Pharyngeal Proprioceptors in the Dogfish Squalus Acanthias L.

In this species, if respiration is slowed, the increase in cycle length is achieved by a prolongation of the filling phase and pause; the expulsive phase changes little. Slowing of respiration may occur naturally as in the cycles that follow a cough or spout (Satchell, 1959). It may be induced experimentally by restricting the rate of water flow through the pharynx. This results in an increase in the depth of respiration and a fall in the rate (Satchell, 1961). Inflating a balloon in the pharynx causes a transient inhibition of respiration (Satchell, 1959) as does mechanical pressure on the outside of the pharynx. Curarization sufficient almost to paralyse respiratory movement abolishes the respiratory slowing caused by a cough or by restriction of water flow. These findings are in accord with the hypothesis that there exists in the pharynx some system of proprioceptors which are fired during the contraction of the oro- and para-branchial cavities, or by artificial mechanical stimulation, and whose output is inhibitory to the onset of the next respiration.

Our attention was drawn to the branchial processes lining the internal openings of the gill pouches as they appear to be well situated to bear proprioceptors. In this paper the responses of receptors associated with the branchial processes will be described and their possible significance discussed.

Trawled Squalus acanthias of approximately 3 lb. wt. were stunned by a blow on the head. The branchial nerves were dissected free and cut at their junction with the vagus, and the gill septa with their attached filaments were removed. A gill was placed in a Perspex chamber in oxygenated Ringer’s fluid (Keynes & Martins-Ferriera, 1953) and the post-trematic branch of the branchial nerve was systematically subdivided with fine forceps under × 10 magnification. Strands were examined with an audio amplifier for their content of receptor fibres firing to mechanical stimulation of the branchial processes. Suitable strands were split further until unitary responses were obtained. The fine strands were placed across platinum recording electrodes in a layer of paraffin oil floating on the surface of the Ringer’s fluid. Temperatures were maintained within 2° C. during any one experiment but varied during the course of the investigation from 9° to 18° C. Nerve discharges were amplified with an a.c. coupled differential amplifier, displayed on the upper beam of a double-beam oscilloscope and photographed with a Grass C4D camera.

The movement of the branchial process was effected with a fine wire ending in a loop that could be placed around a process. The wire was attached to the anode of a RCA 5734 mechano-transducer tube which served to monitor the deflecting force. Tube and wire were moved with a Prior micromanipulator and the tube output, after d.c. amplification, was displayed on the lower beam of the oscilloscope. The stimulus was calibrated by attaching balance weights to the loop and photographing the deflexion.

All four gills have been studied but as no significant differences could be detected between them the last two were most commonly used as they provide the longest length of nerve. The nerve fibres are bound together with connective tissue and the harvest of successful preparations was not great. Responses of many fibres were observed up to the stage of the final splitting of the strands. In all, fifty-six fibre preparations were photographed, of which twenty survived long enough to enable the responses to between 50 and 100 deflexions, differing in magnitude, duration, and speed of onset, to be recorded.

These finger-like projections of the gill bar have each a cartilage support and are covered with an epithelium bearing placoid scales. Their number varies with the size of the fish. Pl. 1 shows the processes of a 3 lb. fish as viewed from inside the pharynx; there are 8, 10, 11, 9 and 5 of them on the posterior margins of the 1st-5th gill slits respectively. They point rostrally and are bent against the anterior margin of the slit as the oro- and para-branchial cavities are contracted in each respiratory cycle. The movement of each process is thus a medial one, its natural resilience causing it to return to its neutral position as the pharynx is expanded. These movements have been observed with a cystoscope telescope inserted through the gill slits of one side to visualize the processes of the other. There are in addition two or three backwardly directed processes on the last two gill bars.

Frozen sections of the branchial processes have been cut and stained by the Schofield modification of the Bielschowsky technique (Schofield, 1960). The processes are richly supplied with free nerve endings and with differentiated end organs. There is no evidence to link any particular receptor structure with any type of response, and they will not be described here. A separate paper on the histology of these processes is to be published.

A variety of quickly adapting, low-threshold receptor responses was encountered when nerve strands were placed across the electrodes, but such fibres were not sufficiently numerous and did not last long enough to enable other than fragmentary information to be obtained about them. The most sensitive had thresholds below the sensitivity of the RCA transducer tube and could be fired by chance vibrations in the room communicated through the apparatus to the wire loop touching the process.

Others were fired by as little as 8 mg. wt. deflecting force. The few receptors of this type that were explored had very limited receptive fields. One was fired only by stimulation of the tip of a process ; another had its receptive field of approximately 2 mm.² extent, confined to one side of a process. Other low-threshold receptors were located on the epithelium of the branchial arch. All the rapidly adapting receptors on the processes responded to contact stimulation with a glass rod; they fired a brief burst of 1–10 impulses at the start of stimulation, and sometimes a second briefer burst as the rod was removed. Osmicated preparations of the nerve branch supplying the processes showed no fibres larger than 10 μ and many much smaller than this. It may be that the rapidly-adapting receptors are linked to small-diameter fibres and that the paucity of satisfactory records reflects the difficulties of isolating them.

Slowly adapting receptors of medium to high threshold were the most commonly encountered and yielded preparations that lasted longer. Within this designation are included receptors whose response to a sustained deflexion ranged from a 3 sec. burst of impulses to a discharge that endured for as long as the process was deflected.

In the isolated gill preparations these receptors could be caused to fire by either an upward or a downward deflexion of the process. This corresponds in the intact fish to a movement towards or away from midline such as would occur in normal respiration. Some could be fired only by the deflexion of a single process. Others responded to movements of the process on either side as well. These slowly adapting receptors have been located at the bases of every one of the processes of the anterior row on the 4th gill bar. Thresholds varied from 46 to 580 mg. wt. deflecting force. All could be fired by light pressure exerted around the bases of the processes with a glass rod.

The response to a sustained deflexion always showed an initial more rapid discharge at the onset of the stimulus; this phasic discharge was at a rate of 14–36/sec. The rate then declined steadily; in some, firing ceased altogether after a shorter (3 sec.) or longer (8 sec.) train. Others achieved a steady rate of 6–7/sec. and continued firing as long as the process was deflected (Text-fig. 1A, 2). The phasic component in the response was also seen as a further acceleration of firing superimposed on the static discharge (Text-fig. 1 B) if the process was further deflected. Some but not all showed an acceleration of discharge when the process was returned towards its normal position (Text-fig. 1 A and 2 B). The phasic discharge evoked by stimulus onset tended to show an initial acceleration, the briefest period between two impulses being between the second and third or third and fourth of the train (Text-fig. 1 A). In some (Text-fig. 1 A) there was a period of receptor inactivity between the phasic and static components in the discharge.

Increase in stimulus amplitude caused an increased rate of discharge for both the phasic and static components of the response (Text-fig. 1C). In Text-fig. 3, the maximum discharge rate calculated from the shortest time interval in which five impulses were fired is plotted against the logarithm of the stimulus in mg. wt. The calculated regression line has been plotted (r = +0·69, P ⪡0·001, n = 57). A linear relation is shown as has been demonstrated in other mechanoreceptors such as the frog muscle spindle (Matthews, 1931). This author also discusses the departure from linearity which commonly occurs at near-threshold stimulus levels, and is seen in Text-fig. 3. This linear relation has been noted in many receptors and is believed (Granit, 1955) to be the manifestation at the receptor level, of the Weber-Fechner law of sensation.

The discharge rate of the phasic component of the response was related to the rate of onset of the stimulus. Deflexions of a given magnitude evoked lower initial discharge rates if they were slow in onset (Text-fig. 1D). In Text-fig. 4 the rate of rise of the stimulus in g. wt./sec. is plotted against discharge rate as calculated from the first five impulses. The curve flattens at 5 g. wt./sec. with a discharge rate of 25/sec. This plateau may reflect a limitation placed on the spatial distortion of the receptor by the elastic properties of the process rather than an upper limit of discharge rate.

When the rate of stimulus-onset is rapid the first impulse is fired at a lower stimulus intensity (Text-fig. 1D). This phenomenon has been observed in other mechanoreceptors such as the cat vibrissa (Fitzgerald, 1940) and reflects the adaptation that is occurring during the time of stimulus onset.

In the introduction some respiratory reflexes in the dogfish are described and the suggestion is made that they are all mediated by a system of pharyngeal proprioceptors the activity of which is inhibitory to the onset of the next respiratory cycle. It is now proposed to discuss this hypothesis further and to examine the possibility that the slowly adapting receptors described in this paper are these proprioceptors. The following points can be made.

It is likely that the slowly adapting receptors do fire impulses up their branchial nerve fibres whether they are deflected naturally, in the course of a respiration or a cough, or artificially when a balloon inflation presses them against the gill bars. Visual inspection with a cystoscope telescope showed that they undergo movements as extensive as those which initiate discharges in the isolated preparation. Moreover, records made in intact respiring fish from branchial afferent fibres demonstrated (Satchell, 1959) that nerve discharges pass up them at the time that the oro- and para-branchial cavities are passing through the peak of their contraction.

Respiratory rates in Squalus acanthias lie between 25 and 60/min. The period during which the branchial process would be undergoingde flexion by pressing against the gill bar in front, and then returning to its neutral position, would not exceed one second and would probably be much less than this. Thus the slowly adapting receptors would be operating largely on the phasic component of their response which endures for approximately 0·8 sec. They would be well able to present a continuous report of the deflexion of a process throughout this phase of a respiratory cycle even at the lower rates of respiration.

Some of the slowly adapting receptors showed an acceleration of discharge both as the process was deflected and again as it returned to the neutral position. Moreover, these phasic responses could be written on to a background of maintained firing evoked by a degree of maintained deflexion. This ability is uncommon in mechanoreceptors in other animals; it enables one receptor to signal both static and dynamic mechanical displacements. It appears well suited to report both the amplitude of the pharyngeal movements associated with the expulsive phase of the respiratory cycle, and any maintained displacements of the pharyngeal wall such as might occur in fast forward swimming.

It is established that electrical stimulation of the central ends of branchial nerves inhibits respiration (Satchell, 1959). Moreover, whilst there is some fragmentary evidence (Satchell, 1958) for the existence of an acceleratory respiratory reflex, electrical stimulation of branchial nerves evokes inhibition at all stimulus strengths that will evoke a response at all. It may thus be conjectured that the inhibito-respiratory afferents are low-threshold ones, and this may correlate with the greater ease with which the slowly-adapting receptor fibres have been isolated. Fibre diameter and threshold are usually inversely related, and larger fibres are more easily isolated.

The inflation of a balloon in the pharynx has been shown to cause a burst of action potentials in branchial afferent fibres at the start and stop of inflation, and a less intense activity during it (Satchell, 1959). Similarly, respiration is inhibited more strongly during the start and stop of an inflation, and tends to break through during it. Both the afferent fibre discharge and the respiratory inhibition recall the phasic and static components in the response of a slowly adapting receptor to a sustained deflexion.

Finally the changes in depth and rate of respiration that occur when the rate at which water flows through the pharynx (the respiratory minute volume) is artificially changed may in part be due to these receptors. When the minute volume is increased the amplitude of respiration falls and the rate rises. Conversely, when the minute volume is curtailed respiration becomes deeper and slower. The action of curare (Satchell, 1961) demonstrates that two reflexes are concerned in these changes in amplitude and in rate. It is possible to curarize a fish to a depth at which movement of the branchial skeleton ceases but the valves guarding the external gill openings still undergo a feeble movement sufficient to actuate a sensitive strain gauge. In such a fish changes in minute volume still evoke changes in the amplitude of respiration but these are unaccompanied by any change in rate. The receptors concerned in mediating the change in amplitude are unknown. But the failure of the respiratory rate to follow changes in minute volume suggests that this reflex depends on movements of normal amplitude. The slowly adapting receptors may again be the source of the afferent input; in a curarized fish the branchial processes are plainly still. It may thus be that these slowly adapting receptors are primarily concerned in a breath-by-breath regulation of respiratory rate, the duration of the filling phase and pause being proportionate to the amplitude of the deflexion of the branchial processes during the preceding expulsive phase of the respiratory cycle.

1. The responses of mechanoreceptors present in the branchial process of Squalus acanthias have been studied oscillographically; unitary responses were obtained by subdivision of the post-trematic branch of the branchial nerve. The deflexion of the branchial processes was monitored with a mechano-transducer tube.

2. Slowly adapting mechanoreceptors of medium to high threshold were most frequently isolated; nerve discharges were readily evoked by deflexion of the processes. Some receptors continued firing as long as the process was deflected. The discharge consisted of a phasic and static component. In some receptors acceleration of discharge occurred as the process was deflected and again as it was returned to the neutral position.

3. Discharge rate was found to be linearly related to the logarithm of the stimulus amplitude. The discharge rate of the phasic component increased as the rate of stimulus onset increased.

4. The role of these receptors in the respiration of the dogfish is discussed and it is suggested that they may play a part in relating the rate of respiration to its amplitude.

It is a pleasure to acknowledge our indebtedness to Mr Vic Hansen who has supplied the dogfish, and to Prof. A. K. McIntyre and Prof. J. R. Robinson, who have read the manuscript. Thanks are also due to the Nuffield Foundation who provided a grant towards equipment, and to the Medical Research Council who have supported the project.