Respiratory neurones of the ventrolateral nucleus of the solitary tract of cat: Vagal input, spinal connections and morphological identification

Brain Research, 61 (1973) 1-22

1

© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

Research Reports

R E S P I R A T O R Y N E U R O N E S OF T H E V E N T R O L A T E R A L N U C L E U S OF T H E SOLITARY T R A C T OF CAT: V A G A L INPUT, SPINAL C O N N E C T I O N S AND MORPHOLOGICAL IDENTIFICATION

C. VON EULER, J. N. HAYWARD*, IRJA MARTTILA AND R. J. WYMAN** The Nobel Institute for Neurophysiology, Karolinska Institutet, S-104 O1 Stockholm 60 (Sweden}

(Accepted April 5th, 1973)

SUMMARY (1) Electrical activity o f single cells was recorded extracellularly in the vicinity of the solitary tract complex in the medulla oblongata ofpentobarbital anaesthetized, paralysed cats. The anatomical location and gross morphological properties of some of the neurones were determined by a fluorescent dye (Procion Yellow) injection technique. (2) Of a total of 279 cells studied, 261 ceils fired rhythmically with some phase of the respiratory cycle, i.e., respiratory neurones. Most respiratory neurones (238 cells) discharged in phase with the phrenic, i.e., they were inspiratory neurones. (3) Most of the 73 anatomically identified cells were found in the ventrolateral nucleus of the solitary tract which proved a somewhat heterogeneous nucleus with a roughly equal mixture of 'inspiratory-vagal', 'inspiratory-non-vagal-spinal' cells along with a few 'inspiratory-expiratory', expiratory and non-respiratory neurones. (4) Inspiratory neurones could be divided into 2 main groups on the basis of their vagal and spinal connections: (1) inspiratory neurones driven by electrical stimuli to the cervical vagus and influenced by lung inflations; (2) inspiratory neurones not influenced by vagal afferents but driven with short latency from the ventrolateral columns of the contralateral side of the spinal cord. (5) The 'inspiratory-vagal' cells showed inspiratory activity even when lung expansion in the inspiratory phase was prohibited, thus resembling the R/~ cells of von Baumgarten and Kanzow 6. * On leave from Departments of Neurology and Anatomy, UCLA School of Medicine, Los Angeles, Calif. 90024 and supported by USPHS Grants NS-05638 and NS-02277. ** On leave from Department of Biology, Yale University, New Haven, Conn. 06520, U.S.A., and supported by USPHS Grant NS-07314.

2

c. VON EULERet al.

(6) The 'inspiratory-vagal' cells exhibited large changes in excitability to vagal stimulation with time during the respiratory cycle, the responsiveness being low in the expiratory phase and early in inspiration and increasing progressively during the course of inspiration. (7) We suggest that these 'vagal-inspiratory' neurones determine the time course of the Hering-Breuer threshold curve described by Clark and yon Euler t4. INTRODUCTION

Vagal reflex control of the rate and depth of breathing depends, in part, upon the slowly adapting pulmonary stretch receptors which are activated by increased lung volumO, a° (ref. 42 for further references) and upon the cyclic changes in reflex excitability of the central medullary neural mechanism 11,29,34. The vagal inflation reflexes which both inhibit inspiratory activity and prolong the expiratory phase were first elucidated by the studies of Breued 2. While the medullary neurones involved in the Hering-Breuer inflation reflex are not known it would seem reasonable to assume that these neurones should lie near the vagal afferent receiving station, i.e., the nuclear complex of the solitary tract. The work of Boyd and MaaskO ~ and the recent results from this laboratory by Clark and von Euled 4 suggest that these neurones should exhibit properties corresponding to the steep decrease in the volume threshold for inspiratory termination which occurs with time from the onset of inspiration. In our search for such neurones involved in the inflation reflex and the shaping of the time course of the Hering-Breuer threshold curve we focused our attention on the cells of the nuclear complex of the solitary tract. Evidence from the literature indicates that vagal sensory fibres project to the intermediate and caudal portions of the solitary nucleus ~3,17,zl,28,26, with pulmonary afferents probably projecting predominantly to the caudal region of the solitary nucleus z,a,1°,34 (see also ref. 43). In contrast, the cardio-vascular afferents seem to project somewhat more rostrally 3,~°, heading mainly for the intermediate area of the solitary nucleus s,9,~s,22,2s,31,3v,3s. In the region ventral or ventrolateral to the solitary tract, 'inspiratory' neurones have been described, some of which are reported to be influenced by the pulmonary afferents and some to have spinal projections ~,7,1a,16,32,33. In the present study we have searched for and recorded spontaneous discharge patterns of single neurones in the nuclear complex of the solitary tract and examined their respiratory (or non-respiratory) characteristics and their responsiveness to lung inflations, to electrical stimulation of the vagus nerves and of the spinal cord. Respiratory neurones which were successfully penetrated by the electrode were marked by intracellular electrophoretic ejection of the fluorescent dye Procion Yellow a9 for determination of their anatomical location and their morphological configuration. The functional and structural characteristics of these inspiratory neurones suggest that the ventrolateral nucleus of the solitary tract is involved in the Hering-Breuer inflation reflex and provide the time dependent change in threshold of this reflex ~4. A preliminary account of some of these results has been presented elsewherO 9.

RESPIRATORY SOLITARY TRACT NEURONES. I

3

METHODS

Experimentalprocedures Animal preparation. Experiments were performed on 35 cats (2.0-3.5 kg b.w.) of both sexes and anaesthetized with pentobarbitone (Nembutal, Abbott: 35 mg/kg i.p.). Glucose and supplementary doses of pentobarbitone were given regularly by i.v. injection. Gallamine triethiodide (Flaxedil, May and Baker) was given intravenously in the amount required for complete respiratory paralysis and supplemented regularly. The animals were suspended prone in a frame by vertebral clamps at L3 and C7 and by a head-holder. The latter kept the head flexed ventrally allowing the floor of the IVth ventricle to assume a roughly horizontal position. An occipital craniotomy and partial removal of cerebellum exposed the medulla and the caudal half of IVth ventricle. To provide the possibility for a 'closed chamber recording technique' a special aluminum chamber with a venting duct was centered over the points along the intermediolateral sulcus 0-2 mm in front of obex and cemented in place with an acrylic resin to the occipital bone and the two uppermost cervical vertebrae. The chamber was filled with warm mineral oil, capped with a clear lucite disc with a central gasket through which the glass micropipette could be passed. For subsequent electrical stimulation the upper cervical spinal cord (C3-4) was exposed by laminectomy and removal of the dura and covered with warm mineral oil. To provide stability the spine from C1-5 was immobilized by strong acrylic bridges to the skull. Body temperature was monitored with a rectal thermocouple probe and body temperature maintained between 37-38 °C by a ventral heating pad and a dorsal radiant heat source. Recording techniques. Gross efferent phrenic activity was recorded from the phrenic C5 root using a small tube-shape indwelling electrode ensemble attached to the scalenus muscle. The desheathed nerve, when placed on the bipolar silver electrodes, was protected by vaseline. The phrenic activity was 'integrated' by means of a rectifier and RC network. For recording neuronal activity in the medulla we have used glass micropipettes, broken to an outer diameter of 1.2-2.0 #m and filled with a nearly saturated (about 5 ~o) solution of the fluorescent dye Procion Yellow, M-4R, ICI. They had a resistance of 15-35 Mf~ which proved suitable for both recording and electrophoretic ejection of dye. The electrodes were connected through a silver-silver chloride junction to the probe of an 'electrometer amplifier' (W-P Instruments, M4ARM) and to a stimulator which served as the source of current for the depositing of dye. The microelectrode was guided by an electrode carrier driven by an electronically controlled stepmotor (AB Transvertex Co., Sweden). A reference electrode (chlorided silver wire) was embedded in the muscles of the neck. Stability of recording was improved by using a 'closed chamber' recording technique (see above). The need to relate imposed volume changes to the functional residual capacity (FRC) did not permit us to use pneumothorax as a means of improving stability.

4

c. VON EULERet al.

Strain gauge manometers were used for monitoring tracheal and arterial pressures. End-tidal CO2 levels were monitored with a catheter in the endotracheal tube and an infrared CO2 analyser (Beckman, LB-1). The output signals from the electrometer, the phrenic neurogram (with or without 'integration'), endotracheal pressure and electronic instantaneous rate-meter were displayed, after adequate amplification, on the face of a 4-channel CRO and photographed on moving film. Stimulating techniques. The cervical vagus nerves were separated from the sympathetic trunk and placed, intact, in indwelling bipolar stimulating electrodes firmly attached to the deep neck muscles. For intraspinal stimulation we used tungsten electrodes etched down to point diameters of 5-10/~m and insulated except for the tip. The electrodes were guided by a micromanipulator. Square wave pulses of either polarity with a duration of 0.05-0.2 msec were delivered through isolation transformers. Marking technique. The location of some physiologically studied neurones was determined by electrophoretic ejection intracellularly of an anionic fluorescent dye (Procion Yellow, M-4R, ICI) according to the technique of Stretton and Kravitz39 (cf. also refs. 24 and 25). When a neurone had been studied by extracellular recordings, attempts were made to penetrate the cell membrane and to eject Procion Yellow electrophoretically from the electrode into the cell by passing a constant hyperpolarizing current of 2 × 10-8 A for 3-10 min. At the end of the experiment, in the animals with dye-filled cells, the brain was perfused with Ringer's solution, removed and preserved in the cold (1-3 °C) for 36 h prior to fixation in formalin (10%). After dehydration in ethyl alcohol, clearing in xylol and embedding in paraffin, the medullas were cut and the serial sections (15 #m thick) mounted, unstained, on glass slides, and examined under the fluorescence microscope (Zeiss filters, BG 38, BG 12 and BG 47 for the exciting and emitted light, respectively). Anatomical considerations. The yellow-orange of the Procion Yellow-filled cells contrasted well with the yellow-green background autofluorescence which also provided satisfactory detail of other medullary structures. As our zero reference point, the lower lateral extent of the solitary tract was used (see Figs. 4-6). This 'zero point' was obtained by extending an imaginary line from the clearly defined upper medial border of the solitary tract 500/zm diagonally through the coarse fibre portion of the tract to this lower, lateral zone. All cells were related to this 'zero point' in order to allow comparisons and composite drawings. Experimental procedures. As mentioned above, the floor of the IVth ventricle was roughly horizontal. For the orientation of the electrode, the obex was our main reference point. As pointed out by von Baumgarten et al), the intermediolateral sulcus, marking on the exterior the border between the cuneate and gracilis nuclei, was, when visible, a very useful guide towards the solitary tract complex. For the mediolateral orientation in cases when the sulcus was not clearly seen, it proved useful to determine physiologically whether the electrode on moving downward met cuneate or gracilis neurones (by trying to localize the corresponding receptors). During the further step-wise advance of the electrode (2--4 #m steps) single shock

RESPIRATORYSOLITARYTRACT NEURONES. I

5

stimuli were applied to the ipsilateral cervical vagus nerve. When spontaneous activity of possible respiratory rhythmicity or vagally driven spikes were encountered, a closer study was made. Extracellular (occasionally intracellular) action potentials were recorded with the respirator switched off, or with pulses of inflation or deflation delivered at any preset time in the respiratory cycle. When possible the responsiveness to single shock stimulation to the vagus nerves and the spinal cord at different phases of the respiratory cycle was studied. At the end of the experiment vagal and spinal conduction distances were measured from the stimulating electrodes to the recording site. RESULTS

Types of neurones in the ventrolateral nucleus of the solitary complex Extracellular records were obtained from a total of 279 cells in the vicinity of the solitary tract of the pentobarbitone anaesthetized, paralysed cat ventilated artificially with a 'servo-respirator' driven by the 'integrated' phrenic activity. Firing patterns were grossly classified according to their relationship to the phrenic nerve discharge, and grouped into 3 general types: (1) inspiratory exemplified by Figs. 1 and 2; (2) other respiratory (inspiratory-expiratory, expiratory as in Fig. 3, expiratory -inspiratory); and (3) non-respiratory neurones. The phrenic activity is, however,

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Fig. 1. Inspiratory activity from 'vagal' neurone (upper tracing). Below in order: phrenic neurogram, intratracheal pressure and integrated phrenic activity. A and B consecutiverecordings. The respirator was driven by 'integrated' phrenic activity. With the respirator off (the second inspiration in A) the inspiratory activity was prolonged and the discharge rate of the neurone was lower than with lungs normally inflated during inspiration. In C respirator was driven by a sine-wave generator. The cyclic inflations influenced markedly the discharge rate of the neurone. This neurone could be driven by single shocks to the ipsilateral vagus nerve at a latency of 3.6 msec (conducting distance 65 mm).

6

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Fig. 2. lnspiratory, 'non-vagal spinal' neurone. Upper tracing in A and B neurone discharge. Below in order: phrenic neurogram, 'integrated' phrenic activity and intratracheal pressure. In A the respirator, driven by the integrated phrenic activity, was switched off during the middle of the 3 inspirations with no change in the initial rate of increase of activity: only prolongation of inspiration due to the lack of volume feedback. In B respirator was driven by sine-wave generator: no influence by inflations and deflations on the discharge rate. This neurone could not be driven by electric stimulation of vagus nerves. C: driven spike in response to single shocks to the contralateral ventrolateral funiculus of the cervical cord at C4: latency 1.1-1.4 msec at a conduction distance of 42 ram.

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Fig. 3. Neurone with mainly expiratory discharge pattern (upper tracing). Below in order: phrenic neurogram, intratracheal pressure (dotted line), and integrated phrenic activity. Upper and lower recordings consecutive. This neurone could he driven by single shocks to ipsilateral vagus nerve at a latency of 3.5 msec (conduction distance 62 ram).

RESPIRATORY SOLITARY TRACT NEURONES. I

7

usually not restricted to the phase of inspiration if the latter is defined from lung volume or airflow criteria, but is often found to linger on at a falling rate far into the expiratory phase. This is a commonly overlooked phenomenon, the control about which we know very little. Since the classification of the neurones was according to their relationship to phrenic activity, neurones referred to as 'inspiratory' usually showed an inspiratory-expiratory firing pattern in the same manner as the phrenic motoneurones did (see Fig. 1A). In a few cases where the activity in the expiratory phase was considerably more dominant or showed a definitely different time course than that of the simultaneously recorded phrenic activity the pattern has been classified as mainly expiratory (see Fig. 3) or as inspiratory-expiratory and referred to the group of 'other respiratory' neurones. The encountered neurones have been grouped also according to whether they showed signs of a vagal influence as in the case of Figs. 1, 8, 9 and 11, and whether they could be driven with short latency in response to single shocks to the ventrolateral funiculi of the contralateral side of the spinal cord as in the case of Fig. 2 and in the neurones dealt with in the subsequent paper.

TABLE I RESPIRATORY AND NON-RESPIRATORY NEURONES IN THE DORSOMEDIAL MEDULLA OF THE CA T

Cell type

Inspiratory Vagal, non-spinal Vagal, not test spinal Total inspiratory vagal Spinal, non-vagal (Spinal, vagal) Spinal, not test vagal Total inspiratory spinal Non-vagal, non-spinal Non-vagal, not test spinal Not test vagal or spinal Total inspiratory

Other respiralory Vagal, non-spinal Vagal, not test spinal Spinal, non-vagal Non-vagal, not test spinal Not test vagal or spinal Total 'other respiratory'

Non-respiratory Vagal, non-spinal Vagal, not test spinal Total 'non-respiratory'

Total cells studied

A. Functionally defined cells

B. Functionally defined and anatomically localized cells

16 72 88 34 (1 ?) 6 40 3 67 40 238

8 19 27 18 0 3 21 0 10 8 66

3 2 0 7 11 23

1 0 0 0 0 1

5 13 18

1 2 3

279

70

8

c . VON EULER e t

al.

Table I presents the whole material and its subdivision into different groups. The vast majority of the neurones or 238 out of a total of 279 cells were classified as 'inspiratory' whereas only 23 were referred to as 'other respiratory'. In addition to the 18 'non-respiratory' neurones included in our material (see Table I) because of their response to vagal stimulation and proximity to respiratory neurones we encountered some 170 other 'non-respiratory' neurones which, when tested, did not respond to electrical stimulation of the vagus nerves. These were of too little interest in the present context to be included in the material. Vagal afferent connections were studied both by the driven responses to electrical shocks to the cervical vagus nerve (at a level below the superior laryngeal nerve) and by the responses to lung inflation. Since the animal was paralysed with gallamine, lung volume information could momentarily be eliminated. This could be done without affecting significantly the chemical respiratory drive by switching off the servorespirator for one or two breaths 14. Sinusoidal inflations were also applied. Of the 238 inspiratory neurones 192 were adequately tested for vagal input: 88 responded, 104 did not. Twelve 'other respiratory' neurones were tested for vagal connections, 5 with positive and 7 with negative result. The latencies for vagal driving of the neurones ranged from 2.1 to 8.0 msec with an average of 3.6 msec. An average conduction velocity of 33 m/sec 35,a6 and an extracephalic conduction distance of about 50 mm gives an intracephalic delay of about 2.1 msec. During the probing of the structures around the solitary tract we also searched for field potentials evoked by single shocks to the ipsilateral vagus nerves. This was done in order to obtain an indication of the arrival of the vagal volley. The evoked potentials were located in or near the solitary tract as shown in Fig. 4, where the areas with evoked potentials (hatched) and the neurones (symbols) for 4 electrode tracks 100 ~m apart are reproduced. The position of the tracks in relation to the solitary tract is given by a Procion Yellow marked cell (P.Y.). The evoked potentials had latencies ranging from 2.0 to 4.4 msec with an average of 3.2 msec. The gross similarity between the average latencies for the evoked potential in the solitary tract and the 'inspiratory-vagal' neurones suggests that a considerable part of the intracephalic delay may be due to slow conduction velocity in the terminal course of the afferents. Spinal connections. Fifty-nine 'inspiratory' and 3 'other respiratory' neurones were tested for their connections with the ventrolateral spinal columns on the contralateral side of the cervical cord 7,32. Of these 62 cells 40 responded in a one-to-one fashion to spinal shocks; all of them were 'inspiratory'. A subsequent paper 2° deals with the properties of these spinal connections. For 53 of the 59 inspiratory neurones tested for spinal connections we succeeded in determining the sensitivity to vagal stimulation. With one possible exception it was found that if a neurone could be driven from the spinal stimulus sites it could not be driven from the vagus (34 neurones); correspondingly it was found that if a neurone could be driven from the vagus it could not be driven from the spinal cord (16 'inspiratory' and 3 'other respiratory' neurones). Concerning the possible ex-

RESPIRATORY SOLITARY TRACT NEURONES.

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Fig. 4. Region of evoked potentials to vagal stimulation and localization of respiratory and nonrespiratory neurones encountered in 4 successive electrode tracks shown in relation to the solitary tract (TS) in the left dorsomedial part of medulla. The same electrode used in all 4 tracks. Calibration: each division 100 Fm with the lower lateral extent of the tractus solitarius as point zero (see Methods). The inspiratory vagal neurone (block triangle) marked P.Y. was marked by intracellular injection of Procion Yellow.

ception we were not certain whether we actually held the same cell during both the vagal and the spinal tests. In summary it thus appears that the inspiratory neurones o f the solitary complex fall into two separate groups: (I) the 'inspiratory-vagar neurones which could not be driven with short latency from the ventral and lateral columns o f the contralateral side o f the spinal cord, and (2) the 'inspiratory non-vagar neurones with direct, rapidly conducting spinal connections.

Anatomical localization Successful penetrations permitting electrophoretic injection o f Procion Yellow were made with 17 physiologically studied neurones, 16 o f which were recovered and histologically identified. In two experiments, in which we were unable to penetrate successfully any o f the many respiratory neurones studied extracellularly, we deposited Procion Yellow extracellularly. In both cases we were later able to identify histologically the diffuse yellow-orange fluorescent spot (roughly 100 # m in diameter). Using

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RESPIRATORYSOLITARY TRACT NEURONES. I

11

TABLE II DISTRIBUTION OF THE 7 0 ANATOMICALLY DEFINED FUNCTIONAL CELL TYPES AT

14 FRONTAL

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MEDULLA OF THE CAT

Frontal Actual plane frontal diagram plane (Fig. 5)

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Fr. 1

Fr. 2

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Total

Functional cell types Insp. vagal

lnsp. nonvagal spinal

---0.15 0.00 +0.18

7 2 4

+0.60 +0.70 +0.90 + 1.05 +1.26 +1.30 +1.33 + 1.40

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5

2 6 2

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1

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lnsp. spinal not test vagal

lnsp. not further studied

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1 4

12

1 27

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2 18

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70

these 16 n e u r o n e s a n d 2 extraceUular spots as markers it proved possible to reconstruct the respective electrode tracks a n d determine the a n a t o m i c a l positions o f a total o f 70 n e u r o n e s : 66 ' i n s p i r a t o r y ' neurones, 1 ' o t h e r respiratory' n e u r o n e a n d 3 ' n o n respiratory' n e u r o n e s all of which are projected o n t o the 3 charts of Fig. 5 pred o m i n a n t l y in the ventrolateral nucleus o f the solitary tract a n d within 300 # m of o u r 'zero p o i n t ' (see Methods). Fig. 5 is a composite d i a g r a m where the cells are projected o n t o the nearest of the 3 frontal planes at obex, 1 m m a n d 2 m m in f r o n t o f obex. It can be seen that the largest n u m b e r of cells (40) were f o u n d a r o u n d the level 1 m m in f r o n t o f obex (Ft. 1) a n d 18 cells at the level of obex (Ft. 0) a n d 12 cells a r o u n d

Fig. 5. Anatomical localization of respiratory and non-respiratory neurones in the dorsomedial medulla of the cat. Composite diagram with the cells from 14 different frontal levels projected on the nearest of 3 frontal planes: at obex (bottom), including --4).15 to +0.18; 1 mm rostral to obex (middle), including +0.6 to +1.4; 2 mm rostral to obex, including +1.6 to +2.34 (see Table II). Right: cross-section of the left hemi-medulla with the area of study, tractus solitarius and nucleus, indicated within the box (dashed lines). Left: 10-fold magnification of these areas showing positions of 73 anatomically identified and functionally studied cells. Note the preponderance of inspiratory neurones lying within a 300/tm radius of 'point zero' (see Methods) of the ventrolateral nucleus of the solitary tract. The heterogeneity of this nucleus is indicated by the intermixing of 'vagal' and 'non-vagal' but spinally driven inspiratory cells, 'other respiratory' cells and 'non-respiratory' cells. Calibration: each division 100/~m with the lower lateral extent of the tractus solitarius as point zero. Symbols: to be interpreted by combinations of explanations given in the key.

12

c. VON EULERet al.

2 mm in front of obex (Fr. 2). The actual distribution can be seen in Table II. Fig. 5 further demonstrates the heterogeneity of the neurone population within the ventrolateral nucleus of the solitary complex. 'Inspiratory-vagal', 'inspiratory non-vagal but spinal', 'inspiratory-expiratory', 'expiratory' and 'non-respiratory' cells are distributed throughout the nucleus with no definite grouping according to function. The distribution of cells in Fig. 5 and Table II suggests, however, that cells with spinal connections may have some preferential location immediately around 'point zero' from Fr. 0.7 to 1.4, while cells with vagal input may have a tendency to be located closer to 'point zero' at rostral and caudal levels, but more in the periphery in the intermediate sections (Fr. 0.6-1.4). The overall distribution of the different functional cell types in the region of the solitary complex (see column A of Table I) appears similar to the anatomically selected sample (Table I, column B).

Morphology Out of a total of 16 cells which were histologically identified by their fluorescence from the intracellularly deposited Procion Yellow, we were able to draw, photograph and reconstruct 10 cells as shown in Fig. 6. Fig. 7A and B are montages of photomicrographs showing two of the cells of Fig. 6 (nos. 2 and 8). The drawings of the photomicrographs show soma and nucleus in all cells except no. 3, the proximal dendritic tree up to a radius of about 200 #m, and the proximal part of the presumed axon (a) in cells nos. 1, 3 and 8. Axons were identified tentatively by the very thin initial part and the uniform diameter of the process24, 25. These inspiratory neurones of the ventrolateral nucleus of the solitary tract seem to range in diameter from 20 to 60 #m. All stained cells except no. 7 were located within a 300/~m radius of 'zero point' of the solitary tract. The 3 'axons' were oriented in a dorsoventral direction, cell no. 1 ventrally, cells nos. 3 and 8 dorsally. Dendrites extend in a mediolateral and a dorsoventral direction in a radius of up to 200-250 #m in some cells (nos. 2, 4, 7 and 8) and into direct contact with the fibres of the solitary tract (nos. 2, 3, 6 and 8). From our limited sample of inspiratory neurones there is no clear distinction between physiological and morphological cell types. Functional aspects of the inspiratory neurones with vagal input Of the 88 'inspiratory-vagal' neurones 70 showed marked changes in their discharge rates in response to volume changes of the lungs. In view of the aim of this investigation it was of primary interest to study the functional significance of these neurones and their possible role for the Hering-Breuer reflex inhibition of inspiration. The first question to be answered was whether the vagal response to lung expansion was obligatory for the firing of such neurones or whether they had some other input that by itself was sufficient to fire the cell. In 67 of the 70 neurones with obvious pulmonary mechanoreceptor input the discharge activity retained some respiratory rhythmicity when the respirator was momentarily switched off, as first described by von Baumgarten and Kanzow e for their R/~ cells and later confirmed by others (e.g. refs. 7, 15 and 16). Only 3 neurones stopped their respiratory rhythmicity all together

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Fig. 6. Composite drawings of 10 'inspiratory' cells in the left ventrolateral nucleus of the solitary tract injected with Procion Yellow. The drawings of each cell were made by superimposing camera lucida tracings from a number of consecutive sections. The neurones are from different frontal levels (+0.15 to +2.34) but are here projected on the same frontal plane in relation to the point zero (see Methods) of the solitary tract TS as in Figs. 4 and 5. Note heterogeneity of inspiratory cells in the nucleus, vagal and non-vagal spinal, large and small cells, dendrites oriented toward and away from the tractus solitarius. An initial portion of the axon was tentatively identified in 3 inspiratory cells (nos. 1, 4 and 8) with dorsoventral orientation. Trajectory of dendrites both dorsoventral and mediolateral. Perhaps the two inspiratory vagal cells (nos. 2 and 4), which are larger and stellate, represent some morphological specialization when compared with the smaller, elongated inspiratory, nonvagal, spinal cells (nos. 8 and 10). The variability of cell size and the limited number of cells visualized prevent further generalizations at this time. Calibration: 100/~m divisions from point zero. The diagram oriented so that the right side is medial and parallel with the midline.

m

15

RESPIRATORY SOLITARY TRACT NEURONES. I

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Isec Fig. 8. Inspiratory, 'vagal, non-spinal' neurone. From above: 'instantaneous' frequency record, discharge from the neurone, 'integrated' phrenic activity and, at the bottom, intratracheal pressure. In the first two inspiratory discharges the respirator was driven by the 'integrated' phrenic activity (with some time lag). During the last inspiration respirator was switched off so that the neurone received only CIE input giving a slower rate of increase of inspiratory activity. It reaches roughly the same value as in the first two breaths, but after a longer time. Inspiratory termination thus occurs when the discharge rate has reached approximately the same level whether volume feedback or not.

when the lung movements were stopped. Thus most o f the neurones in our population received a respiratorily modulated excitation, other than that from the p u l m o n a r y stretch receptors, sufficient to produce an inspiratory pattern o f firing. This input we have chosen to call 'Central Inspiratory Excitation' or C i E as opposed to peripheral excitation arising f r o m lung receptors. The relative contributions o f the p u l m o n a r y afferent and C I E inputs varied f r o m neurone to neurone, A typical neurone is depicted in Fig. 8. Here the 'instantaneous frequency' records (top tracing) show that the rate o f increase in discharge rate was steeper in the first two inspirations during which the lungs were normally inflated and the neurone received an excitatory contribution also f r o m the vagal volume receptors. In the last inspiratory discharge when the respirator was switched off, the cell received no volume related input. Due to the lengthening o f the inspiratory phase in the absence o f vagai volume feedback the firing nevertheless reached the same final discharge rate at the m o m e n t o f inspiratory arrest. Changes in vagal responsiveness during the inspiratory phase. The variation during the breathing cycle o f the threshold for vagal responses to electrical stimulation was studied in detail in 12 cells. The vagus nerve was stimulated with single shocks at a rate permitting 4 or 5 shocks to be delivered during the course o f each inspiration.

Fig. 7. Montages of photomicrograms of 2 inspiratory neurones of the solitary tract complex filled with Procion Yellow and photographed under UV light. A: inspiratory-vagal cell (no. 2 in Fig. 6). Montage from 7 serial sections (15/~m). Note radial distribution of dendrites. Axon not clearly visualized. B: inspiratory-spinal, non-vagal cell (no. 8 in Fig. 6). Montage from 6 serial sections (l 5 pm). a, axon.

16

c. VON EULER et al.

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Fig. 9. Inspiratory 'vagal, non-spinal' neurone driven only in the inspiratory phase by single shocks to the ipsilateral vagus nerve. Driven evoked potentials and spikes displayed on the sweep records with shock artefacts at the upper end of the sweeps. Continuous record of phrenic activity signifying the phases of inspiration. A stimulus strength at threshold for driving. B stimulus strength increased 2 dB above A. C stimulus strength increased 4 dB above A. For further explanation see text.

The intensity of the single shocks was always near the threshold for cutting short the inspiratory phase when shocks of this intensity were applied in a tetanus of 205/sec. It was adjusted until the cell could just be driven at some point in the cycle. As illustrated in Fig. 9 a driven spike was most easily evoked at the end of inspiration. On slight increase of stimulus strength the cell (B of Fig. 9) responded during a somewhat longer portion of the inspiratory phase but there was still a definite preference for the responses to occur toward the end of inspiration. As intensity was increased further (C) the cells would respond earlier in the inspiration. After the end of the inspiratory phase the responsiveness to vagal shocks decreased along a time course similar to that of the declining phrenic activity, and usually no responses could be obtained in the last 2/3-1/2 of the expiratory phase. For graphical representation of the time dependence of the responsiveness of the neurones (see Fig. 10), responses were recorded for about 25 breaths at each stimulus intensity. The onset of the phase of inspiration, as judged from the phrenic activity, was taken as zero time. In each experiment the inspiratory phases were divided in 4 or 5 equal divisions. For each division of time the presence or absence of a fixed latency response was used to construct histograms examples of which are shown in Fig. 10. The values of the bars in the histograms represent the average number of spikes of the neurone in response per stimulus shock given as per cent. The probability of eliciting a spike response increases steeply with time as inspiration proceeds. Values above 100 in D of Fig. 10 indicate the occurrence of double or

RESPIRATORY SOLITARY TRACT NEURONES. I

17

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Fig. 10. Response histograms from 4 different inspiratory, 'vagal, non-spinal' solitary tract neurones. The histograms show the probability for a response in terms of driven spikes to single shocks to the ipsilateral vagus nerve at different times during the phase of inspiration. Values greater than 1.0 denote that a single shock may elicit more than one spike. For further explanation see text.

triple firing to a single vagal shock. A marked increase in responsiveness with time during the inspiratory phase was seen in the majority of the neurones which showed a pronounced CIE input, whereas little or no significant change in responsiveness could be demonstrated in the 3 neurones mentioned earlier, with no clear manifestation of central inspiratory excitatory input. It thus would seem that the magnitude and time course of the CIE input are the main factors causing the time dependent change to vagal responsiveness. Responsiveness to inflation. In spite of the 'closed chamber' recording technique employed in this study stability was not good enough to 'keep' the cell in the presence of sudden changes in intrathoracic pressure. (As mentioned in Methods the use of pneumothorax for improving stability of recording was not compatible with the goal of the experiment.) Therefore the responsiveness of these neurones to inflations has not been studied in a systematic way similar to the responsiveness to electrical stim-

C. VON EULER et al.

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Fig. ] ]. Record from an Jnspiratory 'vagai, non-spinal' solitary tract neurone (upper tracing) under conditions when the phrenic nerve showed some residual activity also during the whole of the expiratory phase. The two middle tracings show phrenic neurogram and 'integrated' phrenic activity. ]Note that under these conditions the neurone shows some response to imposed inflations (signalled by the intratracheal pressure record) also during the phases of expiration.

ulation of the vagus nerve. However, when inflations were imposed out of phase with the phrenic activity these neurones with a combined CIE and pulmonary mechanoreceptor input showed little or no response to volume changes in the latter half of the expiratory phase, i.e., when there was no phrenic activity and presumably no CIE input. In a few cases, however, with some tonic phrenic activity remaining during the whole of the expiratory phase, inflation induced responses also during the expiratory phase, an example of which is shown in Fig. 1 I. DISCUSSION

Allowing for possibility of some bias of sampling and of some slight damage to the vagal input we can conclude that in our sample of inspiratory neurones in the ventrolateral nucleus of the solitary tract complex the 'vagal' (88) and the 'non-vagal' cells (104) constitute roughly equal populations. Of considerable importance in that respect is our finding that none of the 'vagal-inspiratory' neurones showed any signs of short latency responses from the ventrolateral funiculi of the contralateral cervical cord. Such responses could only be demonstrated for neurones belonging to the group of 'non-vagal' neurones (with the one uncertain exception). This is not in full agreement with some earlier work 7,3z in which, however, coarser and less discriminative electrodes were employed. In addition to these two groups of 'inspiratory' neurones we found also 'other respiratory' and 'non-respiratory' cells. Neurones of different characteristics were often found to be close neighbours. The ventrolateral solitary nucleus thus appears fairly heterogeneous with regard to firing patterns and connections. This, however, does not mean that it is heterogeneous from an overall functional and integrative point of view. The 'vagal-inspiratory' neurones. The main problem dealt with in the present paper concerns the extent to which the 'vagal-inspiratory' neurones are responsible to the reflex characteristics of the Hering-Breuer termination of inspiration, especially the strong dependence of its volume threshold on time from the onset of inspiration 14. In addition to their input from pulmonary afferents these neurones have at least one

RESPIRATORY SOLITARY TRACT NEURONES. I

19

other major source of input which is inspiratory in character and of central origin 6. Our results indicate that it is through the slowly increasing CIE input that these cells acquire their time dependent change in responsiveness to their vagal input. We therefore suggest that the 'vagal-inspiratory' neurones are the mediators of the Hering -Breuer inspiratory inhibitory reflex and are responsible for the time dependent decrease in the volume threshold of the 'all or nothing' inhibition of inspiration as described by Clark and von Euler 14. As compared to the volume-threshold curve of the Hering-Breuer termination of inspiration described by them the time course of the responsiveness of these cells to vagal stimulation appears less steep early in inspiration and less 'curved'. In the present experiments, however, the electrical mode of stimulation excluded the mechanoreceptors of the lungs with their curvilinear transfer characteristics between volume and discharge rate14,4~, whereas in the work of Clark and von Euler the receptor properties contributed to the shape of the volume-time relationship. As discussed already in their paper it was therefore to be expected that the time course of the increasing responsiveness to electrical stimulation should follow a somewhat straighter line than that of the volume threshold. In all cases, however, the threshold of the respiratory neurone to vagal input decreased very markedly with time as the inspiratory phase proceeded. Are the 'vagal-inspiratory' neurones of the ventrolateral solitary nucleus the second order neurones of the pulmonary afferent pathway? This is a question which is difficult to settle from the present data. Histological evidence suggests such a possibility. Afferent terminal degeneration within the ventrolateral solitary nucleus (in addition to such degeneration seen in the medial solitary tract nucleus) has been described following intracranial sections of vagal rootlets carrying pulmonary stretch receptors 4. In many of the neurones of the present work the latencies of spikes driven by vagal shocks were as short as those obtained for the evoked response recorded among the fibres in the tractus solitarius itself (cf. Fig. 4). This is in concord with the possibility of these neurones being second order pulmonary afferent neurones. Such a view may, however, be questioned on other functional grounds. For instance, cardiovascular neurones with similarly short latency but with a fairly simple type of primary afferent firing pattern have been described in the medial nucleus of the solitary tract complex9,22,28 and Koepchen et al. 28, working on dogs, have briefly reported on the presence of neurones located in the medial nucleus (very close to but somewhat caudal relative to the cardio-vascular neurones) which exhibited a firing pattern like that of pulmonary stretch receptors and which exhibited no rhythmic firing when respiratory movements were stopped. Since the ceils of the dorsomedial nucleus are small it is very likely that our fine, high impedance (Procion Yellow filled) glass microelectrodes missed them. Certainly our electrodes have passed through this nucleus; only a few active neurones were encountered there but none of the requisite type. We have found only 3 neurones exhibiting a fairly unprocessed vagal volume information. Whether they represented earlier order pulmonary afferent neurones than the others or whether they represent one extreme of the 'vagal-inspiratory' neurones with only relatively small central inspiratory excitatory inputs, as suggested earlier in this paper, cannot be decided, it does not seem unlikely that there would exist a pool of second order

20

c. VON EULER et al.

neurones the activity of which would still retain the afferent information in a relatively undistorted way and which could supply this information to the two different aspects o f the inflation reflex, i.e., termination o f inspiration and prolongation o f expiration. These two reflexes have been found to utilize the same information quite differently and probably require different mechanisms14, 27. It is entirely possible, however, that there exist two separate complex second order neurone pools, one for each aspect of the inflation reflex 43. The spinal inspiratory neurones. F r o m the w o r k by N a k a y a m a and von Baumgarten a2 and by Bianchi 7 it was expected that the short latency spinal driving in our experiments would prove to represent antidromic activation o f the cells. This was expected also from the work of Torvik 4° who demonstrated the existence o f a crossed pathway descending in the ventrolateral q u a d r a n t o f the cervical cord which, when cut, caused retrograde degeneration in the large and medium sized cells o f the contralateral ventrolateral nucleus o f the solitary tract. Performance o f a critically time-controlled collision test, however, has revealed that both o r t h o d r o m i c and antidromic pathways o f similar conduction time were involved. This will be dealt with separately in a subsequent paper 2° in which we further conclude that there is an inhibitory loop connecting the ortho- and antidromic pathways. This may be significant also for the discharge characteristics o f the inspiratory neurones under discussion.

REFERENCES l ADRIAN, E. D., Afferent impulses in the vagus and their effect on respiration, J. Physiol. (Lond.),

79 (1933) 332-358. 2 ANDERSON, F. D., AND BERRY, C. M., An oscillographic study of the central pathways of the

vagus nerve in the cat, J. comp. Neurol., 106 (1956) 265-283. 3 BAUMGARTEN, R. VON, V ARANDA CODDOU, L., Distribuci6n de las aferencias cardiovasculares

y respiratorias on las raices bulbares del nervio vago, Acta neurol, lat.-amer., 5 (1959) 267-278. 4 BAUMGARTEN, R. VON, BALTHASAR, K., UND KOEPCHEN, H.P., Ober ein Substrat atmungsrhythmischer Erregungsbildung im Rautenhirn der Katze, Pfliigers Arch. ges. Physiol., 270 (1960) 504-528. 5 BAUMGARTEN, R. VON, BAUMGARTEN, A. VON, UND SCHAEFER, K. P., Beitrag zur Lokalisationsfrage bulboreticularer respiratorischer Neurone der Katze, Pfliigers Arch. ges. Physiol., 264 (1957) 217-227. 6 BAUMGARTEN,R. VON,AND KANZOW, E., The interaction of two types of inspiratory neurons in the region of the tractus solitarius of the cat, Arch. ital. Biol., 96 (1958) 361-373. 7 BIANCm, A. L., Localisation et 6tude des neurones respiratoires bulbaires. Mise en jeu antidr6mique par stimulation spinale ou vagale, J. Physiol. (Paris), 63 (1971) 5-40. 8 BISCOE,T. J., ANDSAMPSON,S. R., Field potentials evoked in the brain stem of the cat by stimulation of the carotid sinus, glossopharyngeal, aortic and superior laryngeal nerves, J. Physiol. (Lond.), 209 (1970) 341-358. 9 BlSCOE,T. J., AND SAMPSON,S. R., Responses of cells in the brain stem of the cat to stimulation of the sinus, glossopharyngeal, aortic and superior laryngeal nerves, J. Physiol. (Lond.), 209 (1970) 359-373. 10 BONVALLET, M., ET SIGG, B., l~tude 61ectrophysiologique des aff6rences vagales au niveau de leur p6n6tration dans le bulbe, J. Physiol. (Paris), 50 (1958) 63-74. 11 BOYD, T. E., AND MAASKE, C. A., Vagal inhibition of inspiration, and accompanying changes of respiratory rhythm, J. Neurophysiol., 2 (1939) 533-542. 12 BREUER,J., Die Selbsteuerung der Atmung durch den Nervus vagus, S.B. Akad. Wiss. Wien, 58 (1868) 909-937.

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21

13 CAJAL,S. RAM6N Y, Histologie du Systdme Nerveux de l'Homme et des Vertdbrds, VoL 1, Maioine, Paris, 1909, Ch. 26. 14 CLARK, F. J., AND EULER, C. VON, On the regulation of depth and rate of breathing, J. Physiol. (Lond.), 222 (1972) 267-295. 15 COrIEN, M. I., Discharge patterns of brain-stem respiratory neurons during Hering-Breuer reflex evoked by lung inflation, J. Neurophysiol., 32 (1969) 356--374. 16 COaEN, M. I., How respiratory rhythm originates: evidence from discharge patterns of brainstem respiratory neurones. In R. PORTER (Ed.), Breathing: Hering-Breuer Centenary Symposium, Churchill, London, 1970, pp. 125-150. 17 COTTLE, M. K., Degeneration studies of primary afferents of IXth and Xth cranial nerves in the cat, J. comp. Neurol., 122 (1964) 329-345. 18 CRILL, W. E., AND REIS, D. J., Distribution of carotid sinus and depressor nerves in cat brain stem, Amer. J. Physiol., 214 (1968) 269-276. 19 EULER, C. VON, HAYWARD,J. N., MARTTILA,I., AND WYMAN,R. J., Vagal and spinal connections of the inspiratory neurons of the ventrolateral nucleus of the tractus solitarius of cat, Arch. Fisiol., 68 (1971) 327-328. 20 EULER, C. VON, HAYWARD, J. N., MARTTILA,I., AND WYMAN, R. J., The spinal connections of the inspiratory neurones of the ventrolateral nucleus of the tractus solitarius of cat, Brain Research, 61 (1973) 23-33. 21 FOLEY,J. O., AND DUBOIS, F. S., An experimental study of the rootlets of the vagus nerve in the cat, J. comp. Neurol., 60 (1934) 137-159. 22 HUMPHREY, D. R., Neuronal activity in the medulla oblongata of cat evoked by stimulation of the carotid sinus nerve. In P. KEDZI (Ed.), Baroreceptors and Hypertension, Pergamon Press, Oxford, 1967, pp. 131-167. 23 INGRAM,W. R., AND DAWKINS, E. A., The intramedullary course of afferent fibres of the vagus nerve in the cat, J. comp. NeuroL, 32 (1945) 157-168. 24 JANKOWSKA, E., AND LINDSTROM, S., Morphological identification of physiologically defined neurones in the cat spinal cord, Brain Research, 20 (1970) 323-326. 25 JANKOWSKA,E., AND LINDSTRSM,S., Morphological identification of Renshaw cells, Acta physiol. scand., 81 (1971) 428-430. 26 KERR, F. W. L., Facial, vagal and glossopharyngeal nerves in the cat, Arch. Neurol. (Chic.), 6 (1962) 264-281. 27 KNOX, C. K., Characteristics of the inflation and deflation reflexes during expiration in the cat, J. Neurophysiol., (1973) in press. 28 KOEPCHEN,H. P., LANGHORST,P., SELLER,H., POLSTER,J., UND WAGNER,P. H., Neuronale Aktivifiit im unteren Hirnstamm mit Beziehung zum Kreislauf, Pfliigers Arch. ges. PhysioL, 294 (1967) 40-64. 29 LARRABEE,M. G., AND HODES, R., Cyclic changes in the respiratory centers revealed by the effects of afferent impulses, Amer. J. PhysioL, 155 (1948) 147-164. 30 LARRABEE, M. G., AND KNOWLTON, G. C., Excitation and inhibition of phrenic motoneurones by inflation of the lungs, Amer. J. PhysioL, 147 (1946) 90-99. 31 MIURA, M., AND REIS, D. J., Termination and secondary projections of carotid sinus nerve in the cat brain stem, Amer. J. Physiol., 217 (1969) 142-153. 32 NAKAYAMA, S., UND BAUMGARTEN, R. YON, Lokalisierung absteigender Atmungsbahnen im Riickenmark der Katze mittels antidromer Reizung, Pfliigers Arch. ges. Physiol., 281 (1964) 231244. 33 NELSON, J. R., Single unit activity in medullary respiratory centers of cat, J. Neurophysiol., 22 (1959) 590-598. 34 OBERHOLZER,R. J. H., Circulatory centers in medulla and midbrain, PhysioL Rev., 40, Suppl. 4 (1960) 179-195. 35 PAINTAL,A. S., The conduction velocities of respiratory and cardiovascular afferent fibres in the vagus nerve, J. Physiol. (Lond.), 121 (1953) 341-359. 36 PAINTAL,A. S., Vagal afferent fibres, Ergebn. PhysioL, 52 (1963) 74-156. 37 SELLER,H., AND ILLERT, M., The localization of the first synapse in the carotid sinus baroreceptor reflex pathway and its alteration of the afferent input, Pfliigers Arch. ges. Physiol., 306 (1969) 1-19. 38 SMITH, R. S., AND PEARCE, J. W., Microelectrode recordings from the region of the nucleus solitarius in the cat, Canad. J. Biochem., 39 (1961) 933-939. 39 STREYrON, A. O. W., AND KRAVITZ, E. A., Neuronal geometry: determination with a technique of intracellular dye injection, Science, 162 (1968) 132-134.

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40 TORVIK,A., The spinal projection from the nucleus of the solitary tract. An experimental study in the cat, J. Anat. (Lond.), 91 (1957) 314-322. 41 WIDDICOMBE,J. G., Receptors in the trachea and bronchi of the cat, J. Physiol. (Lond.), 123 (1954) 71-104. 42 WIDDlCOMBE, J . G . , Respiratory reflexes. In W. O. FENN AND H. RAHN (Eds.), Handbook of Physiology, Section 3: Respiration, Vol. 1, American Physiological Society, Washington, D.C., 1964, pp. 585-630. 43 WYss, O. A. M., Die nerv6se Steuerung der Atmung, Ergebn. Physiol., 54 (1964) 1-479.