This gives further confidence that the observed patterns are a reflection of biological properties. Finally, we made recordings from anaesthetized mice (Figure 1F and Figure 4—figure supplement 1). In this case movement artefacts cannot be a potential confounding factor. To elicit neural activity, we induced electrographic seizures via intraperitoneal kainic acid injection in mice in the absence of any behavioural seizure movements such as partial (including forelimb or hindlimb) or whole body continuous clonic seizures (Figure 1—video 2). After ~70 min this resulted in perturbations of the WBP trace and correlated Ca2+ activity in the RTN neurons, presumably denoting the invasion of seizures into this nucleus. Detailed examination revealed that the fluorescence changes were of two types: (i) transients with a fast rise and exponential fall; and (ii) slower sustained changes (Figure 1G). This is an important observation as it shows that we expect to see two types of genuine Ca2+ signal in recordings from freely-moving mice: fast transients with an exponential decay phase, that may sum together and slower more gradual rises. Movements of the visual field, evident during most recordings, were corrected for via the Inscopix motion correction software to allow ROI-based measurement of fluorescence to remain in register with the cells during the recording. Where movement artefacts persisted following motion correction, in some cases it was possible to draw additional ROIs over a high contrast area devoid of fluorescent cells, such as the border of a blood vessel. This allowed for a null signal to be subtracted from the GCaMP signal (Figure 7E) thus providing the neuronal Ca2+ transients free of movement artefact. If there was too much uncompensated motion artefact, so that we could not clearly analyze Ca2+ signals, we excluded these recordings from quantitative analysis (details given in Figure 1A). GCaMP6 signals were only accepted for categorization if the following criteria were met: (1) the features of the cell (e.g. soma, large processes) could be clearly seen; (2) they occurred in the absence of movement of the mouse or were unaffected by mouse movement; (3) fluorescence changed relative to the background; (4) the focal plane had remained constant as shown by other nonfluorescent landmarks (e.g. blood vessels). Applying these criteria left 144/194 cells eligible for study. All of the recordings that were included are shown in Figure 1—figure supplements 2–4. The activity of the cells fell into the following categories: Inhibited (I) Displayed spontaneous Ca2+ activity at rest, which was greatly reduced during both 3 and 6% inspired CO2 and could exhibit rebound activity following the end of the hypercapnic episode. Excited - adapting (EA) Silent or with low level activity at rest and showed the greatest Ca2+ activity in response to a change in 3% inspired CO2. Following an initial burst of activity, they were either silent or displayed lower level activity throughout the remainder of the hypercapnic episode. These cells often exhibited rebound activity following the end of the hypercapnic episode due to suppression by higher CO2 levels. These cells essentially encoded the beginning and end of hypercapnia. Excited - graded (EG) Silent or with low level activity at rest and displayed an increase in Ca2+ activity at 3% inspired CO2 with a further increase in activity at 6% that returned to baseline upon removal of the stimulus. These cells encoded the level of inspired CO2. Tonic (T) Displaying spontaneous Ca2+ activity throughout the recording that was unaffected by the hypercapnic episode but could provide tonic drive to the respiratory network. Sniff-coding (Sn) Displayed elevated Ca2+ signals correlated with exploratory sniffing. Expiratory (Exp) Displayed elevated Ca2+ signals correlated with large expiratory events. Non-coding (NC) Displayed low frequency or sporadic Ca2+ activity that was neither tonic in nature nor had any discernable activity that related to the hypercapnic stimulus or any respiratory event. Non-coding respiratory related (NC-RR) Displayed Ca2+ activity that did not code the hypercapnic stimulus, but it was instead related to variability in breathing frequency. Our categorisation of neurons into these types was supported by a two-component analysis in which we plotted the change in Ca2+ activity (from baseline) elicited by 6% inspired CO2 versus the change in Ca2+ activity elicited by 3% inspired CO2. EA neurons as they showed more activity at 3% compared to 6% CO2 should cluster below the line of identity (x=y), whereas EG neurons as they respond more strongly to 6% than 3% should cluster above the line of identity. NC neurons as they did not show a response to hypercapnia should be clustered around the origin (x=y=0), and the I neurons should be clustered in the quadrant where the change in Ca2+ activity to both stimuli is negative. This analysis shows that the different classes of neurons in both the RTN and Raphe do indeed cluster in the appropriate regions of the graph (Figure 1—figure supplement 7). Chemosensory responses in the RTN As the RTN contains chemosensitive glia and neurons (Gourine et al., 2010; Mulkey et al., 2004; Nattie and Li, 1994; Ramanantsoa et al., 2011), and is neuroanatomically diverse (Shi et al., 2017; Stornetta et al., 2009), we began by targeting all neurons of the RTN with a synapsin-GCaMP6s AAV (Figure 2A–C); a necessary step to enable their imaging during free behaviour via a mini-headmounted microscope (Figure 1B). The localization of successful and accurate transduction was confirmed posthoc by using choline acetyltransferase (ChAT) staining to define the facial nucleus and the location of the transduced cells (Figure 2B), with ~7% (9/132) of transduced neurons in the focal plane of the microscope co-labelled with ChAT (Figure 2C). The lens track terminated under the caudal pole of the facial nucleus, containing the highest number of neurons (Figure 2B), with the focal plane 150–450 µm below that, covering the dorso-ventral extent of the RTN (Figure 1—figure supplement 1). Therefore, our viral transduction and lens placement allowed us to record from the RTN. In some mice (n=3), we further checked the identity of transduced neurons by examining whether they expressed neuromedin B (NMB), a marker specific to Phox2b chemosensory neurons in the RTN (Figure 2D, Figure 2—figure supplement 2, Li et al., 2016; Shi et al., 2017). We found 43% (40/93) of NMB + cells in the RTN were also transduced with GCaMP6, and 20% (40/204) of GCaMP + neurons in the RTN were NMB+. Thus, we transduced nearly half of the NMB + neurons, and if the fluorescence imaging sampled transduced cells randomly and without bias to cell type, roughly 1 in 5 of cells recorded would have been of this phenotype. Figure 2 with 4 supplements see all Download asset Open asset Excitatory chemosensory responses of RTN neurons in awake mice. (A) AAV9-Syn-GCaMP6s injection into the RTN. (B) Micrograph of lens placement and viral transduction of neurons (green) relative to the facial nucleus (ChAT +neurons red). (C) Venn diagram of cell counts of GCaMP6s transduced (green), and ChAT+ (red), neurons under the GRIN lens (1 representative section from each of 4 mice). (D) Neurons transduced with GCaMP6s also contain NMB confirming their identity as RTN neurons. (E) Venn diagram of cell counts of GCaMP6s transduced (green), and NMB+ (magenta), neurons under the GRIN lens (1 representative section from each of 3 mice). (F) Recording of mouse whole body plethysmography (WBP) in response to hypercapnia time-matched with Inscopix recorded GCaMP6 signals. These show neurons that gave a graded response to CO2 (EG). (G) Average of the graded neuronal responses aligned to the WBP trace in (F). (H) Examples of neurons that showed an adapting response to a change in inspired CO2 (EA). WBP trace aligned to the GCaMP6 fluorescence and instantaneous VE (minute ventilation) shown to demonstrate how the signals in the EA neurons closely correspond to VE. (I) Average waveform of all EA neurons in the RTN aligned to 0, 3 and 6% inspired CO2 (gray bar). (J) A plot of VT versus F/F0 for the Ca2+ signal during the transition from 0 to 3% CO2 in three individual mice showing positive correlation. Underneath each VT vs F/F0 graph is a plot of change in VT (grey) and average of EA neuronal Ca2+ activity (magenta) from respective mouse during transition from baseline to 3% CO2. Note that there is a transient increase in breathing at the beginning of hypercapnia that corresponds to the activation of the EA neurons. Abbreviations: 7 N, facial motor nucleus; Py, pyramidal tract; MVe, medial vestibular nucleus; sp5, spinal trigeminal nucleus: RTN, retrotrapezoid nucleus; RMg, raphe magnus; RPa, raphe pallidus; pFL, parafacial lateral region. After assessing the quality of recordings, we retained Ca2+ activity traces from 98 RTN neurons in 9 mice (Figure 1A). There was a wide variety of neuronal responses to the hypercapnic challenge (Figure 1—figure supplement 2). While we observed 4/98 neurons exhibited a graded response to hypercapnia (EG, Figure 2F and G, Figure 2—video 1), a much greater number (27/98) were of the adapting subtype (EA, Figure 2H1, Figure 2—video 2). This adapting response may also be physiologically meaningful, as it matched the time course of changes in VE calculated from the WBP records (Figure 2H). In three mice the averaged Ca2+ trace of the EA neurons in each mouse matched the changes in VT for the same mouse and a plot of VT versus F/F0 for the Ca2+ signal gave a positive correlation (Figure 2J, and Supplementary file 1). This correspondence between features of the responses in these neurons and the adaptive ventilatory response, supports the hypothesis that these are a physiologically important class of chemosensitive neurons. An alternative explanation that the rates of Ca2+ sequestration greatly increased during hypercapnia such that Ca2+ levels fell even although firing rates increased can be excluded by examining the dynamics of individual Ca2+ transients. The rise time of these transients reflects the rate of Ca2+ influx, and the exponentially falling decay phase the rate of Ca2+ extrusion or sequestration. Comparison of these transients before, during and after the hypercapnic stimulus shows that these transients do not change in shape (Figure 2H, Figure 2—figure supplement 2). A further 5/98 neurons were inhibited during hypercapnia (Figure 3A and C, blue traces and Figure 3—video 1). These neurons displayed spontaneous Ca2+ activity during normocapnia, but were abruptly silenced during hypercapnia (Figure 3C). We recorded from sniff-coding (Sn) neurons, 3/98, which showed elevated Ca2+ signals correlated with sniffing (Figure 3A and B). In sniff-coding neurons, Ca2+ activity coincided with perturbations of the plethysmography traces (increase in respiratory frequency, increase of tidal volume, presence of expiration, Figure 3B and Figure 3—video 1). Analysis of the onset of the Ca2+ signal relative to the start of the sniff, showed that, on average, activity in these neurons preceded the sniff by 0.4–0.8 s (Figure 3D). The temporal correlation between the sniff-coding neurons and the sniff was further confirmed by averaging the Ca2+ recordings from multiple sniffs (Figure 3E). Interestingly the different functional types of neuron are clustered adjacent to each other (Figure 3F). Figure 3 with 1 supplement see all Download asset Open asset Sniff-coding, and CO2-inhibited RTN neuronal responses in awake mice. (A) Recording of mouse WBP in response to hypercapnia time-matched with Inscopix recorded GCaMP6 signals. CO2-Inhibited (blue) and sniff-coding (eggplant purple) calcium traces correspond to the neurons shown in panel F. (B) Expanded recordings from (A), the start of the Ca2+ transient is shown by the dotted line. (C) Average waveform of RTN inhibited neurons in response to hypercapnia. (D) Sniff-correlation histogram and (E) Spike triggered average (eggplant purple line) of all Ca2+ events (grey lines) temporally correlated to the beginning of sniff activity (dotted verticle line). (F) GCaMP6s fluorescence of transduced RTN neurons in freely behaving mice. Individual regions of interest (ROIs) drawn around CO2-inhibited (blue) and sniff coactivated (eggplant purple) neurons. (Scalebar- 20 μm). Figure 3—source data 1 Data for Figure 3 panels D and E. https://cdn.elifesciences.org/articles/70671/elife-70671-fig3-data1-v2.xlsx Download elife-70671-fig3-data1-v2.xlsx Additionally, 5/98 neurons which were tonically active but did not encode any information about hypercapnia (T, Figure 4A, Figure 4—video 1). Tonically active neurons in the RTN could provide background excitation to the respiratory network. Of 98 neurons, 35 showed sporadic activity that did not correlate with CO2 (NC, Figure 4B) and a further 19/98 neurons that did not encode CO2 levels but displayed Ca2+ signals that were correlated to changes in respiratory frequency and were generally silent when tidal volume was at its greatest (NC-RR, Figure 4C). Figure 4 with 3 supplements see all Download asset Open asset Tonically active, non-coding (NC) and NC-respiratory related (NC-RR) RTN neuronal responses in awake mice. Recording of mouse WBP in response to hypercapnia time-matched with Inscopix recorded GCaMP6 signals (A, B, C). GCaMP fluorescence of RTN (A) tonically active (T; violet blue) (B) non-coding (NC; green) and (C) non-coding respiratory related (NC-RR; shamrock green) neurons. Average of NC-RR neuronal activity (dotted box) expanded under it displayed Ca2+ signals that were correlated to changes in respiratory frequency (fR) and were generally silent when tidal volume (WBP) was at its greatest. In summary for RTN neurons: ~40% (39/98) had activity patterns that were in some way modulated by CO2. The majority of these neurons, 69% (27/39), displayed an adaptive response to CO2, thus marking the onset of hypercapnia. Only a minority of neurons, 10% (4/39), encoded the magnitude of hypercapnia and a similar minority (13%; 5/39) were active and inhibited throughout the hypercapnic episode. Interestingly, we found a small number of sniff-activated neurons, which may be from the most rostral and ventral aspect of the retrofacial nucleus, which overlaps with the most caudal and dorsal aspect of the RTN (Deschênes et al., 2016; Kurnikova et al., 2018). Effect of anaesthesia on RTN chemosensory responses Until now most investigation of RTN chemosensory responses at adult life stages has been in anaesthetised preparations. We therefore examined the effect of deep anaesthesia on the activity of RTN neurons before and during chemosensory stimuli (Figure 4—figure supplement 1). As might be expected, compared to the awake state, urethane anaesthesia had a deeply suppressive effect on the spontaneous activity of all RTN neurons. Whereas almost all neurons displayed spontaneous activity, there was hardly any activity under anaesthesia (Figure 4—figure supplement 1A-B). Furthermore, the responses to hypercapnia were greatly blunted, with many neurons simply being unresponsive. When we examined those neurons that retained a chemosensory response to hypercapnia in the anesthetised state, these responses differed from those that the same neurons showed in the awake state (Figure 4—figure supplement 1C). For ex" @default.
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W4312331490 date "2022-03-14" @default.
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W4312331490 title "Author response: Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice" @default.
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