BRAIN RESEARCH ELSEVIER Brain Research 671 (1995) 314-316 Short communication Role of the parabrachial nuclei in the airway dilation evoked by the Hering-Breuer reflex Ann M. Motekaitis *, Irene C. Solomon, Marc P. Kaufman Division of Cardiovascular Medicine, Departments of Internal Medicine and Human Physiology, University of California, Davis, TB 172, Davis, CA 95616, USA Accepted 22 November 1994 Abstract We tested the hypothesis that blockade of glutamatergic receptors in the parabrachial nucleus (PBN) of chloralose-anesthetized cats attenuated the reflex airway dilation evoked by activation of pulmonary stretch receptors. Unilateral microinjection of kynurenic acid (50 nl, 100 mM) into the PBN reversibly attenuated the reflex relaxation of the trachealis muscle in 7 cats. These findings suggest that the PBN is part of the central pathway mediating the airway dilation component of the Hering-Breuer reflex. Keywords: Airway smooth muscle; Autonomic nervous system; Pneumotaxic center The Hering-Breuer reflex is well known to terminate inspiration. The parabrachial nuclei (PBN), and the Kolliker-Fuse nucleus, which comprise the pontine respiratory group, appear to be part of the central pathway evoking this ventilatory effect. For example, activation of pulmonary stretch receptors by inflation of the lungs, inhibits the discharge of cells in the medial PBN [7]. Further, the medial PBN and the Kolliker-Fuse nucleus have been shown to control the sensitivity of the Hering-Breuer reflex. Electrolytic lesions in these areas increase the lung volume threshold necessary to terminate inspiration [6,8,12]. Another component of the Hering-Breuer reflex is dilation of the airways [13]. Activation of pulmonary stretch receptors, the afferent arm of the HeringBreuer reflex, dilates the airways by withdrawing cholinergic tone to airway smooth muscle [2]. Although the medial PBN has been implicated as essential for full expression of the ventilatory component of the Hering-Breuer reflex, its role in causing the airway dilation component is unknown. Previous work from our laboratory demonstrated that stimulation of the medial and lateral PBN with a glutamate receptor agonist decreased total lung resistance by withdrawing * Corresponding author. Fax: (1) (916) 752-3264. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 3 7 1 - 3 cholinergic tone to the airways [11]. We therefore hypothesized that the PBN may be part of the reflex arc dilating the airways when the Hering-Breuer reflex is evoked by activation of pulmonary stretch receptors. A detailed description of the methods has been published elsewhere [11]. In nine cats, anesthesia was induced by vaporized halothane (5%) and maintained with a-chloralose (initial 35 mg/kg; supplement 5 mg/kg, i.v.). The cats were paralyzed (vecuronium bromide; 0.1 mg/kg, i.v.) and their lungs were mechanically ventilated. The tracheal smooth muscle response to stimulation of pulmonary stretch receptors was recorded before and after unilateral blockade of glutamatergic receptors with kynurenic acid (Kyn; 50 nl, 100 mM) in the PBN. Pulmonary stretch receptors, whose afferent fibers travel in the vagus nerves, were activated with positive end-expiratory pressure (PEEP). This was accomplished by placing the expiratory outlet of the ventilator under an additional 2-6 cm H20. Transverse tension of the trachealis muscle was recorded isometrically [3]. The site of Kyn microinjection into the PBN was selected on the basis that chemical stimulation (DL-homocysteic acid; 25 nl, 100 mM) of this nucleus, decreased tracheal smooth muscle tension and total lung resistance. In two cats, control microinjections of xanthurenic acid (Xan; 50 nl, 100 mM), an inert analog of Kyn, were made prior to Kyn. A.M. Motekaitis et al. / Brain Research 671 (1995) 314-316 315 K /--- P2.1 Trachul ]' TeNdon le (g) 12 20 P3.1 / B, t Tmohoal le Tw~don P4.0 (g) 12 C. it " rr, o~,l Tw~lion Fig. 1. Schematic drawings of coronal sections of the rostral pons showing sites of kynurenic acid microinjection. Numbers on the left side of the figure represent mm posterior (P) to interaural zero [1]. Filled circles represent sites where kynurenic acid was microinjected. Open circles represent sites where xanthurenic acid followed by kynurenic acid was microiniected. Abbreviations: BC, brachium conjuctivum; IC, inferior colliculus; P, pyramidal tract; PBI, lateral parabrachial nucleus; PBm, medial parabrachial nucleus; SO, superior olive; TB, trapezoid body. I n five cats, p h r e n i c n e r v e activity was r e c o r d e d a n d i n t e g r a t e d by the m e t h o d described by E l d r i d g e [5]. Baseline was c a l c u l a t e d by averaging p h r e n i c n e r v e discharge for the o n e m i n u t e p e r i o d p r e c e e d i n g the a p p l i c a t i o n of P E E P . P E E P was i n c r e a s e d for o n e m i n u t e a n d the maximal relaxation of the trachealis muscle was recorded. P h r e n i c nerve activity was i n t e g r a t e d , averaged over this o n e m i n u t e period, a n d t h e n c o m p a r e d with its b a s e l i n e value. All data are r e p o r t e d as m e a n + S.E.M. Statistical significance ( P < 0.05) was d e t e r m i n e d with a repeated measures one-way A N O V A . U n i l a t e r a l m i c r o i n j e c t i o n of Kyn into the m e d i a l ( n = 6) a n d the lateral ( n = 1) P B N (Fig. 1) reversibly a t t e n u a t e d the trachealis muscle r e s p o n s e to a n increase in P E E P (Figs. 2 a n d 3). T h e increase in P E E P also evoked a small (i.e. 8 + 1 m m H g ) , n o n - s i g n i f i c a n t decrease in m e a n arterial p r e s s u r e which did n o t c h a n g e p o s t - K y n microinjection. I n two of these cats, m i c r o i n j e c t i o n of X a n into the P B N did n o t a t t e n u a t e the tracheal relaxation b u t s u b s e q u e n t m i c r o i n j e c t i o n of Kyn into the same site did. T r a c h e a l relaxation was n o t a t t e n u a t e d in two a d d i t i o n a l cats by m i c r o i n j e c t i o n of Kyn into t h r e e different sites o u t s i d e of the P B N (2 sites in o n e cat, 1 site in the other). Histology revealed that two of these sites were c a u d a l a n d o n e was m e d i a l to the p a r a b r a c h i a l n u c l e u s (not shown). I n o n e cat d u r i n g the time of a t t e n u a t i o n of the reflex relaxation of the trachealis muscle, we i n c r e a s e d the stimulus from 2 to 7 cm H 2 0 to test w h e t h e r the airway smooth muscle was still reactive. T h e g r e a t e r stimulus r e s u l t e d (o) \ /--~ / 16 1 + + lSl - - . Fig. 2. Computer-aided reconstruction of tile reflex response of the trachealis muscle to activation of the pulmonary stretch receptors. Arrows indicate an increase in PEEP from 1 to 5 cm H20. Note that with the increase in PEEP, the trachealis muscle relaxed and after the increase in PEEP was removed, returned to baseline tension levels (panel A). Sixty minutes after unilateral microiniection of l~nurenic acid into the parabrachial nucleus, the trachealis did not respond to an increase in PEEP (panel B) but by 90 minutes, recovery occurred (panel C). J ~ • I-I Baseline i~ m mPEEP * e Tracheal Tension (g) tS 10 Control AttenuationRecov~ Atrop~ Fig. 3. Summary data from 7 cats showing the effects of unilateral microinjection of kynurenic acid (KYN; 50 nl, 100 mM) into the parabrachial nucleus on the tracheal smooth muscle response to an increase in PEEP. Open bars represent baseline means and filled bars represent mean responses to an increase in PEEP. Peak attenuation occurred at 33 + 10 min (shown as attenuation). Full recovery of the response occurred at 69 + 9 min (shown as recovery). Asterisks represent statistically significant difference (P < 0.05) between baseline and its corresponding response to PEEP; brackets across the top represent statistically significant difference (P < 0.05) between the mean responses to PEEP. Note that the decrease in tracheal tension evoked by PEEP during lqmurenic injection was significantly less (P < 0.05) than the decrease in tracheal tension evoked by PEEP during the control or recovery periods. Also note that even though baseline muscle tension decreased during the course of the experiment, the trachealis muscle was still capable of further relaxation as indicated by its response to atropine (1 mg/kg, i.v; hatched bar). 316 A.M. Motekaitis et al. /Brain Research 671 (1995) 314-316 in a reflex relaxation of 3.0 g, whereas the smaller stimulus resulted in a reflex relaxation of only 0.4 g. The latency between the increase in PEEP and the start of tracheal muscle relaxation was 7 + 1 s before microinjection of Kyn into the PBN (n = 7). In three cats in which the response was attenuated but not abolished by microinjection of Kyn, the latency increased, non-significantly, to 12 + 4 s. The latency of the response after recovery (11 + 3 s) still tended to be non-significantly longer than the latency of the control response. In five cats, an increase in PEEP evoked a decrease in phrenic nerve activity to 57 + 19% of baseline of nerve activity. Microinjection of Kyn into the PBN did not change the magnitude of this response. For example, at the time of peak attenuation (i.e. 33 min, Fig. 3) of the trachealis muscle response to PEEP, phrenic nerve activity decreased to 58 + 16% of baseline. Likewise, at the time of recovery (i.e. 120 min, Fig. 3), phrenic nerve activity decreased to 60 + 17% of baseline. We have shown that unilateral blockade of glutamatergic receptors in the PBN reversibly attenuated the reflex relaxation of tracheal smooth muscle evoked by P E E P but had no effect on phrenic response to this maneuver. Previous studies have demonstrated that the medial PBN and the Kolliker-Fuse nucleus are essential for the full expression of the ventilatory component of the Hering-Breuer reflex [6,8,12]. The reason that our microinjections of Kyn did not have an effect on the ventilatory component of the Hering-Breuer reflex may have been due to the criterion used in selecting the site in the PBN to be blocked. This criterion was that stimulation evoked a decrease in tracheal tension and a parallel decrease in total lung resistance. The concomitant phrenic response was not a criterion and consisted of neural apnea (n = 2), a decrease (n = 2), or a small increase (n = 1) in phrenic discharge. Our findings agree with previous studies which reported stimulation of the PBN evoked various effects on phrenic nerve discharge [4,9,11]. Another reason that we did not effect the ventilatory component of the Hering-Breuer reflex may be the type and extent of blockade used. Previous studies examining the effect of blockade in the parabrachial nucleus on ventilatory control used large bilateral electrolytic lesions that irreversibly destroyed both cells and fibers of passage [6,8,12]. A recent study examining ventilatory control used large volumes (80-3,000 nl) of a N M D A receptor antagonist, MK-801, which were bilaterally microinjected into the PBN region [10]. This study found a large variation in the magnitude of the prolongation of phrenic inspiratory time evoked by withholding lung inflation. Moreover, the larger the volume and the closer the microinjection was to the Kolliker-Fuse nucleus, the larger the effect on prolonging inspiratory time [10]. In contrast to the above studies, we unilaterally blocked glutamatergic receptors in a small area of the PBN that was shown to relax airway smooth muscle when stimulated with oL-homocysteic acid. Therefore, the possibility exists that a larger a n d / o r different area needs to be blocked to have an effect on the ventilatory component of the Hering-Breuer reflex. The PBN has been demonstrated to be essential for the full expression of the ventilatory component of the Hering-Breuer reflex [6,8,12] but it was not known if this region was also essential for the airway dilation component of this reflex. Previously, we had demonstrated that stimulation of the PBN evoked dilation of the airway smooth muscle by withdrawing cholinergic tone [11]. This study has extended those findings by demonstrating that the PBN plays a role in causing the airway dilation evoked by the Hering-Breuer reflex. The PBN might be an essential relay in the reflex arc causing this dilation or, alternatively, it might function to lower the volume 'threshold' needed to evoke the airway component of this reflex. We thank Mr. Michael Wong for his technical assistance. This work was supported by NIH Grant HL40910. [1] Berman, A.L., A Cytoarchitectronic Atlas with Stereotaxic Coordinates, University of Wisconsin Press, Madison, WI, 1968. [2] Bowes, G.E., Shakin, E.J., Phillipson, E.A. and Zamel, N., An efferent pathway mediating reflex tracheal dilation in awake dogs, J. Appl. Physiol., 57 (1984) 413-418. [3] Brown, J.K., Leff, A.R., Frey, M.J., Reed, B.R. and Gold, W.M., Physiological and pharmacological properties of canine trachealis muscle in vivo, J. Appl. Physiol., 49 (1980) 84-94. [4] Dick, T.E., Haxhiu, M.A. and Cherniack, N.S., Salivary secretion elicited by activation of the parabrachial nuclei in the cat, J. Autonom. NerL,. Syst., 39 (1992) 19-28. [5] Eldridge, F.L., Relationship between phrenic nerve activity and ventilation Am. ~ Physiol., 221 (1971) 535-543. [6] Feldman, J.L. and Gautier, H., The interaction of pulmonary afferents and pneumotaxic center in control of respiratory pattern in cats, J. Neurophysiol., 39 (1976) 31-44. [7] Feldman, J.L., Cohen, M.I. and Wolotsky, P., Powerful inhibition of pontine respiratory neurons by pulmonary afferent activity, Brain Res., 104 (1976) 314-346. [8] Gautier, H. and Bertrand, F., Respiratory effects of pneumotaxic center lesions and subsequent vagotomy in chronic cats, Respir. Physiol., 23 (1975) 71-85. [9] Hade, J.S., Mifflin, S.W., Donta, T.S. and Felder, R.B., Stimulation of parabrachial neurons elicits a sympathetically mediated pressor response in cats, Am. J. Physiol., 255 (1988) H1349H1358. [10] Ling, L., Karius, D.R. and Speck, D.F., Role of N-methyl-i> aspartate receptors in the pontine pneumotaxic mechanism in the cat, J. Appl. Physiol., 76 (1994) 1138-1143. [11] Motekaitis, A.M., Solomon, I.C. and Kaufman, M.P., Stimulation of parabrachial nuclei dilates the airways in cats, Z Appl. Physiol., 76 (1994) 1712-1718. [12] St. John, W.M., Glasser, R.L. and King, R.A., Apneustic breathing after vagotomy in cats with chronic pneumotaxic center lesions, Respir. Physiol., 12 (1971) 239-250. [13] Widdicombe, J.G. and Nadel, J.A., Reflex effects of lung inflation on tracheal volume, Z Appl. Physiol., 52 (1963) 1266-1271.