Role of pre-inspiratory neurons in vestibular and laryngeal reflexes and in swallowing and vomiting

Role of pre-inspiratory neurons in vestibular and laryngeal reflexes and in swallowing and vomiting

Neuroscience Letters 225 (1997) 161–164 Role of pre-inspiratory neurons in vestibular and laryngeal reflexes and in swallowing and vomiting Yu Zheng ...

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Neuroscience Letters 225 (1997) 161–164

Role of pre-inspiratory neurons in vestibular and laryngeal reflexes and in swallowing and vomiting Yu Zheng a, Toshiro Umezaki b, Ken Nakazawa b, Alan D. Miller b ,* a

Department of Physiology, West China University of Medical Sciences, 610044 Chengdu, PR China b The Rockefeller University, 1230 York Ave, New York, NY 10021-6399, USA Received 29 January 1997; revised version received 4 March 1997; accepted 4 March 1997

Abstract Fifteen pre-inspiratory (pre-I) neurons were extracellularly recorded in the pre-Bo¨tzinger complex and their involvements in vestibular (VN) and superior laryngeal (SLN) nerve reflexes and in fictive swallowing and vomiting were tested in decerebrated and artificially ventilated cats. Both type I (1 of 9) and type II (1 of 6) pre-I neurons could project to the contralateral ventral respiratory group region. Pre-I neurons changed their firing during VN and SLN respiratory reflexes and fictive swallowing and vomiting; different response properties were observed among individual pre-I neurons. These results suggest that pre-I neurons are a population of heterogeneous and multifunctional propriobulbar neurons.  1997 Elsevier Science Ireland Ltd. Keywords: Pre-inspiratory neuron; Ventral respiratory group; Pre-Bo¨tzinger complex; Respiration; Vestibular reflex; Deglutition; Emesis; Cat

In addition to the function of ventilation, respiratory muscles are involved in non-respiratory motor behaviors such as swallowing and vomiting, as well as in some reflexes such as vestibular and laryngeal respiratory reflexes [10]. Respiratory rhythmic activity comes from a central pattern generator located in the brainstem, which may also be involved in the production of non-respiratory functions and reflexes, such as those mentioned above. Respiratory rhythmogenesis has been intensely investigated for several decades, and it has recently been suggested that its kernel locus is the pre-Bo¨tzinger complex in the medulla oblongata [18]. A unique type of neuron, the pre-inspiratory (pre-I) neuron, which was originally described as a phase-spanning expiratory-inspiratory neuron [2], located in the preBo¨tzinger complex [4,15] has been proposed to play an essential role in the expiratory to inspiratory phase transition [2,9,15]. It has been suggested on the basis of crosscorrelation analysis that pre-I neurons excite various classes of medullary inspiratory neurons [16]. The present experiments were conducted both to examine the bulbar projection * Corresponding author. Tel.: +1 212 3278598; fax: +1 212 3278530; e-mail: [email protected]

of these pre-I neurons and mainly to analyze their involvement in vestibular and laryngeal respiratory reflexes and in fictive swallowing and vomiting. Data were obtained from 12 adult cats of either sex, which were mid-collicularly decerebrated under isoflurane anesthesia, paralyzed with gallamine triethiodide, and artificially ventilated to maintain end-tidal CO2 at 4–5%. The activities of phrenic, abdominal and pharyngeal vagus nerves were recorded to monitor the respiratory reflexes and fictive swallowing and vomiting. The vestibular nerves were stimulated, as described previously [17], to induce vestibular respiratory reflexes, using trains of five pulses, 0.2 ms pulse width, 3 ms interpulse interval, repetition rate of one stimulus train every 0.8–0.9 s, delivered at random with respect to the respiratory cycle, at an intensity between 75–500 mA (five times the threshold for evoking a volley recorded from the medial longitudinal fasciculus). This intensity was always less than the threshold for current spread to the facial nerve, the closest non-target nerve (tested with trains of 50 pulses before paralysis of the animal). The superior laryngeal nerve (SLN) ipsilateral to the neuron recording side was stimulated to induce both laryngeal

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0208-5

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respiratory reflexes (using single pulses, 0.2 ms pulse width, 100 mA, repetition interval 1.3 s delivered at random with respect to the respiratory cycle) and fictive swallowing (using trains of 0.2 ms pulses, 2–20 Hz, 50–200 mA). Fictive swallowing was identified by the burst activity of the pharyngeal vagus nerve [6], which innervates pharyngeal constrictor muscles. Fictive coughing can also be induced by SLN stimulation; however, this behavior was not studied during the present series of experiments. The vagus nerves were stimulated just rostral to the diaphragm (using trains of 0.8 ms pulses, 25 Hz, 0.5–3 mA) to induce fictive vomiting, which was identified by a characteristic series of bursts of coactivation of phrenic and abdominal nerve discharge that would produce vomiting in non-paralyzed animals [12]. Pre-I neurons were extracellularly recorded in the region of the rostral nucleus ambiguus (in the so-called preBo¨tzinger complex), and fast green dye was deposited through the recording micropipette to localize histologically the recording locations. The ventral respiratory group

(VRG) contralateral to the neuron recording side was identified by multi-unit recording of respiratory neuronal activity and micro-stimulated (0.2 ms pulses, up to 85 mA) using an array of four tungsten microelectrodes placed between 0 and 3 mm rostral to the obex to test if this region received projection from the pre-I neurons (confirmed by collision test). The superior laryngeal and pharyngeal vagus nerves ipsilateral to the recording side were also antidromically stimulated to test if the pre-I neurons were motoneurons. The recording data from the nerves and neurons together with trachea pressure were saved using a Cambridge Electronic Design (CED) 1401-plus data interface and Spike 2 software in conjunction with a Power Macintosh 8100/110 computer. Fifteen pre-I neurons were recorded from a region located 3.0–4.0 mm rostral to the obex, 1.0–1.6 mm caudal to the caudal pole of the facial nucleus, 3.0–4.0 mm lateral to the midline and 3.8–5.0 mm deep from the dorsal surface of the medulla oblongata, corresponding to the pre-Bo¨tzinger complex as described previously in the adult cat [4,15].

Fig. 1. Type I (A) and type II (B) pre-I neurons and their responses to vestibular (VN) and superior laryngeal (SLN) nerve stimulation. (a) Unit recording (UNIT) and histogram (HIST) of discharge pattern of the pre-I neuron in relation to phrenic nerve activity (PHR); inspiratory onset (dashed line) triggered histogram (20 ms/bin, 13 sweeps in (A) and 16 sweeps in (B)). (b) Peri-stimulation (upward arrows) histograms of responses of pre-I neuron to VN and SLN stimulation; in (A), 1 ms/bin and 122, 121 and 116 sweeps for ipsi-VN, contra-VN and ipsi-SLN stimulation, respectively; in (B), 5 ms/bin and 143, 142 and 110 sweeps, respectively. The stimulation artifacts were canceled in the last two cases. These neurons were not antidromically activated from the contralateral VRG.

Y. Zheng et al. / Neuroscience Letters 225 (1997) 161–164

The neurons could be subclassified into two types, type I (n = 9) and type II (n = 6). Type I exhibited a peak frequency of discharge during the expiratory-inspiratory phase transition (Fig. 1Aa), and type II had a second discharge peak during the late inspiratory period (Fig. 1Ba), as previously described [15]. One of the nine type I pre-I neurons could be antidromically activated from the contralateral VRG (Fig. 2Aa insert). It was activated from two stimulating points, one at the obex level with a latency of 1.8 ms and a threshold of 12 mA and the other 2 mm rostral to the obex with a longer latency, 4.4 ms, and a lower threshold, 5 mA, suggesting the presence of axonal collaterals. One of the six type II pre-I neurons could also be antidromically activated from the contralateral VRG 2 mm rostral to the obex with a latency of 2.6 ms. No pre-I neurons could be antidromically activated from the superior laryngeal or pharyngeal vagus nerves. Three out of nine type I (Fig. 1Ab) and two out of five type II (Fig. 1Bb) pre-I neurons tested were inhibited by stimulation of the vestibular nerve on either side with latencies between 14 and 35 ms from the onset of stimulation and durations between 85 and 240 ms. One type I cell was excited by stimulation of contralateral vestibular nerve. Of the two neurons antidromically activated from the contralateral VRG, the type II neuron was not affected by vestibular input, and possible effects on the type I neuron were not tested. Five of seven type I (Fig. 1Ab) and all six type II (Fig. 1Bb) pre-I neurons were inhibited by stimulation of the ipsilateral SLN, with latencies between 3 and 10 ms and durations between 4 and 125 ms. The type I neuron that projected to the contralateral VRG was not tested. Three of five type I and three type II pre-I neurons tested, including the two contralaterally projecting neurons, were inhibited or non-activated during fictive swallowing induced by SLN stimulation (Fig. 2Aa). Another two type I pre-I neurons were excited during the period of high intensity activity, but not before the onset, of swallowing (Fig. 2Ab). Changes in activity of four type I and two type II pre-I neurons, including the two neurons with contralateral VRG projections, were examined during fictive vomiting. The neurons exhibited a variety of vomiting-related firing patterns. Two type I neurons were active between bursts of phrenic and abdominal coactivation. One of these, which exhibited reduced firing during vomiting, is illustrated in Fig. 2B. The third type I neuron was active during bursts of phrenic and abdominal coactivation. The remaining type I neuron, which was antidromically activated from the VRG, was active both during phrenic and abdominal bursts and during the last 2/3 of the period between these bursts. The two type II pre-I neurons, one of which projected to the contralateral VRG, were either silent or largely inhibited during vomiting. The present study confirmed that two types of pre-I neurons, type I and type II, exist in the pre-Bo¨tzinger complex.

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We demonstrated contralateral VRG projections for one neuron of each type, and showed that none were pharyngeal vagus or superior laryngeal motoneurons. Although it is possible that these neurons may be motoneurons for other cranial nerves, we suggest that they are primarily brainstem interneurons, as previously described [15]. These findings suggest that pre-I neurons may play an important role in connection and coordination of the activities of the central pattern generators in the two sides of the brainstem. Pre-I neurons have also been reported in the dorsal respiratory group (DRG) in the nucleus of the solitary tract [3], but these pre-I neurons have received less attention in the literature since the pre-Bo¨tzinger region, as opposed to the DRG, is thought to be more important for generation of the respiratory rhythm. Pre-I neurons responded to inputs from the vestibular and superior laryngeal nerves and changed their activities during swallowing and vomiting. These results indicate that pre-I

Fig. 2. Behavior of pre-I neurons during fictive swallowing (A) and vomiting (B). (Aa,b) Examples, respectively, of neurons not activated and activated during fictive swallowing (FS). (A) left, Control discharge patterns; dashed lines in (a) indicate the phase relationship between the neuronal firing and phrenic activity. (A) right, behavior of the two neurons during FS induced by SLN stimulation. Insert in (a) antidromic collision test of the neuron; the neurons in (Ab) and in (B) were not antidromically activated. In (B), the dashed line indicates the phase relationship between the neuronal firing and nerve bursts of fictive vomiting induced by vagus nerve stimulation; the expulsion phase is delimited by the two dotted lines at the end of the episode. The neuron in (B) is the same one shown in (Ab). ABD, abdominal nerve; PH-X, pharyngeal vagus nerve; PHR, phrenic nerve; UNIT, neuronal activity.

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neurons are involved, not only in the control of breathing, but also in the control of some other motor activities and in their integration with breathing. Thus, pre-I neurons can be considered to be multifunctional neurons [5]. Moreover, pre-I neurons are a heterogeneous population of neurons on the basis, not only of two different firing patterns during respiration, but also of the different responses of individual pre-I neurons during the behaviors investigated in the present study. In addition, we were able to antidromically activate a subset of, but not all, pre-I neurons using an array of electrodes placed in the contralateral VRG. Vestibular input is known to reach many VRG bulbospinal neurons [13,17]. However, this study is the first report of vestibular input affecting pre-I neurons. Vestibular respiratory reflexes are thought to help maintain airway patency and compensate for mechanical constraints placed on respiration due to movements and changes in posture [19]. The activity of various types of respiratory-related neurons in the pre-Bo¨tzinger region is known to be affected by stimulation of the SLN [1,8]; however, this is the first report that we know of to demonstrate that SLN input affects pre-I neurons. SLN stimulation inhibited or did not affect, but never excited, the activity of pre-I neurons. However, some pre-I neurons were active during fictive swallowing induced by SLN stimulation, as are some other types of propriobulbar neurons [14]. Pre-I neurons exhibited different response patterns during vomiting, a phenomenon that has also been described for other types of propriobulbar as well as bulbospinal respiratory neurons [7,11,12]. In conclusion, a heterogeneous, multifunctional population of pre-I neurons exists in the pre-Bo¨tzinger complex of cats. They may be classified into two types according to their firing pattern during respiration, each type with contralateral projections. This work was supported by NIH grants NS20585 and DC02644. [1] Bianchi, A.L., Gre´lot, L., Iscoe, S. and Remmers, J.E., Electrophysical properties of rostral medullary respiratory neurones in the cat: an intracellular study, J. Physiol., 407 (1988) 293–310. [2] Cohen, M.I., Discharge patterns of brain-stem respiratory neurons during Hering-Breuer reflex evoked by lung inflation, J. Neurophysiol., 32 (1969) 356–374. [3] Cohen, M.I. and Feldman, J.L., Discharge properties of dorsal medullary inspiratory neurons: relation to pulmonary afferent and phrenic nerve efferent discharge, J. Neurophysiol., 51 (1984) 753– 776.

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