Lateral parabrachial FoxP2 neurons regulate respiratory responses to hypercapnia

Abstract

Although CGRP neurons in the external lateral parabrachial nucleus (PBel^CGRP neurons) are critical for cortical arousal in response to hypercapnia, activating them has little effect on respiration. However, deletion of all Vglut2 expressing neurons in the PBel region suppresses both the respiratory and arousal response to high CO2. We identified a second population of non-CGRP neurons adjacent to the PBel^CGRP group in the central lateral, lateral crescent and Kölliker-Fuse parabrachial subnuclei that are also activated by CO2 and project to the motor and premotor neurons that innvervate respiratory sites in the medulla and spinal cord. We hypothesize that these neurons may in part mediate the respiratory response to CO2 and that they may express the transcription factor, Fork head Box protein 2 (FoxP2), which has recently been found in this region. To test this, we examined the role of the PB^FoxP2 neurons in respiration and arousal response to CO2, and found that they show cFos expression in response to CO2 exposure as well as increased intracellular calcium activity during spontaneous sleep-wake and exposure to CO2. We also found that optogenetically photo-activating PB^FoxP2 neurons increases respiration and that photo-inhibition using archaerhodopsin T (ArchT) reduced the respiratory response to CO2 stimulation without preventing awakening. Our results indicate that PB^FoxP2 neurons play an important role in the respiratory response to CO2 exposure during NREM sleep, and indicate that other pathways that also contribute to the response cannot compensate for the loss of the PB^FoxP2 neurons. Our findings suggest that augmentation of the PB^FoxP2 response to CO2 in patients with sleep apnea in combination with inhibition of the PBel^CGRP neurons may avoid hypoventilation and minimize EEG arousals.

Figure 1. CO2 activates cFos expression in the PB^FoxP2 neurons:

Photomicrographs in columns a-d represent sections at the rostral (level 1, first row), middle (level 2, second row) and more caudal (level 3, third row) portions of the PB labeled immunohistochemically for both the immediate early gene cFos (green) and FoxP2 (red), from a mouse that was exposed to 2h of either normocapnic room air (columns a and b) or 10% CO2 (columns c and d). The insets in a and c demarcate the areas that are magnified in the b and d. Double-labeling (yellow nuclei) was prominent in the KF and in the central lateral FoxP2 clusters in mice exposed to CO2 but not those breathing normocapnic air. The arrow heads in b and d point to doubly-labeled neurons, while the arrows mark neurons that were only labeled for cFos (green), a large cluster of which represent the CGRP neurons in the PBel (c2, c3). The graphs in e and f compare the percentage of the cFos cells that also expressed FoxP2 (e), and the percentage of Foxp2 cells that were also labeled for cFos (f) in the PBcl and KF areas, after exposure to 10% CO2 (hypercapnia, n=5) or room air (normocapnia, n=3). The groups were analyzed using a one-way ANOVA, where ***- P<0.001; **- P<0.01. Scale in c= 200μm; d= 20 μm. Abbreviations: KF- Kölliker Fuse PB subnucleus; PBcl- central lateral PB subnucleus; PBel- external lateral PB subnucleus; scp- superior cerebellar peduncle; vsct- ventral spino-cerebellar tract.

Figure 2. In vivo intracellular calcium imaging of PB^FoxP2 neurons by fiber photometry during exposure to CO2:

Adeno-associated virus (AAV) expressing Cre-dependent GCaMP6s was injected into the lateral PB of FoxP2-Cre mice (a), resulting in green GCaMP6s expression in the PB^FoxP2 neurons (red) (b). The track of the implanted optical fiber just dorsal to GCaMP-expressing PB^FoxP2 neurons is outlined in b and allowed us to record their calcium activity as shown in the schematic in c. A representative fiber photometry recording (d) shows the activity profile of the PB^FoxP2 neurons (ΔF/F), with simultaneous EEG/EMG signals and the respiratory rate (RR) and minute ventilation (MV = RR × tidal volume, V_T), both of which increased significantly in each CO2 trial. The control UV signal (isobestic at 405nm) was also recorded. Note that the GCaMP signal increases slowly during CO2 exposure, but more sharply as the animal wakes up (abrupt change in EMG and EEG about 25 sec after CO2 onset- marked by red arrow). The ΔF/F from 59 trials from n=5 mice is depicted in e as a heat-map illustrating activity 15 s before and after onset of CO2 exposure. The graphs below show the mean + SEM ΔF/F normalized to the pre-CO2 values (f), % CO2 in the plethysmograph (g), respiratory rate (RR, h) and tidal volume (V_T, i) across the same trials. Two way ANOVA compared the changes in ΔF/F, RR and V_T during CO2 exposure compared to the pre-CO2 baseline, where ***- P<0.001; **- P<0.01 and *- P<0.05. Scale in b, 200μm (lower left) and 20μm (lower right). Scale in b= top right 100μm; bottom right= 20 μm. Abbreviations: PBcl- central lateral PB subnucleus; PBel- external lateral PB subnucleus; scp- superior cerebellar peduncle.

Figure 3. In-vivo activity of individual PB^FoxP2 neurons during CO2 exposure:

Gradient-index (GRIN) lens were implanted above the injection sites in the PB of FoxP2-cre mice injected with Cre-dependent AAV-GCaMP6s (a) and the calcium activity profiles (ΔF/F) of individual PB^Foxp2 neurons in response to the CO2 was acquired. Representative intracellular calcium activity of 3 neurons is shown for 30 sec before and 60 sec after onset of CO2 (b) and in the same trial for 10 sec before and 60 sec after onset of CO2 (c). Two cells (marked by green and yellow arrowheads whose activity profiles are also plotted in green and yellow) shown in b had fluorescence that peaked at about 17–19 sec after exposure, before the maximal changes in respiration. A third cell (marked by a magenta arrow and activity profile) peaked roughly 50 sec after onset of the CO2 trial (c), but while CO2 levels were still high. A representative trial that showed no cortical arousal during CO2 exposure but had a clear respiratory response to the 8% CO2 as seen by a gradual rise in the respiratory (RR) and the minute ventilation (MV) is shown in d. The ΔF/F from 9 cells is also plotted, showing the overall increase in intracellular calcium across the population, with most cells showing peaks at the time of maximal respiration, but with substantial variability across neurons (d). A heat map of the mean ΔF/F over 4–7 trials (during which animals exposed to 8% CO2 did not awaken) is shown for all 28 cells in e (blue to red- shows low to high ΔF/F). Note that although the RR and MV summated over these trials increased relatively smoothly (f), the activation of the PB^FoxP2 neurons occurred in waves, and of the 17 cells that showed 3 peaks, 10 of them showed second and third peaks that were 17–19 sec apart and were synchronous in time after CO2 exposure, but that not every neuron participated in each wave of excitation. Two way ANOVA compared the changes in RR and MV post CO2 to the Pre-Co2 baseline, where ***- P<0.001.

Figure 4. Effect of photoactivation of PB^FoxP2 neurons on respiration:

FoxP2-cre mice were injected bilaterally targeting the PB with cre-dependent AAV-Flex-ChR2-mCherry (red a, b; also immunostained for FoxP2 (green florescence, b2,3,5) and implanted for EEG/EMG recording and with bilateral glass optical fibers to target illumination of the ChR2-expressing FoxP2 neurons (b1,2). The right side of the coronal section in b1, is also shown in b2 with immune-labeling for Foxp2 (green). The area within the box in b2 is shown at higher magnification in b3–b5, and the doubly labeled neurons (yellow) are shown at higher magnification in the inset in b5 (scale= 50 μm). Scale in b1- b5=100μm. Abbreviations: PBel- external lateral PB subnucleus; scp- superior cerebellar peduncle. Respiration was analyzed for 5 breaths pre-stimulation, then for 5 breaths just before the end of stimulation or before cortical arousal and for 5 breaths after cortical arousal when stable breathing was attained without EMG artifacts. A representative trial with stimulation at 20 Hz (in normocapnic air) showed gradually increasing respiration which preceded the cortical arousal that occurred at 4.5 sec in this trial (c). In trials with 5 sec stimulation, the animals on an average woke up around 15 sec after the stimulation stopped (d), suggesting that the awakening was not due to the stimulation of the PB^FoxP2 themselves, but may have been elicited by some aspect of the response to stimulation (e.g., respiratory efforts). Trials with stimulation for 10 sec usually caused EEG arousal either just before or after the termination of stimulation. Arousal latency was decreased dramatically (by 83%) by exposing the mice to continuous 2% CO2 during opto-stimulation (d, shown in yellow rectangle). A representative trial of stimulation at 20Hz for 10sec with continuous 2% CO2 is shown in e. Graphs in f compare the RR, V_T and MV in mice subjected to laser stimulation at 5, 10 or 20Hz vs. no laser (Laser-OFF) for either 5 or 10s during normocapnia. Graphs in g compare the RR, V_T and MV parameters in the mice subjected to laser stimulation at 20Hz vs. no laser (Laser-OFF) for 10s, in mice continuously exposed to 2% CO2 (as shown in e). The average values (mean ± SEM) are plotted for 5 breaths before onset of stimulation, during stimulation but before cortical arousal and in the post stimulation period after the cortical arousal when stable breathing is attained without EMG artifacts (e-g). Two-way ANOVA was used for statistical comparison, where **= P<0.01 (vs. pre-stimulation/Laser OFF); ***= P<0.001 (vs. pre-stimulation/Laser OFF), #= P<0.05 (vs. Laser-ON at normocapnia).

Figure 5. Descending projections of the PB^FoxP2 neurons:

Bilateral injections of Cre-dependent AAV-ChR2 (magenta) into the PB in a FoxP2-Cre mouse (a1) is shown with immuno-labeled for FoxP2 (green); nearly all of the ChR2-labeled cell bodies are doubly labeled (white) in the area along the dorsal margin of the PBel (a, magnified view of injection site on the right side). The inset in a, is a magnified view of the area marked by the dashed-rectangle. Descending fibers and terminals in the medulla are shown in magenta in b and c. Note that inferior olive neurons (green) also express FoxP2. Scale in a= 100μm, in c=500μm. The photomicrograph in d shows the injection site of the retrograde tracer cholera toxin subunit b (CTb −0.2%, magenta) in the pre-Bötzinger area (PBZ, d) (n=3; scale= 500μm). The injection of CTb in PBZ (d) retrogradely labeled many FoxP2 neurons in the KF (e1–e4) and the PB area (f1–g3), especially in the lateral crescent and the central lateral PB (PBcl) areas that surround the PBel subnucleus. The arrow heads in e4, f3 and g3 mark the cells doubly labeled for FoxP2 and CTb (white), while the arrows mark cells labeled only with CTb (magenta). The insets in f3 and g3 are 2x magnified views of the areas encompassed by dashed rectangles, highlighting the double labeled cells. Scale in e1= 100 μm; in e3= 50 μm and in e4= 30 μm. Scale in g1 and g3= 100 μm. Abbreviations: CVLM- caudal ventrolateral medulla; KF- Kölliker Fuse PB subnucleus; NTS, nucleus tractus solitarii; PBZ- pre-Bötzinger area; PBcl- central lateral PB subnucleus; PBel- external lateral PB subnucleus; RVLM – rostral ventrolateral medulla; scp- superior cerebellar peduncle; vsct- ventral spino-cerebellar tract; XIIn – hypoglossal nucleus.

Figure 6. Acute optogenetic silencing of the PB^FoxP2neurons blocks hypercapnia-induced respiratory drive:

FoxP2-Cre mice were injected with AAV-FLEX-ArchT (a), and implanted for EEG/EMG and with bilateral optical fibers targeting PB^FoxP2 neurons (c1, 2). There was virtually complete co-expresion of FoxP2 (red) and ArchT (green, c3–5). The experimental strategy to test respiratory responses to 10% CO2 given for 30s every 300s, with and without photo-inhibition of the PB^FoxP2 neurons is shown as schematic in b. Representative examples of a laser-ON and a Laser-OFF trial are shown in d and e, with magnified views of the bounding box from each trial shown in d₁ and e₁. For each stimulation, the laser was switched ON for 60s beginning 20 sec prior and extended for 10 sec after the CO2 stimulus (30 sec, illustrated by yellow box) as shown in e. Photoinhibition of PB^FoxP2 neurons significantly reduced the increases in V_T (g) and MV (h) but not RR (f) caused by CO2 exposure. Two way ANOVA, with *- P<0.05, compared to the pre-stimulation. The photo-inhibition of PB^FoxP2 neurons did not change the latency to arousal (i).

Figure 7. Neural circuit for mediation of hypercapnia induced increase in ventilation:

PB neurons receive information about CO2 levels via relays from the carotid body through the NTS (nucleus of the solitary tract), and directly from chemosensory neurons including the RTN (retrotrapezoid nucleus)^,,,, and medullary (Med R) and dorsal (DR) raphe nuclei^,,. The projections of the PB^FoxP2 neurons to the preBötzinger complex (PBötZ), rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM) may mediate the increase in ventilation by increasing respiratory rate (RR)^,, and tidal volume (V_T) or directly by the projections from the Kölliker-Fuse FoxP2 neurons to the phrenic motor^,,. Mechanical sensory feedback by thoracic stretch receptors during increased ventilatory effort may activate PBel^CGRP neurons to cause cortical arousal, via activation of neurons in the basal forebrain (BF), central nucleus of amygdala (CeA), and lateral hypothalamic area (LHA)^,. Selective activation of PB^FoxP2 neurons may be effective for augmenting breathing in apneas, if combined with ways to suppress the EEG arousal.