ABSTRACT
An account is given of the muscles and skeleton of the trout head and of the mechanisms coupling the different functional components of the skull.
The activity of the main respiratory muscles has been recorded electromyo-graphically and simultaneously with pressure and movement recordings from the buccal and opercular cavities.
The muscles may be divided into two main groups according to whether they are active during the expansion or contraction phase of the pumps. The protractor hyoideus (geniohyoideus) was found to be active only during the contraction phase.
There are differences in the muscles that are active, depending upon the depth of ventilation. Shallow ventilation is maintained by the adductor mandibulae, adductor arcus palatini et operculi, and levator hyomandibulae et arcus palatini. During deeper ventilation the stemohyoideus, protractor hyoideus and hyohyoideus muscles come into action. Only during strong ventilation does contraction of the dilator operculi play a part in opercular abduction.
There are variations in the pattern of muscular activity in different individuals and also in the same individual at different times. Such differences in muscular activity are not clearly reflected in the movement and pressure recordings, because of the complex couplings between different parts of the pumping mechanism.
As contraction of most muscles affects both the opercular and buccal cavities it is concluded that a model of teleost ventilation based upon a double pumping mechanism must incorporate the couplings between these pumps.
INTRODUCTION
The respiratory pumps of a teleost consist of a buccal cavity and two opercular cavities, separated by the gills (Hughes, i960; Hughes & Shelton, 1958, 1962; Woskoboinikoff, 1932). Volume changes of the cavities, caused by movements of bone arches and the operculi, result in the pumping action of the system.
In these pumps a fairly large number of muscles are working on a complex mechanism of bones, ligaments and articulations. Several authors (Baglioni, 1910; v. Dobben, 1937; Henschel, 1939, 1941; Holmquist, 1910; Hughes & Shelton, 1962; Kirchhoff, 1958; Willem, 1931, 1940, 1947; Willem & de Bersaques-Willem, 1927; Woskoboinikoff, 1932) have described this system and made an attempt to understand its working principles. Many theories have been developed to indicate which muscles take part in respiration and the role they play. In most papers the evidence for the theory is purely anatomical : the position and insertion of a muscle are used to explain its role. As, however, the moving parts of the teleost head skeleton articulate with one another in a complex way and are also coupled by means of ligaments, a great deal of interaction takes place and consequently contraction of one of several muscles can give rise to the same, or almost the same movement. Experiments in which muscles or ligaments are cut (Henschel, 1939,1941 ; Holmquist, 1910) give more direct evidence. The possibility exists, however, that the function of one muscle is taken over by another that is inactive during normal respiration, so that from experiments of this kind the only valid conclusion is whether a given muscle can or cannot be missed.
The contraction of a muscle is associated with electric potential changes which can be led off with a pair of fine electrodes put into the muscle. The recording of this electromyogram makes it possible to be more certain if, and precisely when, a muscle is active.
With this technique an analysis has been made of the activity pattern in the head muscles of the trout in relation to the movements and pressures recorded simultaneously from the buccal and opercular cavities.
MATERIALS AND METHODS
The trout used were obtained from a hatchery (Nailsworth, Gloucestershire). They were kept in stock tanks with running tap-water in the basement of the laboratory. The temperature was about 13°C.
During the experiments the animals were put in a glass trough containing 10 1. M.S. 222 solution at 0·03−0·05 %,,, and when anaesthetized they were fixed in a clamp. At the beginning of each experiment the temperature of the solution was the same as in the stock tanks and it was not allowed to rise above about 16°C. After an experiment the fish, brought back to the stock tank, very quickly recovered and showed normal activity.
During an experiment simultaneous recordings were made of the movement of the lower jaw, the movement of the operculum, the pressure in the buccal cavity, the pressure in the opercular cavity, and the electromyograms of four muscles.
Fig. 1 shows a block diagram of the experimental set-up. The movements were converted into an electric signal with R.C.A. 5734 mechano-electric transducers (1) and a bridge circuit (2), and the pressures converted with Hansen condenser manometers (3) and a capacity transducer circuit (4) (Hughes & Shelton, 1958). These signals were recorded on a four-channel Ediswan pen recorder with a frequency response up to 90 cyc./sec. (5).
The potentials of each muscle were led off with a pair of stainless-steel electrodes, insulated with araldite except for the tip.
After amplification with Tektronix type 122 preamplifiers (6), the myograms of four muscles were displayed on a Tektronix 532 oscilloscope (7) through a five-channel coincidence switching unit (8). On the fifth channel, the movement of either mouth or operculum was displayed. It served to correlate the pen recordings made simultaneously with the oscillograph records, filmed with a Dumont oscillograph camera (9).
RESULTS
Before considering the results of the electromyographic, movement and pressure recordings it is necessary to be clear about the morphology of the skeleton and musculature of the trout head. The account which follows is partly based upon our own observations but it is also necessary to review previous work, most of which was done with purely morphological methods.
1. The skeleton
In Fig. 2 a schematic diagram is given of the bones and muscles of a trout head. The skeleton is composed of the following units:
(a) The palatal complex, the major components of which are hyomandibula, quadrate and palato-pterygoid, is suspended from the neurocranium at two points. Anteriorly it articulates with the ethmoidal region, posteriorly with the otic capsule. The connexion between metapterygoid-hyomandibula and quadrate-symplectic consists of rather flexible cartilage.
(b) A second functional unit is the lower jaw, which articulates posteriorly with the quadrate. Anteriorly its two rami have a flexible connexion, so that besides opening and closing movements it can expand laterally when the palatal complex expands.
(c) The third unit will be referred to as the hyoid and is composed of the ventral elements (ceratohyal, basihyal, copula) of the hyoid arch. These elements support the floor of the buccal cavity and are connected to the hyomandibula through an intermediate bone, the stylohyal. The stylohyal articulates with both the hyomandibula and the ventral elements of the hyoid arch and the latter articulates also with the interoperculum. The ventral junction of the two halves of the hyoid is flexible. The branchiostegal rays are implanted along the hyoid.
(d) The branchial arches are stretched between the most ventral element of the hyoid, the basihyal, and the neurocranium.
(e) The operculum consists of four parts: the preopercular, interopercular, subopercular and opercular bones. The latter articulates with the hyomandibula, and the interoperculum articulates with the stylohyal-hyoid junction.
(f) The cleithrum forms the posterior border of the opercular cavities.
2. Muscles
The muscles working this system can be divided into two groups, of which one expands the respiratory cavities and the other reduces their volume. Essentially every moving element has its own abductor and adductor muscles, but through the coupling between the elements a given muscle can affect a greater part of the system than only the bones to which it is attached.
The levator hyomandibulae et arcus palatini lifts and abducts the palatal complex and so gives rise in the first place to a lateral expansion of the buccal cavity, of which it forms the sides. As, however, the lower jaw, the hyoid and the operculum are connected to the hyomandibula-quadrate part of the palatal complex, expansion of the latter also results in abduction of the operculi and in lateral expansion of the jaw and hyoid.
The antagonist of the levator hyomandibulae et arcus palatini in the trout is a strip of muscle fibres, inserted on one side of the neurocranium and on the other side running along the inside of the palato-pterygoid, the hyomandibula, the operculum and the dorsal rim of the operculum. In other species this muscular sheet is divided into different muscles: the adductor arcus palatini et hyomandibulae, the adductor operculi and the levator operculi (Dietz, 1912). In the following account this sheet is referred to as adductor arcus palatini et operculi. Besides adducting the palatal complex it adducts and lifts the operculum.
The abductor of the operculum is the dilator operculi muscle. Some authors (van Dobben, 1937; Henschel, 1941; Holmquist, 1910) believed that the levator operculi lifts the operculum synchronously with its abduction by the dilator operculi. The fact, however, that in trout the adductor operculi and levator operculi are one strip of muscle suggests the contrary. Indeed, our records show that activity in the adductor and levator regions of this muscular strip is synchronous and alternates with contraction of the dilator operculi.
Movements of the operculum are transmitted to the lower jaw by the mandibulo-interopercular ligament that connects the interopercular bone with a process of the lower jaw behind the articulation with the quadrate (van Dobben, 1937; Holmquist, 1910). Consequently levation of the operculum by the levator operculi part of the strip also produces depression of the lower jaw. According to Holmquist and van Dobben, this movement is reinforced by contraction of the dilator operculi.
The protractor hyoideus is sometimes called geniohyoideus, but according to Edgeworth (1931) the development of this muscle in Lepidosteus and the Teleostei does not justify the use of this name. It is attached anteriorly on the lower jaw and posteriorly on the hyoid and has been regarded as the abductor muscle of the lower jaw (Allis, 1897; Baglioni, 1907; Vetter, 1878). Even the double function of abducting the lower jaw and adducting the hyoid arch has been ascribed to it, but Holmquist (1910) concludes from his extirpation experiments that it only adducts the hyoid arch. This view was confirmed by Hughes (Hughes & Shelton, 1962) using electromyography. The lower jaw thus has no abductor muscle of its own. It is, however, abducted through levation of the operculum and also through movements of the hyoid which will be described later.
A large muscle, the adductor mandibulae, connects the lower jaw with the hyomandibula bone. It shuts the mouth.
Contraction of the sternohyoideus muscle, which connects the basal part of the hyoid arch with the cleithra, retracts and abducts the hyoid arch and so lowers the floor of the mouth. This also produces a force along the hyoid bones (Figs. 2, 3) working against the stylohyal and interoperculum. This force can be resolved into two components: one abducting the interoperculum, stylohyal, and through the latter the palatal complex (Henschel, 1941); the second component results in a caudal movement of the interoperculum. This caudal movement is transferred to the lower jaw by the mandibulo-interopercular ligament and lowers the mandible. Besides this there are the following direct connexions between the hyoid arch and the lower jaw : the hyoideo-mandibular ligament, the protractor hyoidei, and the skin. These elements are stretched during more extensive abductions and exert a direct traction on the lower jaw (van Dobben, 1937; Holmquist, 1910; Kirchhoff, 1958).
As mentioned above, the protractor hyoideus adducts the hyoid arch. According to Holmquist (1910) the lower jaw is fixed by the adductor mandibulae when the protractor hyoideus contracts so that the hyoid arch is adducted and the floor of the mouth raised by this muscle.
Evidence in the literature regarding the function of the hyohyoideus muscles which interconnect the branchiostegal rays and cross ventrally to the contralateral side is not unanimous. Borcea (1906) and Henschel (1941) both divide this muscle for a flatfish into an inspiratory and an expiratory part. Holmquist (1910) thinks that expansion or contraction of the branchiostegal membrane through this muscle depends on the position of the mouth and the direction of the contraction wave in the muscle. Hughes & Shelton (1962) conclude that in some fishes, during opercular expansion, the opercular membrane is actively held close to the cleithral girdle, dorsally through contraction of the levator operculi and ventrally through contraction of the hyo-hyoideus.
Skeletal couplings
The units which make up the teleost skull are connected with one another in a complex way. The following account is based on a study of the skeleton both in isolation and with the muscles and ligaments in situ. The results of the electromyo-graphic work have also been of value in this connexion.
The couplings (Fig. 4) between skeletal units which play an important part in the trout mechanism can be summarized as follows :
Three-dimensional diagram of the couplings. The numbering refers to the description in the text. Bone is stippled, muscle striped and ligaments black.
Coupling brought into action by contraction of the levator operculi part of the adductor arcus palatini et operculi:
(A) Lowering of the jaw. Levation of the operculum produces depression of the lower j aw by way of the operculum, the inter-operculum and the ligament to the angular which lies behind the articulation of the lower jaw with the quadrate, and therefore gives depression.
Couplings brought into action by contraction of the sternohyoideus:
(A) Lowering of the jaw may involve three couplings: (a) The sternohyoideus depresses the hyoid, which is connected by the ligamentum hyoidei-mandibularis to the mandible, and this produces a lowering of the jaw. (6) Depression of the hyoid is also accompanied by its retraction and the force along the hyoid may be resolved into two components. One of them presses backwards on the inter-operculum, which is connected by a ligament to the angular bone, which then depresses the lower jaw (coupling 1). (c) The sternohyoideus action also lowers the mandible when the hyoid has been pulled a good distance back because the skin becomes stretched and the passive elastic properties of the protractor hyoidei also help in this action.
(B) Expansion of the operculum results from contraction of the sternohyoideus in two ways: (a) Owing to the flexibihty of the anterior parts of the hyoid it expands laterally when the sternohyoideus contracts. This produces expansion of the operculum through the inter-opercular bones. (b) There is a mechanism for expansion of the operculum as a result of movement of the hyoid because the operculum is attached to the hyomandibula and so expands with it.
(C) Expansion of the palatal complex. Lateral expansion of the hyomandibula, produced above, also results in expansion of the whole palatal complex of which it forms a part.
(D) Increase in the size of the mouth. The lower jaw articulates with the quadrate, which is connected to the hyomandibula, and therefore the posterior ends of the lower jaw expand laterally, and as the junction of the two rami is flexible this produces a widening of the mouth. This mechanism plays no part in respiration but is probably important in feeding as it results in an increase in the effective crosssectional area of the mouth.
(E) Expansion of the branchial arches. The branchial arches are joined ventrally to the copula, which is connected to the hyoid. Consequently, retraction of the hyoid by the action of the stemohyoideus not only affects the copula but also produces a backward movement and consequent expansion of the branchial arches. In this way further expansion of the oro-branchial cavity is produced.
(B) Couplings brought into action by the levator hyomandibulae et arcus palatini :
(A) Lateral expansion of the operculum. Expansion of the palatal complex by contraction of this muscle results in abduction of the hyomandibula, which produces opercular expansion.
(B) Lateral expansion of the lower jaw because it articulates with the quadrate, which is connected to the hyomandibula and adducts with it.
(C) Lateral expansion of the hyoid, which is connected to the hyomandibula through the stylohyal.
Couplings brought into action by the adductor mandibulae:
(A) When this raises the lower jaw it has the effect of raising the hyoid through the action of the hyoideo-mandibular ligament and protracting it because the floor of the mouth is stretched. As a result the hyomandibula is adducted and with it the operculum.
Couplings brought into action by the hyohyoideus (constrictor in origin):
(A) When this muscle contracts it effectively raises the copula and results in a folding of the branchial arches and more or less helps to adduct the operculum.
This, then, is a summary of some of the main couplings found in the trout.
It cannot be overemphasized that, although the skeletal couplings are similar in many species of fish, the muscle co-ordination is not necessarily the same. From the results obtained so far it has been found that, for the same species and even the same individual, many different patterns of muscle action are possible, each of which can be effective in producing normal ventilation. Clearly the interspecific differences may be quite marked.
3. Electromyography
By correlating the pressure and movement records from the pen recorder with the electromyographic records from the oscilloscope (Fig. 5), a composite picture has been obtained of the activity of several head muscles as related to the operation of the respiratory pumps.
In Fig. 6 such correlated records are reproduced for two sets of respiratory muscles. Fig. 7 gives a summary of all the muscles that were recorded from and forms a basis for the following account.
Correlated records of electromyograms, respiratory movements and water pressures in buccal and opercular cavities. For abbreviations see Fig. 2.
Time relations between muscle activity and the movements and pressures of the respiratory pumps. For abbreviations see Fig. 2.
To the left of the diagram (Fig. 7) the mouth is open and the operculi are abducted, or nearly abducted. Contraction of the adductor mandibulae starts the cycle with adduction of the lower jaw. When the respiratory movements are strong, the protractor hyoideus and the hyohyoideus contract synchronously or slightly after the adductor mandibulae. As the lower jaw is fixed by the latter muscle, the protractor hyoideus moves the hyoid arch forwards and upwards and so raises the floor of the mouth. The hyohyoideus contracts the branchiostegal apparatus and also raises the hyoid arch, because its fibres cross to the opposite side. This lifting of the hyoid arch adducts the hyomandibula (Henschel, 1941) and the interoperculum, with which it articulates, and so initiates adduction of the palatal complex and operculum. Contraction of the branchiostegal membrane reinforces the adduction of the operculum.
When depth and frequency of respiration are low the protractor hyoideus and the hyohyoideus are not active. Adduction of the lower jaw, however, results in lifting of the hyoid arch through the hyoideo-mandibular ligament, so that all the abovementioned movements can take place, but with less force.
The adductor arcus palatini et operculi often commences its activity during the last part of the adductor mandibulae contraction. Its activity, however, is low at first, but as soon as the adductor mandibulae stops firing its activity reaches a high level. It adducts and lifts the operculum, pulling it laterally and ventrally close to the body. The lifting motion pulls the interoperculum in a dorso-caudal direction and this action, because of the mandibulo-interopercular ligament, results in abduction of the lower jaw (Holmquist, 1910; van Dobben, 1937).
When the ventilation is strong the sternohyoideus becomes active at this time and supports the abduction of the lower jaw; it lowers the hyoid arch and moves it backwards ; this pushes the interopercular bone in a dorso-caudal direction, resulting through mediation of the mandibulo-interopercular ligament in depression of the lower jaw. Besides that, the hyoideo-mandibular ligament, and also, as soon as it is stretched, the protractor hyoid muscle and the skin of the floor of the mouth, all exert a traction on the lower jaw, opening the mouth. The role of the protractor hyoideus is an entirely passive one; activity could never be detected during this phase of the movements.
When electrical activity in the adductor arcus palatini et operculi stops, that in the levator hyomandibulae et arcus palatini begins and produces abduction of the palatal complex and opercula. In two of our records activity in this muscle was much earlier, approximately coinciding with the activity in the adductor mandibulae. This could be due to extreme variability in the timing of the levator hyomandibulae et arcus palatini.
The possibility, however, that the electrodes were not leading-off potentials from the levator hyomandibulae et arcus palatini, but rather those of the adductor mandibulae, is supported by the following facts:
(a) both muscles are close together and the adductor mandibulae is a very big muscle partly overlying the other;
(b) in the movement records no abductor component can be detected during activity in this muscle ;
(c) one of our records of the levator hyomandibulae et arcus palatini shows double activity : one burst of high amplitude when this muscle is normally active, and another of low amplitude, synchronous with the adductor mandibulae and obviously recorded from that muscle (for instance through a leak in the electrode insulation).
With stronger respiratory movements the levator hyomandibulae et arcus palatini is also supported by contraction of the stemohyoideus ; abduction of the hyoid abducts the hyomandibula as well (Henschel, 1941), as soon as it is no longer held adducted by the adductor arcus palatini et operculi. The palatal complex and the operculum are abducted because of their connexion with the hyomandibula.
Only during strong breathing movements is abduction of the operculum reinforced by contraction of the dilator operculi.
DISCUSSION
As a result of this work it appears that the muscular basis of the trout respiratory pumps is relatively simple, but this simplicity results from the intricate nature of the couplings between different parts of the skeletal system. Thus a particular movement of part of the skeleton may be produced by several different patterns of muscular co-ordination. Correspondingly, the contraction of a given muscle affects many different parts of the system. These general conclusions emphasize the value of electromyography as a technique for the elucidation of the activity of muscles during rhythmic movements. Furthermore, because of the complex interaction between parts of the teleost head skeleton it is important to realize that when a given muscle is active it may prevent the action of certain couplings, as well as operating others. Clearly the total pattern of muscular activity is what is most important. By comparison of the muscle action potentials in relation to the movement records it has been possible to recognize those couplings which are most important under different circumstances (Figs. 5-7). And it is now possible to give an account of the different patterns of ventilation in fish showing different intensities of pumping. As yet no quantitative measurements have been made of the ventilation volume but a relative estimate has been made by reference to the amplitude and frequency of the respiratory movements.
The respiratory cycle
In the trout three muscles are of prime importance: the adductor mandibulae and the ‘palatal complex muscles’ levator hyomandibulae et arcus palatini and adductor arcus palatini et operculi. During shallow ventilation electrical activity can be recorded only in these muscles. The cycle starts with activity in the adductor mandibulae alone and this results, often after a short delay, in a closing movement of the mouth. Through coupling 4 A the hyoid is protracted and the hyomandibula is adducted. The operculum although connected to the hyomandibula, does not adduct immediately and may even go on expanding because it is forced open by the passage of the exhalant water current. The pressure in both buccal and opercular cavities is positive as a result of the reduction in volume produced by the closing action of the jaws and operculum.
Following the activity of the adductor mandibulae, the adductor arcus palatini et operculi begins to contract. Its primary action is to lift and adduct the operculum, drawing it close to the body (often the movement record becomes steeper when this muscle becomes active), and it adducts the palatal complex, thereby compressing the buccal cavity laterally. Secondarily it depresses the lower jaw through coupling 1A; that is : operculum → interoperculum → mandibulo-interopercular ligament → lower jaw. The pressure in the opercular cavity reaches a maximum when the operculum is maximally adducted. The buccal pressure initially rises steeply as a result of the lateral compression by the palatal complex and then returns to zero as the mouth opens. The last part of the activity in the adductor arcus palatini et operculi normally overlaps the commencement of activity in its antagonist, the levator hyomandibulae et operculi. This ensures a gradual and smooth reversal in the direction of movement as the palatal complex expands and widens the buccal cavity. It is accompanied by expansion of the opercular cavity because the opercular bones articulate with the hyomandibula, which is a part of the palatal complex. The buccal pressure falls below zero, but the opercular pressure becomes more negative because the flap valves of the expanding opercula remain in contact with the sides of the fish, supported by the cleithral girdle. Consequently water can only enter the opercular cavities through the gill resistances. As a result of these pressure changes a current of water flows across the gills throughout the cycle (Hughes & Shelton, 1957, 1958, 1962).
During the last part of the opercular expansion there is often a complete absence of electrical activity in any of the muscles. Presumably the expanded palatal complex begins to adduct because of elastic and gravitational forces and the resulting water current may even abduct the operculum.
After this brief pause in muscular activity, during which the mouth is often not completely open, the cycle starts again with contraction of the adductor mandibulae.
During stronger ventilation more of the head muscles become active, the frequency increases and excursions of the moving parts become greater, but no consistent changes in the overall pattern of movement can be detected. This is not surprising because the effect of increased activity in any muscle would benefit a large part of the system through the numerous couplings. Three muscles that come into action with stronger ventilation are stemohyoideus, protractor hyoideus and hyohyoideus. The adductor arcus palatini et operculi now shows low amplitude activity during the latter part of the adductor mandibulae contraction.
The influence of the sternohyoideus is very widespread. It abducts the hyoid and through couplings 2 A-E it lowers the jaw, expands the branchial arches and abducts the palatal complex and operculum as soon as they are no longer adducted by the adductor arcus palatini et operculi. Thus it increases the size of both the buccal and the opercular cavities.
The activity in the adductor mandibulae, protractor hyoideus and hyohyoideus is almost synchronous, with a tendency for each muscle to start its activity a little later than the one before in this order. The protractor hyoideus is clearly an antagonist of the sternohyoideus and primarily it protracts the hyoid. The hyomandibula, as a part of the hyoid arch, is also adducted as a result of its contraction and with it the rest of the palatal complex (reverse of coupling 2C). The operculum, through its articulation with the hyomandibula, also takes part in the adduction as soon as it is no longer forced open by the passing water current (reverse of coupling 2B (b)). Finally, activity of the adductor mandibulae also results in the branchial arches being contracted (reverse of coupling 2E). The volume of all the respiratory cavities is decreased by these movements.
The hyohyoideus contracts the floor of the mouth, folds up the branchiostegal apparatus and reinforces opercular adduction and thus helps decrease the volume of the buccal and opercular cavities. The dilator operculi reinforces the opercular abduction. Appreciable activity in this muscle is only found, however, during strong ventilation.
It is apparent from this account that ventilation in a bony fish involves a pumping mechanism on either side of the gill resistance but that the two pumps are coupled together to such an extent by the skeletal mechanism that the action of almost all the muscles affects both pumps. In terms of the model originally suggested (Hughes, i960) it is clearly necessary that the two pistons should be coupled together by a spring of varying stiffness (Hughes, 1964).
Similar conclusions have also been reached as a result of a further investigation of the respiratory pumps of the dogfish (Hughes & Ballintijn, 1965). In the dogfish the presence of elastic components in the walls of the pumps also plays an important part, Such components are also involved in the teleost but do not fulfil so important a role. In the trout they are less important than in fish with a very flexible wall to the opercular cavity, such as in eels. It is of interest that for both the trout and the dogfish normal quiet respiration is produced by activity which is restricted to some lateral plate muscles. During more intense ventilation muscles in the ventral part of the head come into action. In the dogfish these are of myotomic origin and some of those in the trout are also of this type. From an evolutionary point of view this is what might be expected, as the lateral plate muscles are primarily concerned with the visceral skeleton and only later in evolution is it supposed that the hypobranchial musculature became involved in their movements.
