Absence of viscerosomatic inhibition with injections of lobeline designed to activate human pulmonary C fibres

Abstract

1. Activation of pulmonary C fibres (J receptors) in animals produces inhibition of spinal motoneurones. Intravenous bolus injections of lobeline are believed to activate pulmonary C fibres (J receptors) in human subjects and to produce characteristic sensations and cardiorespiratory responses. This study quantified the respiratory sensations evoked by such injections and then used a range of suprathreshold doses of lobeline and tested for the presence of reflex or descending inhibition of motoneuronal output.

2. Injections of lobeline produced dose-dependent sensations of respiratory discomfort referred to the throat and upper chest beginning within about 10 s and often associated with coughing. As the dose increased the latency for the sensations decreased while their duration and intensity increased. Reflex changes in blood pressure, heart rate and ventilation also occurred.

3. Injections of lobeline at doses sufficient to evoke respiratory discomfort lasting 25-32 s (37-73 μg kg⁻¹) increased the size of the H reflex in soleus with an onset latency of about 10 s and lasting about 20 s.

4. The size of EMG responses evoked in upper limb muscles by transcranial magnetic stimulation of the motor cortex increased shortly after injections and remained elevated for about 30-35 s.

5. Injections of lobeline during sustained voluntary contractions of the elbow flexors at submaximal or maximal levels did not impair the ability to produce force.

6. Walking was not disrupted by repeated suprathreshold doses of lobeline.

7. It is concluded that injections of lobeline sufficient to evoke cardiorespiratory reflexes and sensations of severe respiratory discomfort are not associated with functionally important inhibition of motor performance. The results cast doubt on the ability of the J reflex to limit exercise in humans.

The lungs are richly endowed with unmyelinated vagal afferents which terminate in receptors known either as juxtapulmonary capillary receptors (J receptors; Paintal, 1973) or pulmonary C fibres (Coleridge & Coleridge, 1984). It is believed that the cardiovascular and respiratory changes which follow shortly after injection of lobeline in human subjects and patients are due to their activation (Jain et al. 1972; see also Bevan & Murray, 1963). Injections of lobeline also induce respiratory sensations in the throat and cause subjects to cough (Paintal, 1986; see also Raj et al. 1995).

In the cat, activation of pulmonary C fibres produces powerful reflex effects on motoneurones. Based on assessment of monosynaptic reflexes in the anaesthetized cat, there is a transient and powerful inhibition of spinal motoneurones which has a similar time course to that of the accompanying cardiorespiratory reflexes (Deshpande & Devanandan, 1970; Paintal, 1970; Anand & Paintal, 1980). In addition, the cough reflex is suppressed (Tatar et al. 1988). This inhibition of motoneurones can sometimes be demonstrated in decerebrate preparations depending on the level of decerebration (Deshpande & Devanandan, 1970; Schiemann & Schomburg, 1972; Ginzel, 1973; Kalia et al. 1973). During locomotion in the mesencephalic cat, activation of pulmonary receptors through increased pulmonary capillary pressure generated by a balloon in the left atrium, or by intravenous injection of phenyldiguanide, can attenuate or abolish walking (Kalia et al. 1973; Pickar et al. 1993). There is a brief report of a reduction in the H reflex following lobeline injections in human subjects (Raj & Agrawal, 1997).

Given that the pulmonary C fibres are activated by rises in interstitial pressure (Paintal, 1969; Coleridge & Coleridge, 1984), and that rises in this pressure accompany exercise, it has been hypothesized that activation of pulmonary C fibres may not only generate sensations of breathlessness but also actively inhibit exercise through the J reflex (e.g. Paintal, 1970; Paintal, 1973; Anand & Paintal, 1980; Pickar et al. 1993). In a recent study, Paintal and colleagues (Raj et al. 1995) measured the doses of intravenous lobeline required to elicit threshold respiratory sensations and to induce coughing. These doses produced both respiratory sensations and reflex changes in respiration which were temporally consistent with an afferent input accessed via the pulmonary circulation. This fits with observations that injections of lobeline into the left ventricle do not elicit respiratory sensations (Stern et al. 1966).

The present study was designed to document the time course and magnitude of sensations induced by intravenous injections of lobeline in humans and then to determine whether lobeline altered motoneuronal output. To assess the apparent excitability of motoneurones, we examined changes in their reflex responses to muscle afferent stimulation via the H reflex. Because this method cannot reveal the effects of lobeline acting through the motor cortex, responses to transcranial stimulation of the motor cortex were also measured during the respiratory sensations and cardiorespiratory reflexes. To assess voluntary performance, changes in maximal and submaximal isometric forces were measured following lobeline injections. In addition, lobeline was injected during walking. Preliminary results have been presented (Gandevia et al. 1997).

METHODS

Studies were conducted on four subjects (1 female, 3 males; age range, 37-46 years) who were healthy and did not have a history of any significant respiratory or neurological illness. Their heights ranged from 1.70 to 1.90 m (mean, 1.77 m) and weights from 56 to 83 kg (mean, 71 kg). Subjects were comfortably seated for all procedures. Eight main experimental sessions were conducted and one subject was studied on five occasions. Informed written consent was obtained, and the procedures conformed to the Declaration of Helsinki and were approved by the local ethics committee.

Experimental procedures

All subjects were familiar with the major measurements being made in the study. They arrived in the laboratory having fasted for 6 h. A cannula was inserted in the antecubital vein at the right elbow and extension tubing connected (1.1 ml deadspace) so that injections could be made remotely. The lobeline HCl (Clinalfa, Switzerland) was prepared in saline at a concentration of 1 mg ml⁻¹. The test injections were either saline or lobeline and the deadspace of the tubing was primed with the appropriate solution in advance of injection without the subject's knowledge. The timing and duration of injections were marked using a switch activated by an experimenter. Latencies were measured from the onset of injections. The following Doppler method was used to estimate the transit time for the lobeline to reach the right atrium from the injection site at the elbow (see Michenfelder et al. 1972). The right atrium was targeted with a dual-frequency ultrasound probe positioned parasternally in the right third intercostal space (model 915-AC; Parks Medical Electronics, Aloha, OR, USA). A characteristic change in the auditory signal occurred shortly after injection of a mixture of 1-3 ml of saline and 0.5 ml of air. This occurred at 2.3-3.5 s following injection in the different subjects (mean, 3 s).

During the procedures a number of variables were monitored including blood pressure, heart rate and ventilation. Blood pressure was measured continuously using a plethysmograph-based system from a finger of the right hand (Ohmeda 2300, Finapres, Wisconsin, USA). Ventilation was measured non-invasively with a pair of calibrated inductance bands positioned around the upper chest and abdomen. In addition, oxygen saturation was measured with an oximeter which also provided heart rate (averaged over 5 beats). End-tidal P_CO2 was monitored with a small catheter inserted into the left nostril. Because it was important to ensure that the level of attention was similar for each injection and for each subject, instructions to subjects were read from a prepared script and all extraneous visual and auditory stimuli were minimized. Subjects were reminded that they would receive lobeline or saline and were given specific instructions (see below) depending on the motor and sensory responses being measured during that particular injection. An interval of 5-10 min separated injections and up to twenty injections were performed in a session. Unless indicated the subjects were blindfolded.

Measurements of sensory responses

Subjects were asked to signal any sensations associated with breathing following the warning that an injection would occur within the next 1-2 min. Subjects were read instructions from a script to ‘signal any definite sensations, signal with the potentiometer… and to use it to signal the intensity of any sensations. ‘Injections were begun at 0.4 mg and increased by 0.2-0.4 mg until definite respiratory sensations were evoked. The dosage was then elevated until coughing also occurred. Additional doses were given and a dose selected for the studies of motor responses. To determine the onset and intensity of sensations, the subject signalled with a rotary potentiometer operated by the left hand. In some studies, the onset of other sensations associated with the injections (such as nausea) was signalled by movement of a finger or by an additional transient movement of the potentiometer. At least 2 min after each injection the subject described any respiratory sensations verbally and they were later transcribed from tape. The subject then selected evoked sensations from a list (based on sensations reported by Raj et al. 1995) and indicated their apparent location (nose, throat, larynx, upper or lower chest, or elsewhere). The listed sensations included: ‘air hunger’, ‘tightness in the chest or elsewhere’, ‘difficulty or discomfort with breathing’, ‘choking’, ‘wheezing’, ‘pain or burning in the chest or elsewhere’, ‘smoke in the throat’, ‘a need to cough’, and ‘any other sensations’. To estimate the peak level of respiratory discomfort, subjects selected a number from a modified ten-point scale (Borg, 1982; see Table 1 for details) as used in other studies of respiratory discomfort (e.g. Gandevia et al. 1993). After every injection subjects selected descriptors for the sensations and gave an intensity rating for ‘respiratory discomfort’. Subjects did not signal with the potentiometer during studies involving transcranial stimulation of the motor cortex or voluntary contractions or during the main studies of the H reflex.

Table 1.

Sensations induced by injection of lobeline in individual subjects

Subject	Sensation threshold dose (μg kg−1)	Cough threshold dose (μg kg−1)	*Motor dose (μg kg−1)	†Sensations	Sensation duration (s)	Locations	Mean Borg score
1	14.5	36.4	72.7	2,3,4,6,7,8,9	29.3	C,D,E	7.3
2	24.6	38.5	46.2	4,6,7,8,9	27.9	D,E	5.0
3	14.6	29.3	36.6	1,2,3,4,6,7,8,9,10	32.0	A,C,D,E	3.3
4	19.5	39.0	48.2	1,2,4,6,7,8,9,10	25.4	D,E	5.8

^*Dose of lobeline used in main study of motor responses.

^†Data obtained with motor dose of lobeline. Sensations: 1, air hunger; 2, tightness in the chest or elsewhere; 3, difficulty/discomfort with breathing; 4, choking; 5, wheezing; 6, pain or burning in the chest; 7, swallowing; 8, ‘smoke’ in the throat; 9, need to cough; 10, other sensations. Location: A, mouth; B, nose; C, larynx; D, throat; E, upper chest; F, lower chest; G, elsewhere. Borg scale: 10, extremely large (maximal); 9, …; 8, …; 7, very large; 6, …; 5, large; 4, considerable; 3, moderate; 2, mild; 1, slight; 0.5, just noticeable; 0, infinitely small.

H reflex testing

H reflexes were elicited in the soleus muscle via a cathode in the popliteal fossa over the tibial nerve and an anode on the patella in three subjects. Constant-current stimuli were used with a duration of 1 ms. Electromyographic responses (EMG) were recorded from the soleus muscle with surface electrodes. Subjects were reminded to remain relaxed. Subjects were read the following instructions from a script before each injection. ‘Try to stay completely relaxed during the test period. During this time we will measure the H reflex while you stay relaxed. ‘To ensure that responses were reproducible the stimuli were delivered continuously at regular intervals (0.5 Hz). The intensity was set to produce reflex responses of 15-20 % of the maximal evoked motor response (M wave). In two subjects the H reflex was also recorded in the flexor carpi radialis muscle using stimulation of the median nerve at the elbow and surface EMG recordings over the muscle in the forearm. The main measures reported for the H reflex were made following injections for which sensations were not signalled because signalling itself may induce arousal and thereby may alter the H reflex responses. However, H reflexes were also recorded during the initial runs when the timing and intensity of sensations were signalled by the subject. Changes in the H reflex during these runs were similar to the changes recorded when sensations were not specifically signalled.

Additional control studies were conducted to determine the effects of voluntary coughing and the effects of inhalation of smelling salts (sal ammoniac) on the size of the H reflex in soleus. In three subjects, coughing was initiated voluntarily by the subject when the baseline H reflexes (evoked on the left and right sides by alternate stimulation, each at 0.5 Hz) were stable. The bout of coughing lasted about 4-10 s and was designed to mimic the most severe coughing induced by the lobeline injections. In three subjects, smelling salts were inhaled through the nose to total lung capacity while the H reflexes were elicited. The inhalation was designed to mimic the intensity of discomfort produced by large injections of lobeline. These interventions were repeated 2-6 times for each subject.

Transcranial stimulation of the motor cortex

For some injections the responses to transcranial stimulation of the motor cortex were measured. Subjects were given instructions from a script which read ‘Try to stay completely relaxed during the test period. During this time we will stimulate the cortex while you stay relaxed’. EMG responses were recorded from elbow flexor muscles (surface electrodes over biceps brachii) and from the first dorsal interosseous muscle on the left side with the arm relaxed and held comfortably in a padded frame. Stimuli were delivered regularly at 0.4 or 0.5 Hz from a pair of stimulators (Magstim 200, Magstim Co., Dyfed, UK) via a single large circular coil (14 cm diameter). The exact site of the coil over the vertex was chosen based on the presence, in both muscles at rest, of reproducible responses which increased substantially during a deliberate voluntary contraction (peak-to-peak amplitude ∼2 mV). In each subject, magnetic stimulation was performed during three runs: in two the subject received lobeline and in the other saline was injected.

Voluntary contractions

Three subjects performed isometric contractions of the left elbow flexors lasting about 40 s during which they attempted to maintain a constant torque (20 % maximum). The elbow was flexed to 90 deg and the supinated wrist was strapped to a vertical myograph (Fig. 1A). Visual feedback of elbow flexor torque was provided. Three contractions were performed. Lobeline was injected during two of them and saline during one, with injections given about 10 s after the start of the contraction. Before each contraction subjects were read the following instructions from a script. ‘You will be warned when to begin the voluntary contraction. Watch the force feedback. You may be given saline or lobeline during the contraction. Try to maintain the force throughout the effort. ‘Finally, each subject performed a maximal voluntary contraction of the left elbow flexor muscles lasting about 60 s during which they received verbal encouragement and visual feedback of torque. Injections of lobeline were made about 10-30 s after the initial peak of the contraction.

Figure 1. Experimental arrangement and example of sensory and cardiorespiratory responses to an intravenous injection of lobeline.

A, experimental arrangement to show the parameters measured. Note that the injections of lobeline were made via a vein at the right elbow and that the right arm is not shown. B, example of typical responses to a suprathreshold injection of lobeline during the initial injections when thresholds were being determined. Top trace shows the timing of the injection and the occurrence of coughing. Second trace is from the potentiometer used to signal sensation (from left to right: first arrow, onset of burning in throat and upper chest; second arrow, onset of ‘air hunger’; third arrow, end of ‘throat’ sensation; fourth arrow, onset of nausea; fifth arrow, ‘back to normal’). Note that the onset of respiratory sensations induced by lobeline was accompanied by a brief hypopnoea prior to coughing and that the later sensation of ‘air hunger’ was associated with hyperventilation (see signals of ribcage and abdominal movement fifth and sixth traces, respectively). The magnitude of the hyperventilation is evident in the reduction of end-tidal CO₂ beginning about 35 s after the injection. Abbreviations: BP, blood pressure; HR, heart rate (bpm = beats min⁻¹); SaO₂, arterial oxygen saturation.

Walking

Injections of saline and lobeline were delivered in random order to one subject on two occasions while walking continuously at a self-selected pace around a circular grass track (circumference, 16 m). An experimenter walked behind the subject on the inside of the track and gave the injections in the usual way (see above). The track was marked at intervals of 0.5 m. Injections were given without warning and the subject signalled the onset and severity of sensations by raising the left hand. All sequences were captured on videotape for analysis. The mean velocity of walking and the average stride length were calculated for each 8 m walked.

Analysis of responses

In all studies except those involving locomotion, signals were recorded on tape (Vetter PCM) and simultaneously stored to disk (Cambridge Electronic Design 1401+, Cambridge, UK). For the sensory responses, the latency of the onset of sensations after the start of the injection and the time course of sensations were derived from the potentiometer signal. Peak intensity of the sensations was estimated from the 10-point Borg scale. For the H reflexes and responses to transcranial stimulation, the areas of the evoked muscle action potentials were determined. Data were averaged when two or more similar runs were performed in one subject.

Statistical analyses

Correlations between the doses of lobeline and sensory responses were assessed with Pearson correlation coefficients. To determine whether the size of H reflex responses and cortically evoked responses were altered by lobeline, changes were considered to be significant when two or more consecutive responses were outside two standard deviations from the mean of responses immediately before the injection. Statistics for measurements of H reflexes and responses to transcranial stimulation were based on data pooled from each run in each subject. Statistical significance was set at the 5 % level.

RESULTS

Sensory and cardiorespiratory responses

In all subjects the injection of lobeline at sufficient dosage produced transient sensations involving the throat, larynx and upper chest. The estimated doses for threshold respiratory sensations and for the production of coughing are given in Table 1. All doses which were suprathreshold for sensation were reliably detected with no false positive reports of lobeline-induced sensations when saline had been injected or no injection had occurred. If the same doses of lobeline were delivered, the evoked sensations were similar and there was no diminution in the intensity of the sensations with the dosage interval used here (5-10 min). The only exception occurred if injections were given during strong sustained voluntary contractions when the sensations were reported as somewhat less intense although of similar duration. The total doses of lobeline in a session ranged from 28.8 to 41.8 mg and there were no cumulative effects.

In individual subjects, the higher the dosage of lobeline, the longer the duration of respiratory discomfort. Data for the onset latency of initial sensations and their duration are given in Fig. 2A. For the group of subjects, as doses increased there was a significant reduction in latency for the onset of sensations (P < 0.01; r², 0.45) and an increase in the duration of sensations (P < 0.001; r², 0.50). For the group of subjects, across the range of suprathreshold doses, the change in latency was about 7 s. Furthermore, there was an increase in the peak intensity of the lobeline-evoked sensation as the dose increased. This trend was statistically significant for the group (P < 0.001; r², 0.76; Fig. 2B).

Figure 2. Effect of the dose of lobeline on the latency, duration and intensity of evoked sensation.

A, latency of the initial response (○) and the duration of sensation (•) plotted against the dose of lobeline for the group of subjects. Latencies and durations derived from the potentiometer signals. Lines indicate the appropriate linear regressions. B, peak intensity of the sensations plotted against the dose of lobeline. Peak intensities derived from the Borg scores for respiratory discomfort (scale of 0-10 with verbal descriptors, see Methods and Table 1).

The doses selected for the studies of motor performance (see below) produced sensations of respiratory discomfort which were rated at a mean level of 5.4 on the 10-point Borg scale with a range 3-7. This ranges from ‘considerable’ to ‘very large’ in the descriptors accompanying the scale (see Table 1). On average, these doses produced respiratory discomfort which lasted between 25 and 32 s in the different subjects (mean, 29 s). This estimate did not include the more extended sensory experiences reported by some subjects including late hyperventilation and nausea (see below). In all subjects, the doses used for the main motor studies were above the levels required to induce coughing. The cough began at a mean of 17 s after the injection (range, 13-25 s).

In addition to the sensory changes, there were changes in ventilation, blood pressure and heart rate. A brief hypopnoea or apnoea lasting about 2-4 s commonly occurred at about the same time as the subjects indicated that sensations had been evoked with suprathreshold doses. It was often associated with a swallowing movement and was followed by the large inspiratory and expiratory movements accompanying a cough. Subjects were unaware of the initial apnoea at the time the respiratory sensations developed but they did report the desire to swallow and to cough. When coughing occurred, it began when the sensation was at its highest intensity.

For suprathreshold doses of lobeline there was a dose-dependent involuntary increase in ventilation beginning on average 27 s after the injection (range, 26-29 s). This presumably reflects the late excitation of arterial chemoreceptors by lobeline. The hyperventilation was accompanied by a sensation of ‘air hunger’ in two subjects. Ventilation returned to its usual level about 1-2 min after the hyperventilation began. With the hyperventilation some subjects felt transiently ‘light-headed’ and the hyperventilation in one subject was sufficient to induce paraesthesia. Two subjects reported feeling nauseated after the initial respiratory sensations (Fig. 1B). In all subjects, mean blood pressure fell shortly after the lobeline-induced sensations began (i.e. about 15-20 s after the injection), reached a minimum in the succeeding 5-10 s and recovered with a similar time course. The average reduction in mean arterial pressure was 40 mmHg (range, 27-57 mmHg). Although not studied formally, some of the reduction in blood pressure is likely to be secondary to the haemodynamic effects of coughing. Beat-to-beat heart rate declined transiently in each subject by 15 % (range, 10-21 %) although this was not evident with each injection. The onset of the decline occurred with the reduction in blood pressure but heart rate recovered more quickly than blood pressure.

Changes in H reflexes and the responses to cortical stimulation

In three subjects the changes in the size of the H reflex of the soleus muscle were monitored with stimuli delivered at intervals of 2 s. The tibial nerve stimuli were set to produce a reflex response of 15-20 % of the maximal motor response. This intensity ensured that the responses were reasonably stable and could be readily increased or decreased. The mean latency of the H reflex response was 33.9 ms (range, 31.6-38.0 ms).

H reflexes from a single experiment in one subject are shown in Fig. 3 and measurements for the group in Fig. 4. In each of the three subjects the area (and amplitude) of the H reflex increased significantly, beginning about 10 s after the injection. This corresponded to the time at which the same doses of lobeline initiated sensations of respiratory discomfort. This increase in the H reflex is unlikely to be an artefact because the direct motor response was stable and the increase was not observed when injections of saline were given. Lobeline injections produced similar changes in the size of the H reflex in runs in which the subjects also signalled their sensory responses. In two of the subjects an additional H reflex was evoked in flexor carpi radialis. This response was less stable than that in soleus but it also showed no evidence that lobeline reduced the amplitude of the H reflex.

Figure 3. Effect of lobeline on the soleus H reflex in one subject.

Traces of the H reflexes in soleus from a single run are shown superimposed for the control period before injection and then for successive periods of 4-10 s after the injection of lobeline. Here and in Fig. 5, the horizontal bar denotes the period during which respiratory sensations were evoked by lobeline; the timing for these sensations was taken from previous injections with an identical dose in the same subject. Horizontal dashed lines show the peak-to-peak amplitude of the control responses prior to injection. A small increase in reflex size occurred 17-21 s after the injection (see asterisk). Note that the amplitude of the direct motor response (M wave) was stable. The relatively small amplitude of the H reflex reflected the high frequency of stimulation. The dose of lobeline was 48 μg kg⁻¹ and the subject did not signal during the run.

Figure 4. Effect of lobeline on the H reflex for the group.

Mean data from three subjects for the H reflex (•) and the M wave (○) during suprathreshold injections of lobeline (A) and injections of saline (B) when no signalling was required. Results have been averaged from individual subjects from runs made under identical conditions. Subjects were asked to remain relaxed. Data have been normalized to the size of the H reflex in the period just prior to injection (means ±s.e.m.). With the lobeline injection, there was an initial significant increase in H reflex which could clearly not be explained by a change in the M wave. For both panels the horizontal dashed lines represent ± 2 s.d. of the pre-injection size of the H reflex. The injections were delivered at the arrow (time 0).

A small increase in H reflex size also occurred when doses of lobeline were given which were sufficient to cause a respiratory sensation but not induce coughing. As the H reflex also increased when subjects inhaled smelling salts which caused sensations of similar intensity and also no cough, the increase presumably reflects the arousal associated with the noxious sensations. Control studies revealed that voluntary coughing always reduced the size of the H reflex by about 45 % (range, 40-60 %; 6 runs in 3 subjects). Hence, the increases in the H reflex were not dependent upon the occurrence of coughing. For doses of lobeline which were below the sensation threshold no changes in the H reflex were observed (5 runs in 3 subjects).

The motor cortex was stimulated transcranially every 2-2.5 s with a sufficiently large stimulus to evoke responses in the elbow flexors (biceps brachii) and first dorsal interosseous muscle on the left side with the subject completely relaxed. The latency of the responses in the four subjects was on average 13.8 ms (range, 12.9-14.9 ms) in biceps and 23.6 ms in the first dorsal interosseous (range, 21.2-27.0 ms). These latencies are typical of those found in these muscles when subjects are relaxed. Responses to an injection of lobeline in one subject are given in Fig. 5 and grouped data in Fig. 6. As with the H reflex, the first change in the amplitude of the responses was an increase occurring about 10 s after the injection. The effect was evident in responses in biceps brachii and first dorsal interosseous, although the trend was statistically significant only for biceps in the grouped data. The increase was significant for both muscles in two of the four subjects and for one muscle in another subject. The duration of the increase was 30-35 s.

Figure 5. Effect of lobeline on the EMG responses to transcranial magnetic stimulation of the motor cortex in one subject during relaxation.

Traces of the responses evoked by transcranial stimulation from a single run in the relaxed biceps brachii. Responses are shown superimposed for the control period before injection and then for a periods of 5 or 10 s after the injection of lobeline. An increase in the size of the cortically evoked response occurred from about 12 to 30 s after the injection. Horizontal bar denotes the period of respiratory discomfort.

Figure 6. Data for the group for the changes in the size of the cortically evoked responses in the elbow flexors and first dorsal interosseous muscle.

Mean data from four subjects for the cortically evoked responses (•, biceps brachii; ○, first dorsal interosseous) during suprathreshold injections of lobeline (A) and injections of saline (B) when no signalling was required. Results have been averaged from individual subjects from runs made under identical conditions. Subjects were asked to remain relaxed. Data have been normalized to the size of the response in the period just prior to injection (means ±s.e.m.). There was a significant increase in biceps brachii for the group. For both panels the horizontal lines represent + 2 s.d. of the pre-injection size of the evoked compound muscle action potential (MEP) for biceps (continuous lines) and first dorsal interosseous (dashed lines). The injections were delivered at the arrow (time 0).

Injections during sustained voluntary contractions

In three subjects, injections were delivered at unpredictable times during a sustained voluntary contraction of the elbow flexors which the subjects attempted to maintain with the aid of visual feedback of force at 20 % of maximum. Forces were well maintained despite the sensory and cardiovascular effects of the injections. However, sudden coughing often caused small irregularities in the on-going force. Examples from two subjects are given in Fig. 7A. Control efforts in which the subjects coughed voluntarily showed irregular fluctuations in force which were similar to those in which coughs were produced involuntarily by lobeline. Further evidence for a lack of disruption of motor activities occurred during the runs in which subjects signalled sensation via a potentiometer operated by the left thumb and index finger. Smooth signals were produced with no apparent loss of drive to the relevant intrinsic and extrinsic muscles of the hand.

Figure 7. Changes in voluntary force produced by submaximal and maximal contractions of the elbow flexor muscles during the injection of lobeline.

A, data from two subjects during sustained voluntary contractions of the elbow flexors at 20 % MVC (maximal voluntary contraction) with visual feedback of force. In subject 1 (above) the injection induced coughing and some associated increases in force. Similar increases occurred with voluntary coughing (at right). In subject 2 force was stable. Note that coughing was voluntarily suppressed following the injection. Integrated EMG of the biceps brachii is shown along with the voluntary force. B, force profiles for the other two subjects during a sustained maximal voluntary contraction of the elbow flexors with visual feedback of force. The timing of the injection is indicated by the arrows.

Towards the end of each study, subjects were required to make a sustained maximal contraction of the elbow flexors lasting 1 min (Fig. 7B). Force feedback was provided. Such contractions usually contain some fluctuations in force due to variable drive to the muscles (e.g. Bigland-Ritchie et al. 1978; Gandevia et al. 1996). However, the fluctuations were no more obvious during the period of the lobeline-induced sensations than prior to the injection. No subject reported that more ‘effort’ was needed or that it was subjectively more difficult to generate force voluntarily following the lobeline injections.

Walking

In one subject, lobeline was injected at random times during self-paced locomotion around a circular track. In contrast to the cat, no disruption of walking occurred. Doses of 30 and 60 μg kg⁻¹ were given twice along with an equal number of injections of saline. Both doses induced sensations with the larger doses of lobeline producing ‘severe’ respiratory discomfort at the expected latency. The average stride length and the velocity of walking were not altered by the lobeline injection (Fig. 8) and the subject reported no increase in effort required to walk.

Figure 8. Effects of lobeline injections on walking.

Upper panel shows the velocity of walking with injections of lobeline (60 μg kg⁻¹, □) and saline (▪). Lower panel shows the corresponding stride length (○, lobeline; •, saline). Data from two trials have been averaged for each condition. The arrow indicates the point at which either lobeline or saline was injected and the shaded area indicates the presence of a severe sensation of respiratory discomfort after injections of lobeline.

DISCUSSION

In this study intravenous lobeline was used to induce marked respiratory discomfort but this was not associated with an overt impairment of motor performance as judged by the production of voluntary force. When tested with transcranial magnetic stimulation or H reflexes, motor ‘excitability’ was increased throughout much of the period during which subjects felt respiratory discomfort. This result contrasts with observations which have highlighted the potent viscerosomatic inhibition evoked by J receptors (via pulmonary C fibres) in both the anaesthetized (e.g. Deshpande & Devanandan, 1970; Anand & Paintal, 1980) and the walking mesencephalic cat (Pickar et al. 1993; see also Kalia et al. 1973). Our data must cast substantial doubt on the view that in human subjects pulmonary C fibres (or J receptors) produce inhibition of skeletal muscles, at least for muscles not involved in respiration. No evidence was found that activation of pulmonary C fibres would limit exercise in conscious healthy human subjects.

Given that the present study found no evidence that intravenous injection of lobeline produced a generalized inhibition of motoneurones in conscious subjects, it is important to consider whether the doses of lobeline were likely to have been sufficiently large to have activated pulmonary C fibres. The doses required to produce respiratory sensations and then coughing were within the ranges reported by Paintal and colleagues for a large group of subjects (Raj et al. 1995) and, in the main studies of motor responses to lobeline, we used doses which exceeded those capable of inducing coughing. As these doses, and indeed all those above sensation threshold, could produce transient changes in ventilation beginning during the course of respiratory discomfort, it seems unlikely that they were insufficient to activate pulmonary C fibres.

The sensory reports of subjects were consistent from trial-to-trial if the same dose of lobeline was given. There was no evidence for any habituation, nor were there any false positive detections when only saline was given. The subjective reports agreed very well with those detailed by Raj et al. (1995) (see also Jain et al. 1972). Subjects felt noxious sensations referred to the throat, such as ‘burning’ and ‘choking’ which progressively involved the upper chest with a burning sensation as doses increased. Although the sensations lasted for a predictable period (∼30 s) with the doses used to assess motor performance, subjects rated the respiratory sensations as moderately to severely uncomfortable. Both the duration and the peak intensity of respiratory sensations increased linearly with the lobeline dose, whereas the latency to the onset of respiratory sensation diminished slightly. Some subjects reported that once the initial sensations were subsiding a late feeling of ‘air hunger’ and an awareness of an involuntary hyperventilation developed. These late events could be differentially detected and signalled by subjects (Fig. 1B). Because of their longer and reproducible latency it is probable that these sensations and the reflex increases in ventilation represent the activation of arterial chemoreceptors by lobeline (Coleridge & Coleridge, 1984; see also Jain et al. 1972).

With the higher doses of lobeline, subjects not only reported uncomfortable respiratory sensations but also coughed. Raj et al. (1995) have suggested that this cough is a direct reflex response to activation of pulmonary C fibres. However, stimulation of pulmonary C fibres does not produce a cough in conscious or asleep cats (Kalia et al. 1973; Ginzel & Lucas, 1985) and has been shown to inhibit cough evoked by laryngeal stimulation (Tatar et al. 1988, 1994). Rapidly adapting receptors (RARs) in the lungs may contribute to cough (Karlsson et al. 1988) and in cats, are sensitized by lobeline (Raj et al. 1995). In humans, manoeuvres designed to change the firing of RARs do not alter the threshold for sensations produced by lobeline but the effect of these manoeuvres on the threshold for coughing has not been studied (Raj et al. 1995). The lobeline-induced cough could also be a behavioural response to the noxious sensations in the throat. Coughing occurred when respiratory sensations were at their peak and could be suppressed by some subjects in some trials. In this study, the latency difference between the onset of the earliest respiratory sensations and the occurrence of the cough is several seconds so that we cannot rule out voluntary or reflex mechanisms.

Investigation of the motor effects of stimulation of pulmonary C fibres showed that injections of doses of lobeline above the threshold for sensation significantly increased both the H reflex and the short-latency excitatory response to transcranial magnetic stimulation of the motor cortex. The onset of these effects occurred within 1-2 s of the time that the respiratory discomfort began. The increase in the H reflex indicates an increase in the ‘excitability’ of the spinal motoneurone pool whereas the response to transcranial magnetic stimulation can be increased by increased responsiveness of either cortical cells or the spinal motoneurones that they activate. Thus, an increase in spinal ‘excitability’ could account for the changes of both responses. It is well known that spinal reflexes increase with ‘reinforcement’ manoeuvres such as the contraction of remote muscles, the preparation for movements, and the arousal accompanying sudden sounds or mental arithmetic. Stimulation of pulmonary C fibres is an arousing stimulus. It wakes cats from sleep (Ginzel & Lucas, 1985). Hence in the present study, a general arousal may be produced by the sudden noxious sensations evoked by lobeline and lead to an increase in the ‘excitability’ of spinal motoneurones. Indirect support for this was obtained by observation of an increased H reflex to another arousing stimulus, the inhalation of ammonia. This produced a noxious sensation in the upper airway without the confounding effect of coughing. Further, there was no increase in the H reflex or the response to transcranial magnetic stimulation when lobeline was given in doses subthreshold for any sensation.

It is theoretically possible that an inhibition at a spinal level was produced by lobeline but was overridden by increased descending excitation, either from arousal or from voluntary drive. Irrespective of the precise balance of reflex and supraspinal mechanisms acting to change motoneuronal excitability, the most important result is that the present studies did not reveal a net reduction in the ability of motoneurones to respond to reflex, corticospinal or voluntary excitation.

It has been suggested that one way in which excitation of pulmonary C fibres might decrease motoneuronal excitability is through an inhibition of fusimotor drive (Ginzel et al. 1971). Decreased fusimotor drive will decrease muscle spindle firing and remove reflex afferent support from the motoneurones. In our studies, of H reflexes and responses to transcranial magnetic stimulation, subjects were relaxed and would have had little fusimotor drive. Under these conditions, removal of this drive would have little effect. However, voluntary contractions are accompanied by strong fusimotor drive (Vallbo, 1971; Burke et al. 1979; Edin & Vallbo, 1990; Wilson et al. 1997) and injection of lobeline did not decrease voluntary motor performance when subjects performed submaximal or maximal contractions, or during walking. Thus, it seems unlikely that the level of fusimotor drive was critical in our failure to find inhibition of motoneurones.

There is one report of the behaviour of awake cats following an injection of phenyldiguanide to activate pulmonary C fibres: the animals ceased locomotion and slumped to the ground (Kalia et al. 1973). It is unclear whether this reflected active reflex inhibition of motoneurones or a response to the sensations induced by the injection. In contrast, our observations in awake human subjects did not reveal evidence for a deterioration of voluntary motor performance of muscles in the upper limb despite the presence of strong visceral sensations. Furthermore, the cadence and velocity of walking were not disrupted by lobeline injections despite evoking the expected respiratory discomfort.

In healthy subjects, it is likely that any reduction in motor output associated with the activation of pulmonary C fibres by rises in pulmonary capillary pressure at the termination of exercise is mediated by the evoked sensations rather than by the spinal reflex consequences of the visceral input. Presumably this also applies to pathological conditions such as left ventricular failure and pulmonary embolism. However, the situation is more complex here. Patients with chronic activation of pulmonary C fibres may become habituated to the evoked sensations. In patients with mitral stenosis, cough responses to lobeline are attenuated (Bruderman et al. 1966; Stern et al. 1966). It is possible that with decreased arousal from the sensations, inhibition of motoneurones may be unmasked.

The suggestion that the sensory response, rather than direct motoneuronal inhibition, limits the tolerance of exercise in conscious normal subjects may be consistent with the evolution of reflexes. Encephalization of reflexes associated with primate evolution may mean that supraspinal, presumably cortical structures, retain the ability to activate the spinal motoneurones of limb muscles notwithstanding any motoneuronal inhibition. Thus, a sudden noxious visceral event can be evaluated and the appropriate motor response planned. Investigation of the responses to lobeline injections in subjects during anaesthesia or in patients with neurological lesions may elucidate whether the neural pathways for the inhibitory motor response to activation of pulmonary C fibres are present in human subjects although subject to supraspinal control.