A brainstem circuit for the expression of defensive facial reactions in rat

Abstract

The brainstem houses neuronal circuits that control homeostasis of vital functions. These include the depth and rate of breathing^1,2 and, critically, apnea, a transient cessation of breathing that prevents noxious vapors from entering further into the respiratory tract. Current thinking is that this reflex is mediated by two sensory pathways. One known pathway involves vagal and glossopharyngeal afferents that project to the nucleus of the solitary tract.^3,4,5 Yet, apnea induced by electrical stimulation of the nasal epithelium or delivery of ammonia vapors to the nose persists after brainstem transection at the pontomedullary junction, indicating that the circuitry that mediates this reflex is intrinsic to the medulla.⁶ A second potential pathway, consistent with this observation, involves trigeminal afferents from the nasal cavity that project to the muralis subnucleus of the spinal trigeminal complex.^7,8 Notably, the apneic reflex is not dependent on olfaction as it can be initiated even after disruption of olfactory pathways.⁹ We investigated how subnucleus muralis cells mediate apnea in rat. By means of electrophysiological recordings and lesions in anesthetized rats, we identified a pathway from chemosensors in the nostrils through the muralis subnucleus and onto both the preBötzinger and facial motor nuclei. We then monitored breathing and orofacial reactions upon ammonia delivery near the nostril of alert, head-restrained rats. The apneic reaction was associated with a grimace, characterized by vibrissa protraction, wrinkling of the nose, and squinting of the eyes. Our results show that a brainstem circuit can control facial expressions for nocifensive and potentially pain-inducing stimuli.

Keywords: breathing; chemosensation; ethmoidal nerve; grimace; nasal epithelium; nociception; trigeminus.

Figure 1.. Delivery of a puff of ammonia vapor into the nasal cavity induces apnea.

See Figures S1 and S2 for anatomical substrate of these data and Figure S2 for spiking responses in spinal trigeminal subnucleus muralis. (A) Schema of the general experimental approach with head fixed, anesthetized and tracheotomized rat; see text for details. (B) Representative examples of an apneic reaction (control) and abolition of the apneic reaction following infraorbital nerve cut or lesion of subnucleus muralis (panel C). (C) Representative histology after lesion of spinal trigeminal subnucleus muralis. Abbreviations: SpVC, spinal trigeminal subnucleus caudalis; SpVIc, caudal aspect of the spinal trigeminal subnucleus interpolaris; SpVIr, rostral aspect of the spinal trigeminal subnucleus interpolaris; SpVM, spinal trigeminal subnucleus muralis (D) Prolongation of the respiratory period induced by ammonia is abolished after lesion of subnucleus muralis. (E) Representative histology after an extensive electrolytic lesion of the intertrigeminal region (ITr). Abbreviations: MotV, trigeminal motor nucleus; PrV, principal trigeminal sensory nucleus; sV, sensory root of the trigeminal nerve; tz, trapezoid body. (F) Delivery of ammonia vapors at the entrance of the nares provokes an apneic reaction that persists after an extensive electrolytic lesion of the ITr. (G) Schema of the general experimental approach for electrical stimulation of the nasal epithelium. (H) Electrical stimulation of the nasal epithelium provokes an apneic reaction which persists after an extensive electrolytic lesion of the ITr.

Figure 2.. Change in firing rate of neurons of the ventral respiratory group upon delivery of ammonia vapors into the nasal cavity.

(A) Representative responses of inspiratory and expiratory preBötzinger cells to a puff of ammonia. The red trace is the breathing signal recorded with a thermistor. (B) Deposit of Chicago Sky Blue at the recording site in the preBötzinger complex. (C) Representative raster plots of spiking by Bötzinger and preBötzinger cells before and after the ammonia puff. (D) Plots of firing rate of Bötzinger and preBötzinger cells before and after the ammonia puff. While all inspiratory cells stop firing, expiratory cells display either no significant change (green dots, p < 0.05) or a marked increase in rate (black dots, p < 0.05).

Figure 3.. Facial reactions induced by delivery of ammonia vapors to the right nostril of a head-restrained rat.

See Figure S3 for comparison with response locked to onset of the first breath after the stimulus. (A) Frames from a video sequence that lie before and after the application of an ammonia vapor. Lines indicate how the motion of facial features were parameterized; the letters refer to panels in this figure. See methods for a detailed description of the analysis; note that vibrissae were measured with a separate line-scan camera from above. For representative video recordings, see Video S1 and S2. (B) Representative inspiratory events to the last air puff preceding delivery of room air or ammonia vapor; data across all rats. (C-H) Delivery of an ammonia puff prompts a short apneic reaction (panel C) together with vibrissa protraction (panel D), closure of the eyelids (panel E), curling of the ears (panel F), and wrinkling of the nose (panels G and H). Note that ear displacement following ammonia delivery did not reach significance because movement of the rat in the sac masked landmarks used to estimate ear motion in about 50 % of the trials (32 air puffs and 35 NH₃ puffs). Significance levels p < 0.05.

Figure 4.. Wiring connections for the grimacing reaction induced by delivery of ammonia vapors near the nose of a rat.

Ammonia activates a pool of excitatory spinal trigeminal subnucleus muralis cells that project to the facial nucleus and prompt closure of the nares, and another pool of inhibitory cells that project to the expiratory cells of the preBötzinger complex. The expiratory cells within the preBötzinger complex are glycinergic and are likely to inhibit the whisking oscillator (vIRt), which normally fires in a sustained manner. Inhibition of glycinergic vIRt cells disinhibits IRt glutamatergic cells (red arrow) which project to the motoneurons that control facial pad muscle, hence a grimace.