Classification of vagal afferents firing in phase with breathing in Gallus domesticus

RespirationPhysiology(1974) 22, 57-76; ~Or~h-Uo~~~ndPublishing Company,Amsterdam

CLASSIFICATION OF VAGAL AFFERENTS FIRING IN PHASE WITH BREATHING ZN GALLUS DOMESTICUS’

V. MOLONY ~~p~~nt

of VeterinaryAnatomyLiwpooI Uniuersity,England

Abstract. Forty-five singte *rent units were recorded from the right vagi of 27 acutely deccrebrated hens. Each unit was subjected to a wide range of tests of which four are described. This permit&d the classification of the units into different types. Type 1 (56%) were exquisitely sensitive to CO3 but also showed some responses which have so far only been explained by their having some m~h~o~~~tivity: they showed a wide variety of eupnoeic firing patterns and in some cases changed their firing patterns dramatically. Type IX(20”/0)were sensitive to mechanical stimulation but were not noticeably, sensitive to CO,: they all showed similar firing patterns during eupnoea, tiring throughout inspiration at an almost constant rate and stopping at or near the peak of inspiration. Of the remaining 24% of units no two showed enough similarity to be classed together as a third type. it was concluded that two types of phasic respiratory afferent gbres occur in the vagus of the hen, one from COz-sensitive receptors the other from ~~~or~tors. Birds Carbon dioxide-sensitive receptors Control of Breathing

Mcchanoreceptors Nerve conduction velocity Single unit activity

The importance of vagal afferent activity in the control of breathing of birds has been demonstrated by the effects of vagotomy on eupnoeic breathing (Fedde et al., 1963; King, 1966; Richards, 1969; McLeflsnd, 1970); on the inflation reflex (Graham, 1940; Richards, 1968; Eaton et al., 1971) and on the insufflation reflex (Peterson and Fedde, 1968). Further support is provided by the effects on stimulation of the central end of the cut vagus (Sinha, 1958; McLeHand, 1970). Several workers have investigated respiratory afferent activity in the avian vagus (King et al., 1968; Fedde and Peterson; 1970; Osborne and Burger, 1971; Osborne, 1971; Leitner, 1972). Unlike the situation for mammals, in which three types of pulmonary receptors have been established, pulmonary stretch, irritant, and type J receptors, it is not generally agreed how many types of receptor have been discovered in the avian respiratory system. ’ This paper was presented at the workshop on “Receptors and Control of Respiration in Birds” held May 24-25, 1974, at the Max-Plauck-Institut fit experimentelle Mediin in C&ttingen, Federal Republic of Germany. 57

58

V. MOLONY

Leitner (1972) has proposed that three main types of vagal pulmonary afferents are present, his classification being based mainly on the responses of vagal afferents to inflation and deflation. Our initial attempts to classify units in a similar way (King et al., 1969) proved unhelpful due to the great variety of responses given by units responding to similar stimuli. In addition some units changed in the course of testing from one type to another and units of different types showed great similarity in their responses to other stimuli such as COz. This type of classification depended upon differences in the location within the respiratory system and quantitative differences in the responses to stimuli. In this study an attempt has been made to classify afferent units according to qualitative differences and similarities in their responses to stimuli using a wide range of tests. It has also been possible to put forward some suggestions as to the physiological stimuli, location and roles of the two types of receptor proposed here. Methods Twenty-seven adult hybrid hens in laying condition, weighing from 1.0 to 1.5 kg, were decerebrated just rostra1 to the optic chiasma and tracheotomised at the mid-cervical level under halothane (Fluothane, I.C.I.) anaesthesia. The right cranial thoracic air sac was cannulated between the middle of the third and fourth sternal ribs, and the right caudal thoracic air sac between the ventral ends of the last two vertebral ribs. The right atrium was cannulated via the right brachial vein. Leads

Fig. 1. Schematic drawing of the experimental apparatus. Handrests attached to the baseplate and a Zeiss operating microscope were used in addition to the apparatus shown. Asc 1 and 2: cannulae in the caudal andcranial thoracic air sacs, respectively. At.C.: cannula in the right atrium. Cl: clamp holding wire frame for paraffin bath. 0.B: oesophageal bar at 41 “C. Pl: Perspex plethysmograph. P.R: paper recorder. P.T: pressure transducer. R.E: recording electrode. S.P: 3 inch-steel baseplate. St.E: stimulating electrodes. T.C: tracheal cannula. T: thermometer.

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AFFERENTS

IN THE AVIAN VAGUS

59

(e.c.g.) were attached to the right wing and left leg, and the trunk was sealed into a perspex whole-body plethysmograph by means of a rubber collar stitched to the skin at the base of the neck (fig. 1). Pressure changes in the plethysmograph produced by breathing were monitored by means of a pressure transducer (‘Ether’ UP/2), displayed on a pen recorder (Devices M2) and a dual-beam oscilloscope (Tektronix 502A). A volume calibration was obtained by inflation of the hen with a known volume of air from a large syringe. The hen’s body temperature was monitored throughout by means of a thermistor probe (Yellow Springs Instrument Co.) inserted at least 2 inches into the rectum. Body temperature was maintained at 41 “C by means of a heated copper tube in the oesophagus, inserted as far as the crop; this tube was also used to stabilise the neck and as the indifferent recording electrode. The head was clamped by means of bars in the external auditory meati and the neck was extended with the right vagus uppermost (fig. 1). RECORDING

TECHNIQUE

A bath was made in the side of the neck with skin and a wire frame, the vagus was dissected out for l-2 cm at the cranial and caudal ends of the bath 8-10 cm separating the two sites. The bath was sealed with 4% agar and filled with warm (41 “C) liquid paraffin. Bipolar silver wire (24 S.W.G.) stimulating electrodes were hooked under the vagus at the caudal end of the bath and fibre preparations were dissected from the vagus at the cranial end. The vagus was not transected. Nerve fibres in the hen’s vagus are all ~8.0 pm (Brown et al., 1972). Action potentials were recorded with a monopolar platinum-iridium wire electrode, 0.010 inch diameter, amplified (Tektronix 122 pre-amplifier, bandwidth setting from 80 Hz to 1 kHz), and displayed on the dual beam oscilloscope with breathing. Photographic records were taken from a slave oscilloscope. ‘Single units’ were defined as those units in a filament which could be distinguished from all others at all times and in particular in the compound action potential when the vagus was stimulated at 7 x the threshold (volts) of the most sensitive unit present. Most fibre preparations contained less than five active fibres. SELECTION

AND CHARACTERISATION

OF UNITS

Single units were dissected at random from the right vagus. All units showing a consistent modulation of their firing were selected. Some continuously tiring units showed only very small changes in firing rate at each breath e.g. fig. 2c. Such changes were, however, easily detected from the audiomonitor. Units which lost their respiratory modulation during the testing period were rejected. A battery of tests was applied to each unit including measurement of the conduction velocity, responses to: inflation, deflation, changes in inspired COz concentration (Fb,), rebreathing tubes, occlusion of the trachea, insuhlation, ammonia, halothane, sulphur dioxide, tobacco smoke, veratridine, phenyl diguanide, acetylcholine, histamine, adrenaline and noradrenaline. Some units were lost before all of the tests were completed and some tests were only applied to some of the units.

60

V. MOMNY

The responses of units and of breathing to all of these tests are described elsewhere, Molony (1972); responses to four tests are included here - these show notable differences in the responses of the units described. (1) Responses ,to gas mixtures containing CO,

End-expired air from the operator, 95% 02, 5% CO2 and various mixtures of 0z/C02 were used to replace room air at the tracheal opening. A wide bore (1 inch) rubber tube through which the gas mixture was flowing was placed just over the open end of the trachea. Exposure to these gas mixtures was usually brief (2-3 breaths). ‘The level of CO, in the gas mixtures was calculated from the flow rates measured with rotameter flow meters and in some later experiments it was measured with an infra-red absorption gas analyser (Godart URAS-4). The response delay of CO,-sensitive units was measured. as the time elapsing between the application of CO2 and the arrest of tiring of the unit; tests were arranged so that the “on” coincided with the eupnoeic firing in inspiration. CO,-sensitivity was also investigated by using various mixtures of O2 and COz at several flow rates to insufllate the bird. In this procedure the gas mixture was introduced into the open end of the trachea and allowed to escape via the open air-sac cannulae. (2) Inflation Three techniques were employed: (i) 20-100 ml of air from a large syringe (ii) air from a large (60 L) pressure bottle at pressures from 6 to 18 cm H,O were introduced into the birds via the trachea or (iii) the pressure in the plethysmograph was reduced by 12-15 cm H,O. (3) Occlusion of the trachea The open end of the tracheal cannula was occluded with a damp cotton wool plug for 2-3 breaths. (4) Response to Halothane

A wide bore (1 inch) rubber tube, through which 3% Halothane in O2 from a Fluotec vaporiser (B.O.C.) was flowing at 1.5 L/min was held just over the open end of the trachea for up to :15 breaths. Timing of the “on” and “off’ in all tests was recorded on the tilm by means of a marker light operated by,a foot switch. (5) Conduction velocity

Conduction velocity was measured from the delay of the response to electrical stimulus of the vagus and the distance between stimulating and recording electrodes. Results Forty-live single afferent units which showed a consistent respiratory modulation of firing were characterised. Selection was random, except for any bias which may have

RESPIRATORYAFFTMNTS IN THE AVIAN VAGUS

61

occurred due to a greater ease of dissection of huger fibres and to any topographical organisation within the vagus nerve. The units were classified into different types according to their range of response to the various tests, as described in detail beldw. The most clear-cut difference between the two types was that type I units showed a distinct sensitivity to changes in Fro,,, whereas type II receptors were insensitive to this stimulus. Of the 45 units, 25 (56%) were classified as type I and nine (20%) as type II. No two of the other eleven units showed suffkiently similar responses over the range of tests to be classified together as a third type; these units will therefore not be included in further description. FIRING PAlTERNS DURING EUPNOEA

These units showed a great variety of firing patterns during eupnoeic breathing. Eighteen units (72%) fired mainly in inspiration and seven (28%) mainly in expiration. Ten units (40%) fired separately in both inspiration and expiration and six (24%) fired continuously. Five units changed their firing patterns, often abruptly, and sometimes on several occasions, during the course of the experiment. The essential features are seen in the firing patterns of the four units shown in fig. 2a-d. Those units firing. mainly in inspiration (a, d) often did not fire from the beginning of inspiration but showed a short delay of about 0.3 sec. There was also a deiay in the

i a

rn

Fig. 2. Eupnoeic firing patterns of four type I units (a to d), and two type II units (e and f). Breathing was recorded from the pIetbysmo~ap~ (inspiration, up), the calibration markers indicate 20 ml; the time markers indicate 1.0 sec.

62

V. MOLONY

onset of firing during expiration in units which were not firing continuously (b). In those units that fired during both inspiration and expiration firing in expiration was generally slower than that in inspiration (d). Type I units did not stop firing abruptly at the peak of inspiration. Thirteen units (52%) showed cardiovascular modulation of firing at some time during the course of the experiments. The inspiratory firing of many units showed two phases; an early rapid phase (primary) and a later slower phase (secondary). The secondary phase followed immediately after the primary phase. Some of the type I units which fired continuously showed only small changes in firing rate from breath to breath. The firing rate of type I units did not increase noticeably when deeper breaths occurred. Type II units These units all showed similar firing patterns during eupnoea (fig. 2e, f). They fired almost exclusively in inspiration. They very often fired at a relatively constant rate throughout inspiration, though this was often distorted by a secondary cardiovascular modulation. All type II units showed a secondary cardiovascular modulation at some time and some showed this modulation all of the time. They generally started firing earlier in inspiration than did type I units and stopped abruptly close to the peak of inspiration. Firing increased in rate when deeper breaths occurred. RESPONSES TO GAS MIXTURES CONTAINING

co2

Type 1 units These units showed several different degrees of inhibition by COz included in the inspired air. Thirteen units (52%) stopped firing during the whole of the test period, four (16%) stopped firing in inspiration for the whole of the test period, and six (24%) stopped firing at the beginning of the test but showed a return of firing as the test continued. Two units which fired mainly in expiration showed no response to inspired CO, but did show a response to insulation with OJCOz mixtures. The primary firing in inspiration shown by some type I units was more sensitive to inhibition by CO* than the slower firing occurring later in inspiration and in expiration. This “primary” firing could be stopped by levels of CO* in excess of 2.5%. Six units showing this type of response were investigated further by gradually reducing the level of CO2 in the inspired gas mixture, the most sensitive of these decreased firing with levels of CO, of < 1.0%. Response and recovery times were measured for 10 units. Response times varied from 0.3 to 0.7 set (see fig. 3); the mean and standard deviation of the sample was 0.36kO.15 set (124 tests); recovery times were similar, 0.39kO.15 set (11 tests). Increasing the volume of the upper respiratory dead space by 2-10 ml increased these response times in an approximately linear way. The responses of type I units to insufflation with Oz/COz mixtures were similar

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63

Fig. 3. Responses of three type I units to CO, in the inspired gas. Breathing was recorded from the plethysmograph (inspiration, up). The calibration markers indicates 20 ml; the time markers indicate 1.0 set and the duration of the test is indicated by the horizontal bar. a and d: responses to 95% 0,/S% CO,. b: response to 97.5% 0,/2.5x CO*. c: response to 98.5% 0,/1.5% COz. The same unit is tested with different levels of CO, in b and c. Spikes are retouched in a.

to those seen in response to CO2 included in the inspired air. Six type I units were subjected to supplementary insulllation tests with O2 and O&O2 mixtures at three different flow rates; four units were tested in both paralysed and non-paralysed preparations, e.g. fig. 4 a and b. Two phases of firing occurred; this appeared to be the same effect as seen in inspiration with some units; first, an initial “primary” phase of higher firing at the “on” lasted for about 4.0 sec. This phase was very sensitive to CO, included in the gas mixture; second, a later phase of firing (“secondary”) followed the primary phase directly; firing in this phase continued for as long as the flow of gas was maintained provided that the level of CO2 did not exceed the blocking level (usually about So/J. This phase was less sensitive to CO2 than the “primary” phase. Type II units

All nine of these units were either unaffected by the addition of CO2 to the inspired gas, or they showed a small increase in tiring which coincided with the increase in depth of breathing. Insufflation of the preparation with OJCOz mixtures did not decrease the activity of these units; all either fired continuously with a respiratory modulation or showed periodic firing in phase with inspiration.

Fig. 4. Responses of one type I unit to insuflation with increasing levels of CO2 at flow rates of 1 L/min and 4 L/min in a paralysed preparation. Each point represents the results from one test and indicates the average tiring rate for the preceding second. (a) Responses to insuffiation with both air-sac cannulae open at 1 L/min with 100% O2 V-V-V; 2.8% CO2 in 0, 0-O-O; 4.8% CO, in O2 O-W@; 6.7% CO, in O2 n-Q--c]; 8.4% CO2 in O2 A-A-A. (b) Responses of the same unit as in (a) to insufllation with both air-sac cannulae open at 4 Ljmin with 100% 0, v--v--‘l; 1.0% CO2 in O, O-0-O; 2.6% CO, in O2 O-a-0; 3.6% CO, in O2 0-[7-0; 4.7% CO.* in Ot m-_-II; 7.8% CO, in O2 A-A-A.

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65

INFLATION

Type I units

All of these units were stimulated by inflation with air; they showed firing patterns at the “on” and “off’ which resembled those seen in eupnoea. For example where units tired mainly in expiration, firing was concentrated at the “off’ of inflation; where tiring occurred in both inspiration and expiration, firing occurred at both the “on” and the “off’ of inflation (fig. 5a, b). Only two units (8%) tired at an increased rate throughout inflation. Graded inflations did not give clear-cut graded changes in the rate of firing; in some units, however, the number of action potentials occurring at the “on” increased linearly with increasing inflation volumes. Type II units

All of these units were stimulated by inflation and fired throughout the test (fig. 52, d). Firing was greater when the “on” coincided with inspiration than when it coincided with expiration; a peak of firing usually occurred at the “on”. Breathing and cardiovascular activity continued to modulate tiring of some of these units even

a.

Fig. 5. Responses of two type I units (a and b) and two type II units (c and d) to inflation via the trachea. Breathing was recorded from the plethysmograph. The calibration markers indicate 20 ml; the time markers indicate 1.0 set and the duration of the test is indicated by the horizontal bar. In d the preparation was paralymd and artificial respiration was suspended for the test. a:‘inflation with 50 ml of air. b and c: inflation with 80 ml of air. d: inflation with 150 ml of air.

IIfWl88

nut

@I)

Fig. 6. Response of a type II unit to i&&on of the preparation with increasing volumes of air from a Iarge syringe. Same unit as in fig. 5d. The preparation was parafysed and artifkiat respiration suspended. Firing rates were measured for one test at each voIume. I-T-?3 maximum &ing measured average firing in the Iast 3-5 see of the test. at the height of the peak occurring at the “on”; O-W-*,

during inflations with large volumes or high pressures. With four units (44%) it was necessary to increase the volume of inflation considerably beyond the resting tidal volume to obtain firing of comparably rate to that seen in eupnoea. Graded inflation of type II units consistently produced graded increases in the rate of firing; in one unit tested in a paralysed preparation (Succinyl choline 1 mg/kg) the firing rate of the initial peak increased linearly with increasing volumes of inflation up to 150 ml; later firing however only showed increases in firing rate up to inflation volumes of about 80 ml (see figs. 5d and 6 )* OCCLWSlON OF THE TRACHEA

Type I units

Seven units were tested; they either stopped firing or tired sIowly,

Three units were tested; they continued to fire without noticeable changes in their firing pattern or rate of firing. RESPONSES TO HALOTHANE

Eleven units were tested; all showed marked decreases in firing and some stopped firing completely (fig. 7). In eight of these units the response occurred in

RESPIRATORY AFFERBNTS IN THE AVIAN VAGUS

67

a

c

Fig. 7. Responses of a type I unit (a) and a type II unit (b and halothane, 3%. Breathing was recorded from the closed plethysmograph the plethysmograph with a small outlet open in a, (inspiration, up). 20 ml; the time markers indicate 1.0 set and halothane administration

< 1.0 sec. These responses added to the inspired air.

c almost continuous record), to (inspiration, up) in b, c, and from The calibration marker indicates is indicated by the horizontal bar.

were very similar to those seen when COz was

Type II units

Eight type II units were tested; seven showed marked increases in firing (fig. 7) most of which occurred relatively slowly (in > 5.0 sec.). CONDUCTION VELOCITY

The mean conduction velocities with standard deviations for 25 type I units were 7.1 f 3.1 m/set, and for nine type II units 8.6 &-4.4 m/set. Discussion The maintenance of a high correlation between the firing of vagal afferents and breathing during a prolonged test period has been taken as evidence that units are involved in the control of breathing. Phasic activitv of this type could emanate from sensitive non-respiratory mechanoreceptors anywhere in the neck or coelom, but it is thought unlikely that non-respiratory activity would maintain a suticiently consistent correlation with breathing over periods of 3.X hr and during such a wide variety of tests. In addition, the natural stimuli of non-respiratory receptors would be expected to interfere with any such correlation. The responses of the two types of unit described here are thought to be sufficiently different, in many of the tests, to make it unnecessary to consider them as variants of a single larger group, and the similarity of responses within each type are thought to make it unnecessary to attempt further subdivision.

68

v.

MOMNY

This classification differs from that proposed by Leitner (1972) and it is suggested that it provides a more satisfactory way of describing vagal afferent activity in phase with breathing mainly because the classification is based upon qualitative differences in the sensitivity of the receptors to various stimuli. The two types described here cannot be separated using Leitner’s classification though he does recognise two types, with different responses to COz. The difference in sensitivity to CO2 provides a simple and satisfactory way of separating the two types described here. Other criteria should, however, be employed both to confirm the classification ofa unit and to distinguish these types from other types of afferent units which show activity in phase with breathing. Study of the responses of these two units to a wide variety of stimuli have led to the following suggestions for their physiological stimuli, locations and roles. PEiYsIoLoGIcALSTIMULI The most notable characteristic of type I units was the inhibition of their firing by COz. Vagal afferent units in the hen showing sensitivity to CO2 have also been described by Fedde and Peterson (1970) Osborne (1971) and Leitner (1972). Fedde and Peterson (1970) and Osborne (1971) have described linear changes in the firing of such units when the level of COz. was changed during insufflation. Similar responses have been found in the present study though these appear to constitute only part ofa complex series of responses which individual type I units can show to changes in the fevef of COz. Two different sensitivities can be shown: one in the “primary” phase of firing and the other in the “secondary” phase. In insufflation tests it has been found that sensitivity to CO2 , especially in the secondary phase, can also depend upon the flow rate of the gas mixture (Malony 1972). These results suggest that these receptors may not act simply as monitors of the level of CO2 in different parts of the bird’s respiratory system. It does, however, seem reasonable to propose that changes in the level of CO2 in the respiratory system constitute the main physiological stimulus of these receptors. The great sensitivity of the primary phase of firing to COz and the speed with which the response occurs (4.3 set), suggest that CUP acts directly upon the receptor or the axon close to the receptor. Attempts to change the pH at the receptor with inspired acidic and alkaline gases and with intracardiac injection of acid and alkaline solutions, Molony (1972), did not reproduce the effects of CO,; similar attempts by Osborne (1971) were also unsuccessful. Thus the effectiveness of COz may not be due to changes in pH which it produces at the receptor. Similarities between the responses of type I units to COs and halothane do suggest, however, that an anaesthetic-like property of COz may be responsible for its action. The sensitivity of type I units to COz makes it difficult to determine their sensitivity to mechanical stimulation. It is difficult to exclude with certainty the possibility that the changes in firing, attributed here to mechanical changes in the system, were produced by sudden changes in the level of CO2 at the receptor.

RESPIRATORY

AFFERENTS IN THE AVIAN VAGUS

69

To demonstrate unequivocally the mechanosensitivity of these units it will be necessary to show that firing only occurs when an adequate mechanical stimulus is present. Several lines of evidence for the possible mechanosensitivity of type I receptors were found in this study; they are discussed elsewhere, (Molony, 1972). Avian pulmonary receptors with this dual sensitivity were first proposed by Burger (1968), and Osborne (1971), writing of COz-sensitive receptors in the fowl, said “these pulmonary receptors seem to be sensitive to other modalities than CO,“. Leitner (1972) has also proposed dual sensitivity for avian respiratory receptors. Estavillo and Burger (1973) have described ~rdiova~~ar afferents in the fowl which are both mechano~nsitive and CO,-sensitive and they suggest that such polymodal sensitivity may be a relatively widespread phenomenon. Of the mechanical events monitored during eupnoea in these experiments, changes in firing of type I units were most closely related to changes in the rate of air flow. Firing of many units reached a peak early in inspiration, when the flow rate of inspired air was maximal. On the other hand, the maximal tiring attained by most type I units increased very little when flow rates were increased either during hyperpnoea or insufllation. Some type I units did show small linear increases in firing as flow rates increased and others showed abrupt increases (Molony,..1972). Overall, it appears that any mechanical stimulus to these receptors during eupnoea is closely associated with the flow of air. The extremely complex patterns of flow and pressure gradients developed in the bird’s respiratory system (Brackenbury, 1971; Bretz and Schmidt-Nielsen, 197 1; Scheid et at., 1972) are not’yet well enough described or understood to confirm this hypothesis. It has been demonstrated, however, that the pressure gradients between the airways within the lung and the air sacs, and between the air sacs themselves, are very small (about + 1.0 cm H,O) (Brackenbury, 1972), and it may be easy to exceed the physiological range of mechanosensitivity of type I receptors during insufflation experiments. Many type I units show very little increase in firing rate with increased gas flow in both hyperpnoea and insufflation tests. This could be described as an “all or none” response and suggests that if the receptor is m~hano~nsitive it is concerned more with detecting the presence of a supra~~shoid mechanical stimulus than with measuring its magnitude. Dynamic and static phases of a mechanical stimulus could be responsible for the two phases of tiring shown by type I units, though to confirm this it will be necessary to measure changes in a mechanical stimulus at the receptor. Type II units No evidence was obtained that type II units were directly sensitive to changes in the levels of COz or O2 in the inspired air: all changes in firing have been attributed to changes in the mechanical stimulus to these receptors. The mechanical stimulus to these receptors shows the following c~racteristics: (1) It is present from soon after the beginning until the end of inspiration its onset and ending are fairly abrupt and it appears to be fairly constant except

70

V. MOLONY

for the superimpo~d modulation produced by the mechanical action of the heart or large blood vessels. (2) The stimulus is not noticeably changed when the trachea is occluded and becomes continuous during insulllation with 100% 0, or 100% N,. Increasing the flow rate of insuMation increases the stimulus and the stimulus decreases abruptly at the “off of insufflation. Changes in the stimulus can occur during insulflation with 100% O2 when the preparation appears to be in apnoea. A respiratory modulation of the stimulus can occur when insulating with Oz/C!02 mixtures, even at relatively high flow rates (Molony, 1972). (3) The stimulus is greater during inspiration than during inflation with similar volumes of air or O,, and even during deflation inspira.tion can still produce an adequate stimulus( Molony, 1972). The maximum firing during in~ation, which occurs at the “on”, can be linearly related to the volume of inflation, but later firing may change little when the volume is increased (fig. 6). (4) Histamine and acetylcholine injected into the right side of the heart increase the effectiveness of the stimulus produced by inspiration (Molony, 1972). The main physiological stimulus to these receptors appears to be mechanical, They are slowly adapting and ‘may show differential sensitivity to dynamic and static mechanical stimuli. LOCATION

OF RECEPTORS

Type I units These units show a great variety of eupnoeic firing patterns which suggests that they are located’in a variety of sites. Most of the units are readily accessible to inspired or insufllated gas mixtures and the rapidity with which some respond to changes in the gas mixtures suggests that they may be located in the walls of the airways: this has also been suggested by Leitner (1972). Units which show very rapid tiring early in inspiration are probably exposed to “fresh” air, since this tiring is very sensitive to inspired COz and would be inhibited if COz were present. Airways exposed to “fresh” air during inspiration are the trachea, the primary bronchi and possibly some secondary bronchi, notably the caudodorsals, The mainly negative responses of these units to veratridine (Fedde and Peterson, 1970; Osborne, 1971; Molony, I972) suggest that most of these receptors do not receive their blood supply directly from the pulmonary artery. It has been shown that a few of these units are sensitive to low levels of veratridine injected into the right atrium (Molony, 1972). Most of the type I receptors, therefore, may not be located in the smaller airways of the lung, but may be concentrated in the walls of the major airways or ‘air sacs. Possible afferent endings in the walls of these airways have been described by,Cook and King (1969a, b), Bennett and Malmfors (1970), Cook (1970) and..McLelland (1970, 1972) and Walsh and McLelland (1974). Fedde and Peterson (1972) have shown that axons from CO,-sensitive receptors are found primarily in the cranial and caudal pulmonary branches of the vagus nerve.

RESPIRATORY

AFFERENTS

IN THE AVIAN

71

VAWJS

I L.

-kdl.+

I

Y.

Fig. 8. Model indicating a possible arrangement of the tissues in which type II receptors are located. A relatively inelastic component AB and a relatively elastic component CD lie in parallel and join supports X and Y which can move relative to one another. A receptor R is in series with the elastic component CD and with a muscular component M. Movement apart of X and Y is limited by AB, and the stretch applied to CD, M and R is relatively constant, provided that the forces applied to X and Y do not also stretch AB. The stretch applied to CD (i.e. dl) could provide the stimulus to R. Delays in the onset of tiring could be produced by the time required for X and Y to move through dl. When M contracts or relaxes, it increases or decreases the stretch applied to R. Primary modulation of the activity of R would be achieved by movements of X and Y, and secondary modulation by other forces applied to CD, such as by H.

Type II units

Most type II units show similar eupnoeic firing patterns and may be expected t9 be situated in parts of the respiratory system which are subjected to similar mechanical events. Some of these events can be represented in a simple model (fig. 8). Gross structures which may possess properties similar to those described for this model are the paired bronchaperitoneal membranes (also called the oblique septa): possible afferent nerve endings have been described in these membranes by Bennett and Malmfors (1970) and Groth (1972). The inter-air sac membranes also appear to have some similar properties, but virtually no smooth muscle has been found incorporated in their structure. It does occur, however, along the medial border of the bronchoperitoneal membranes. ROLES OF RECEF’TORS

Type I units

It has been proposed (Fedde and Peterson, 1970) that CO,-sensitive receptors may provide the central nervous system with information on the regional concentration of airway COz in the lungs, and that this information could be used to permit rapid adjustment of respiratory movements to alter the rate of COz elimination. Firing in expiration, and the relatively slow continuous firing of some type I units, appears to be well suited to provide this type of information. Firing of this type appears to be produced in the “secondary* or static phase and could indicate the level of COz (between 0 and 8%) to which the units are exposed. Such informa-

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V. MOLONY

tion may be used to control ventilation and perfusion of the exchange area by changes in the rate and depth of breathing, by changes in flow’ patterns through the airways and blood vessels of the lung, or both. The eupnoeic firing patterns of type I units are complex, however, and may provide considerably more information than is suggested by Fedde and Peterson (1970). The sensitivity of type I units to both dynamic and static stimuli and the differences in the sensitivity of the dynamic and static responses to COz permit different information to be transmitted at different times in the breathing cycle by the same unit. Thus the temporal uttering of activity should be as imprint in this as in the integration of other respiratory afferent activity (Band et al., 1970; Black and Torrance, 1971). Most of the firing of many type I units occurs in inspiration when the stimulus is apparently dynamic and when these receptors are probably exposed to fresh inspired air. It seems unlikely that this firing is mainly concerned with trans~tt~g information about the level of CO2 in the inspired air since the bird will only rarely inspire significant levels of COz from its natural environment: the timing of this tiring in inspiration may, however, be extremely important. In eupnoea, the timing of the abrupt onset of firing depends on the time of arrival of “fresh” air at the receptor, which in turn depends on the rate of inspiration of end-expired air from the upper respiratory dead spaces. An abrupt change in fuing, such as this, may provide the basis for “all or none” pacing of breathing which can be produced by COz oscillations in the fowl (Kunz ef al., 1971; Kunz, A. L., personal communication). These authors have shown that when chickens are artificially ventilated by insuhlation, oscillations in the level of COz in the insufflating gas can pace respiration. Their experiments relating respiratory period and delay in feedback have led them to the h~thesis that “respiratory rate in the chicken is paced by a harmonic oscillator consisting of an information loop between the brain and a peripheral CO2 receptor in the lung”. It thus seems possible that an ongoing breath can be%fluenced by the effectiveness and speed with which inspiration reduces the level of CO,, at, at least, some of the type I receptors. This may be of particular importance in alkalosis when the upper dead spaces are filled with air, containing ~2.5% CO2 at the end of expiration; under these circumstances tiring will commence at the beginning of inspiration. Many type I units showed relatively slow firing late in expiration: the role of this firing is unclear but its timing suggests that it may be concerned with the termination of expiration and the changeover to the next inspiration. The effect on breathing of increased or decreased activity of type I units can be expressed as inhibition or excitation of inspiration or expiration, though it seems unlikely that this descriptive technique will prove subtle enough to explain all the actions of these units. Continuous activity of these units inhibits breathing as shown by Fedde and Peter~n~l970), Osborne (1971) and Molony (1972). A very rapid arrest of breathing occurs when the chicken is insuhlated wih air, loOo/, 0, or 100% N,, but not with 95% OJ5% COz. It is proposed that in this inhibition of breathing the activity of

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IN THE AVIAN

VAGUS

73

type I units may inhibit expiration more than inspiration. The following evidence consistent with’ this hypothesis is fragmentary and is subject to the criticism that in none of the tests carried out were type I units or their axons the only units capable of being excited, inhibited or eliminated. When type I units were made to fire rapidly, by insufflation, veratridine or sulphur dioxide, there was a shift into inspiration with apneusis or rapid shallow breathing. When type I units were inhibited, as by CO2 or halothane, expiration was increased both in depth and duration from.‘the first breath after the “on” (Molony, 1972). Vagotomy produces prolongation of expiration andstimulation of the central end of the cut vagus produces an inspiratory response (McLelland, 1970). Failure of high levels of COz, introduced into the inspired air, to increase noticeably the depth of the first inspiration after the “on” (Molony, 1972) suggests that activity of type I units may not rapidly inhibit inspiration. This test can, however, provoke an early onset and prolongation of the first expiration (Molony, 1972), and this is consistent with the hypothesis that expiration is stimulated in the absence of type I activity. Leitner (1972) has proposed that the sensitivity of “pulmonary mechanoreceptors” to COz may permit reinforcement of inspiration by decreasing the inhibitory effect of CO,-sensitive mechanoreceptors in inspiration. It remains to be demonstrated, however, that thetype I receptors, proposed here, inhibit inspiration; some evidence to the contrary is quoted above. Type II units

It has been shown (Leitner, 1972) that some vagal afferents show linear increases in firing rate with increasing volumes of inflation of the respiratory system. In the present experiments, type II units fired throughout inflation and some showed linear increases in firing rate with increasing volumes of inflation (fig. 6). The tiring patterns of type II units during eupnoea are not consistent, however, with the hypothesis that these receptors monitor changes in the volume of the respiratory system, either in part or as a whole. In most units the firing rate is relatively constant throughout inspiration, and inflation volumes much greater than resting tidal volumes were sometimes necessary to produce firing at the same rate. The physiological stimulus to these receptors thus appears to be associated with an aspect of inspiration which is only poorly reproduced by inflation. Type II units showed increases in firing rate with the development of hyperpnoea, but tiring continued to be at a relatively constant rate throughout inspiration. The effect on breathing of the activity of type II units may also be expressed as stimulation or inhibition of inspiration or expiration. To estimate this effect it is necessary to determine the responses of breathing when these units alone are stimulated or inhibited. It was not possible to achieve such conditions in these experiments, but in some tests it was possible to stimulate the units vigorously by inflation and insufflation with air, lOOo/,02, 100% Nz and 95% 0,/Q CO2 (Molony,

74

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1972). During inflation and insulation with air, O2 and N2 type I units were also stimulated, but during inflation and insufllation with 95%. O&5% COz type I units were generally inhibited. The only evidence that under these circumstances activity of type II units inhibits breathing was that the depth of the first inspiration after the “on” was decreased in 14 of 59 insu~tion tests with 95% OJS% CO2 (Molony, 1972). Eaton et al., (1971) have shown that the first inspiration was decreased in duration and the first expiration. increased in duration after inflation of the hen withgas mixtures containing 6--S% CO,, and Leitner (1972) has described similar responses. These results suggest that inspiration may be inhibited and expiration stimulated by afferent activity which is relatively insensitive to COz in the inflation or insulBation gas mixture: type II activity could be responsible for all or part of this response. If confirmed, this would support a proposal that the function of type II ,receptors may be similar to that of pulmonary stretch receptors in mammals. Further support for homology between these types of unit is given by similarities in their responses to halothane, acetylcholine, histamine and veratridine (Molony, 1972). Type II units may signal the onset and ending of inspiration; these signals, however, appear to follow rather than precede these changes in breathing. They may also give some indication of the enez-gy expended in breathing by changing their firing rate during aspiration, but this remains to be demons~ted. The activity of type II units appears to be much simpler than that of type I units and to resemble more closely that of mammalian pulmonary stretch receptors, though it should be e-mphasised that any homology between type I or type II receptors and mammalian pulmonary stretch receptors remains to be proven. Neither type I nor type II receptors showed a notable resemblance to mammalian respiratory irritant receptors or type J receptors. These two types of mammalian receptor do not show spontaneous activity consistently in phase with eupnoeic breathing {Mills et ai., 1969; Sellick and Widdicombe, 1971;, Armstrong and Luck, 1974) nor have they been described as being sensitive to inspired CO,. It is concluded that two types of phasic respiratory afferent units occur in the vagus of the hen: one is exquisitely sensitive to COz and though most of the evidence for mechanosensitivity of this type of unit may be explained in terms of changes in the level of COz at the receptors it remains to be shown that these units are active in the absence of any mechanical stimulus. The second type of unit is probably mechanosensitive and the firing patterns of this type suggest that they may not be in the lung but in associated parts of the lower respiratory tract. Acknowledgements I should like to thank the British Egg Marketing Board and the Wellcome Trust for financial support, Professor A. S, King for help and encouragement and Dr. M. R. Fedde and Dr. P. Scheid for helpful ~mments during the preparation of this paper.

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McLelland, J. (1972). AlTerent nerve endings in the avian lung: observations with the light microscope. Experientiu 28: 188-189. Mills J. E., H. Sellick and J. G. Widdicombe (1969). Activity of lung irritant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstriction. J. Physiol (London) 203: 337-357.

Molony, V. (1972). A study of vagal afferent activity in phase with breathing and its role in the control of breathing in Gallus domesticus. Ph.D. Thesis, University of Liverpool. Osborne, J. L. (1971). CO,-sensitive pulmonary afferent neurons in Gallus domesticus: Static and dynamic response characteristics. Ph.D. thesis, University of California, Davis. Osborne, J. L. and R. E. Burger (1971). Static and dynamic response characteristics of COzsensitive chemoreceptors in the avian lung. Physiologist 14: 205. Peterson, D. F. and M. R. Fedde (1968). Rece’ptors sensitive to carbon dioxide in lungs of chicken. Science 162: 1499-1501. Richards, S. A. (1968). Vagal control of thermal panting in mammals and birds. J. Physiol. (London) 199: 89-101. Richards, S. A. (1969). Vagal function during respiration and effects of vagotomy in the domestic fowl Gallus domesticus. Comp. Biochem. Physiol. 29: 955-964.

Scheid, P., H. Slama and J. Piiper, (1972). Mechanisms of unidirectional flow in parabronchi of avian lungs; measurements in duck-lung preparations. Respir. PhysioL 14: 83-95. Sellick, H. and J. G. Widdicombe (1971). Stimulation of lung irritant receptors by cigarette smoke, carbon dust and histamine aerosol. J. Appl. Physiol. 31: 15-19. Sinha, M. P. (1958). Vagal control of respiration as studied in the pigeon. Helu. Physiol. Acta 16: 58-72. Walsh, C. and J. Mctilland (1974). Intraepithelial axons in the avian trachea. Z. Zellforsch. 147: 209-217.