Firing pattern and location of respiratory neurons in cat medullary raphe nuclei

Neuroseienee Letters, 16l (1993) 149 152

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@ 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/S 06.00 NSL 09879

Firing pattern and location of respiratory neurons in cat medullary raphe nuclei Masae Hosogai*, Satoshi Matsuo, Shozo Nakao Department o[' Physiology, Faeulo' of Medieine, Tottori L#liversit3; 86 Nishi-maehi, }~mago.62¢3, Japan

(Received 13 May 1993; Revised version received 12 July 1993: Accepted 12 July 19931 Key wor&v Inspiratory neuron; Expiratory neuron: Phase-spanning neuron: Augmenting firing: Decrementing firing: Midline medullary tegmen-

turn: Raphe nuclei Extracellular spikes of single respiratory neurons were explored in the medullary raphe nuclei in spontaneously breathing decerebrated or Nembutal-anesthetized cats. A total of 26 respiratory neurons, whose spikes were confirmed to originate from the cell bodies, was recorded in the raphe obscurus and pallidus. They could be classified into six types based on the relation of their firing to the phases of respiration: Ill respiratory {I) decrementing (I-DEC, n = 14), (2) 1-frequency modulated (n = 3), (3) l-augmenting (I-AUG, n = 2). (4) late I 07 = 2), (5) expiratory (El augmenting (E-AUG. n = 3) and (6) I-E phase-spanning (I-E PS, n = 2). These results suggest the existence of neurons relating to the control of respiration in the medullary raphe nuclei.

A n u m b e r o f studies have shown that electrical or chemical stimulation o f the medullary raphe nuclei, consisting o f the raphe obscurus (RO), raphe pallidus (RP) and raphe m a g n u s (RM), produces inhibitory or excitatory effects on respiratory activity [1, 7, 10, 13, 16, 18]. Recent anatomical studies using the n e u r o a n a t o m i c a l tracing m e t h o d [6, 19] have shown that there are efferent projections from cells in the raphe nuclei to the phrenic m o t o r nucleus and the dorsal and ventral respiratory groups ( D R G , V R G ) , respectively; however, it is unclear whether or not these labeled cells were respiratory neurons. On the other hand, several investigators have recorded neurons firing with a respiratory r h y t h m in the raphe nuclei [1, 4, 5, 8, 11, 12], but there have been few reports on firing patterns and the distribution o f respiratory neurons in the raphe nuclei. Furthermore, there seems to be some uncertainty as to whether the spike activity o f these neurons arises from the cell bodies, since axons o f respiratory neurons in the lateral medulla are k n o w n to cross the raphe nuclei. A n electrophysiological experiment was t h o u g h t to be necessary to confirm m o r e substantially the existence o f the cell bodies o f respiratory neurons in the raphe nuclei. The present study was undertaken to reinvestigate respiratory neurons in cat medullary raphe nuclei with special attention directed to their discharge patterns and distributions. *Corresponding author. Fax: (81) 859-34-8080.

Experiments were performed on 20 adult cats. Ten cats were decerebrated at the precollicular level under ether anesthesia and after that the anesthetic was discontinued. Recordings were always begun at least 2 h after the decerebration. The other 10 cats were anesthetized with pentobarbital sodium (initial dose o f 40 mg/kg and later supplementary doses o f 5 mg/kg/h i.p.). Similar resuits were obtained in both types o f experiments. The animals were tracheotomized and allowed to breath air spontaneously t h r o u g h a tracheal cannula. Blood pressure in the femoral artery, rectal temperature and endtidal C O , were monitored. A pair of enamel-insulated c o p p e r wire electrodes (120 p m diameter) was inserted into the diaphragm. The d i a p h r a g m E M G was passed t h r o u g h an integrator (time constant 0.25 s) and was used as a m o n i t o r o f the central respiratory rhythm. Chest m o v e m e n t was monitored by impedance changes o f a rubber tube filled with saturated zinc sulfate solution placed a r o u n d the thorax o f the animal. The animal's head was m o u n t e d in a stereotaxic frame and kept in the Horsley-Clarke plane. Its b o d y was suspended by vertebral clamps at k 3 and C7. The medial part of the occipital bone and the underlying cerebellum were removed to expose the floor o f the fourth ventricle. Glass capillaries filled with 2 M NaC1 solution saturated with Fast green F C F dye (1 1.5 M.Q) were used for recordings o f extracellular unit spikes and inserted into the brainstcm at a 13 ° angle forward tilt from the vertical. Systematic ex-

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plorations were made for single units with respiratory discharges from the area between the center of the facial genu and I mm caudal to the obex, between the midline and 0.5 mm lateral to the midline, and from the brainstem surface to a depth of 6 mm. At the end of the experiments, the recording sites in the brainstem were marked by electrophoretic ejection of Fast green from the recording electrode. After fixation, serial frozen sections (50 /lm in thickness) of the brainstem in the frontal plane were made and stained with Cresyl violet. Extracellular spikes of 35 units which showed respiratory discharges were recorded in the midline medullary tegmentum. The recorded spikes were carefully distinguished between the activity ofaxons and that of cell bodies, on the basis of previous works [3, 9, 14]. Monophasic positive spikes were considered to originate from axons. On the other hand, if the recorded spikes were either biphasic negative-positive waveforms or predominantly negative spikes having clearly visible IS/SD breaks (cf. Fig. IAh), and if the spikes were held over a relatively long tracking distance (more than 100 /lm), such recorded spikes were considered to originate from cell bodies. A total of 26 unit activities originating from cell bodies was recorded. They were classified into the following six types based on their firing patterns. Decrementing inspiratory (I-DEC) neurons (n = 14): Fig. IA shows a firing pattern from an I-DEC neuron. For 20 consecutive respiratory cycles, each spike of the neuron was represented by a dot and aligned in reference to the onset of the diaphragm EMG burst (Fig. lAt). Fig. IAg shows the averaged spike frequencies of this neuron using the same alignment procedure. This diagram was constructed by averaging the number of spikes for each inspiratory neuron in 50 ms bins for 20 consecutive respiratory cycles. Spike discharges of the neuron started about 50 ms before the onset of the diaphragm activity. An initial high frequency burst attained more than 100 spikes/s, and the rate of increase in frequency was steepest at the onset of the diaphragm EMG. The spikes then gradually decreased in frequency and ceased at the end of the I phase. Fig. IB shows another type of I-DEC neuron. The firing started just before the inspiration, attained an initial high frequency burst, decreased gradually and stopped in mid-inspiration. I-frequency modulated neurons (n = 3): Fig. IC shows the firing pattern of a neuron in another group. This neuron fired tonically during both the I and E phases. This neuron had a lower frequency of firing during the E phase and a higher frequency of firing during the I phase. The firing during the I phase was of the decrementing type. Augmenting I (I-A UG) neurons (n = 2): Fig. 10 shows

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Fig. I. Firing patterns of respiratory neurons recorded from medullary raphe nuclei. A: I-DEC neuron. a: extracellular spikes. b: diaphragm EMG. c: integrated diaphragm EMG. d: end-tidal CO 2 , e: chest pneumograph with an upward deflection indicating inspiration. f: dot plots of spike discharges in 20 consecutive respiratory cycles. g: averaged spike frequencies of f. h: extracellularly recorded negative action potential. Arrow indicates an inflection (IS/SD break) on the rising phase. B: another type of I-DEC neuron. C: I-frequency modulated neuron. D: I-AUG neuron. E: Late I neuron. F: I-E PS neuron. G: E-AUG neuron. a,b,c: same as in A. d: same as in g. Vertical broken line indicates the end of inspiration. The time zero on the abscissa indicates the onset of the diaphragm EMG.

another type of an I neuron. The spike discharges of this neuron started about 100 ms before the onset of the diaphragm EMG burst. The spikes then gradually increased in frequency, attaining the maximum frequency at about 100 spikes/s at the latter half of the I phase. The high frequency firing continued and the spikes decreased rapidly, ceasing at the end of the I phase. Late I neurons (n = 2): Discharges of this neuron started late in the I phase, reached a maximum frequency coincident with the peak of the integrated diaphragm EMG and then declined abruptly (Fig. IE). I-E phase-spanning (I-E PS) neurons (n = 2): This neuron fired at the transition from the I phase to the E phase (Fig. IF). Augmenting expiratory (E-A UG) neurons (n = 3):

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Fig. 2. Frontal plane projection of the brainstem showing distribution of 26 respiratory neurons. Drawings are of cross- sections of the medulla from a level 2.04.5 mm rostral to the obex. Each of the following symbols respectively indicates the sites where different types of respiratory neurons were recorded. Filled circles, I-DEC neuron; circled filled circles, I-frequency modulated neuron: open circles, I-AUG neuron; circled opened circles, Late I neuron; open stars, E-AUG neuron; open triangles, I-E PS neuron. VMN, medial vestibular nucleus; PH, nucleus praepositus hypoglossi: 12, hypoglossal nucleus; 12N, hypoglossal nerve: SM, medial nucleus of the solitary tract; IO, inferior olivary nucleus; RFN, retrofacial nucleus; AMB, nucleus ambiguus.

Their discharges started at the end of the I phase (Fig. 1G) and gradually increased during the E phase. They attained maximum frequency at the latter half of the E phase and ceased just before the I phase. The location of all 26 respiratory neurons is illustrated in Fig. 2 with the aid of a stereotaxic atlas of the brainstem [2]. The region was considered to correspond to RO and R E a subdivision of the nucleus of the raphe [20]. I-DEC neurons were located in RO and RP at the levels of P10.5-P12.0. Most of the I-DEC neurons were concentrated at the level of P10.5 and P11.0. I-AUG neurons, Late I neurons and I-E PS neurons were located in RO and RP at the level of P11.5. E - A U G neurons were located in RO at P9.5, P10.0 and P12.0. Monophasic positive spikes of 9 units which were synchronous with the I phase were recorded in RO and RP at the levels of P11.0-P14.0. These 9 units were presuma-

bly recorded from axons and an analysis showed that 8 units displayed the typical firing pattern of I-AUG neurons and 1 unit displayed the firing pattern characteristic of I-DEC neurons. These are considered to be axons of I neurons in the lateral medulla. In this study, the most numerous neurons were of the I type. The I-DEC were the most abundantly recorded neurons. Hukuhara [8] recorded I and E neurons in the midline of the medulla in anesthetized cat without vagotomy and paralyzation, but the firing patterns were not described. The present recording sites were similar to those in Hukuhara's report. Heym et al. [5] recorded serotonergic and respiratory neurons as well in RP in freely moving cats, although it was not ascertained whether the latter were serotonergic neurons. We also recorded many respiratory neurons in the same region of RP. Bennett and St. John [1] recorded respiratory neurons in the pontomedullary junction. Lindsey et al. [12] recorded midline neurons with respiratory-modulated firing in the pontomedullary junction and caudal medulla. We did not find respiratory neurons in the pontomedullary junction. This discrepancy may be due to the following factors: ( 1) They used the F-test and the Friedman test as the criteria for defining respiratory modulation, and found many tonic neurons with respiratory modulated tiring. But, we did not adopt such a statistical analysis. (2) They used decerebrated or anesthetized, paralyzed, artificially ventilated cats with vagotomy, while we used spontaneously breathing cats with intact vagus nerves. They found an increase in the proportion of neurons with no respiratory modulation of their firing when the end-tidal CO, level was reduced. It may be because in the present study the end-tidal CO~ level of our preparation was lower than that in their study. Previous works [10, 16] showed that electrical stimulation within RP produced an excitatory effect on respiratory movement, Furthermore, electrical stimulation of RO produced either excitatory or inhibitory effects on respiration [16]. Holtman et al. [7] showed that stimulation of cell bodies in RO produced an increase in phrenic nerve activity, although it was not clear whether these cells were respiratory neurons. We found cell bodies of both I and E neurons in RO and many 1 neurons in RP. Therefore, the previously reported inhibitory or excitatory effects on respiratory activity may be produced by stimulation of the cell bodies of respiratory neurons in the medullary raphe nuclei or of passing fibers crossing the midline from either the VRG or DRG. In conclusion, the present experiments show that the cell bodies of six types of respiratory neurons exist in RO and RE These results suggest that these respiratory neurons may relate to respiratoy control by the medullary raphe nuclei.

152 This study was partly supported by a Grant-in-Aid for Scientific R e s e a r c h (no. 04454444)

f r o m the J a p a n e s e

Ministry of Education, Science and Culture.

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