In accordance with expectation, the embryonic chick heart exposes recurrently temperature characteristics of the orders 8,200, 14,200, 16,400, 18,300, 20,500 and 25,200. These are found in both myogenic and neurogenic hearts but not with the same frequencies. These two periods of development also differ in the localisation of critical points. So far μ. = 8,200, 14,200 and 18,300 have been exposed in embryos only by Fundulus’, the remaining three orders however are recurrent also in the embryogeny of Limulus. Two orders not included in this list (5000 and 11,000) are rare, yet, even so, not restricted to any one of the three forms. These facts, together with their implications, suggest for heart rhythm in Limulus, Fundulus and the Chick, an underlying physical-chemical mechanism in structure identical for all three types of heart at all stages of development, and differing in neurogenic stages only by a revision of the myogenic time relations.
Embryonic heart rate as a function of temperature has been analysed recently by Crozier and Stier (1927), by Cohn (1928), and by Glaser (1929),1. In Limulus, prior to innervation by the cardiac ganglion, the heart discloses between 10° and 39° C. well authenticated values of μ. = 11,500, 16,400, 20,000 and 25,500. On the other hand the adult, so far, has yielded only μ = 12,200 (data of Garrey (1921 a, b);,Crozier (1924 a)). However, this contrast of diversity with uniformity must be accepted with caution. In Fundulus the situation is quite the reverse. Here between 4·8° and 29° C. the heart, without nervous control, exposes clearly only two temperature characteristics, 16,300 and 14,300, whereas post-vagus stages usually yield p = 20,900 and sometimes μ = 18,200—values likely to be replaced above 20° C. by the orders 16,000, 14,000 and lower.
Leaving aside momentarily Murray’s (1925) work on isolated fragments of chick auricle and also Cohn’s (loc. cit.) study of the intact heart, the differences between Fundulus and Limulus do not call for structural differences in the catenary systems that underlie their respective rhythms either in myogenic or neurogenic stages of development. Indeed if metabolism in muscle and nerve are fundamentally identical, the onset of nervous control is likely to be documented not by new increments but rather by a revision of existing time relations. Such revisions should externalise as differences in the frequency with which certain increments appear; as differences in the thermal ranges over which they apply; at certain temperatures, as abrupt changes in pulsation rate ; and finally as changes in frequency, localisation, or both, of critical points. As they stand, therefore, the discrepancies between Limulus and Fundulus are quite explicable if we imagine for both hearts and both periods of ontogeny a common physical-chemical mechanism diversified only by circumstances that affect the relative ease with which specific increments emerge (Glaser, 1929).
The heart of the developing chick was chosen in order to test this hypothesis. Certainly the conditions under which it operates differ largely from those in Limulus and Fundulus. In the bird there is a far greater discrepancy between early embryonic mass and total mass, including accessory structures and foods ; respiratory exchange is less immediate and must be interfered with in order to make observations; and finally, we are dealing with an organism which, if not actually equipped with automatic temperature regulation, is genetically homoiothermous and confined within relatively narrow thermal limits to relatively high temperatures.
Among these differences, genetic homoiothermism is theoretically the most important. In fact, if our hypothesis is correct, the chick should afford an opportunity to explore the Limulus-Fundulus mechanism at temperatures above its usual range. Given the knowledge now available, we can definitely expect from myogenic and neurogenic chicks the increments common to the two poikilothermous forms. A temperature characteristic of the order 8,000, so far rare in Fundulus, might appear with heightened frequency in the chick. Orders 14,000, 16,000 and 18,000, which in certain instances apply from upper to lower limits in the fish, at times might do likewise in the bird; on the other hand, μ. = 20,000 and 25,500 should emerge only in the upper ranges. Strictly new increments can neither be expected nor as yet excluded on the basis of experience ; on the other hand, critical points obviously cannot fall at temperatures normal for the other forms and, if they occur at all, may be expected at different regions of the thermal scale during myogenic and neurogenic periods of development.
The eggs used in this study were obtained from a pure and closely inbred strain of Rhode Island Reds, and were selected invariably from the first collection each morning. This procedure reduced differences in developmental age to a minimum.
Subsequent incubation was carried on at precisely 37·5° C. When presumably ready for observation, a small opening was made in the shell, after which the egg was placed in a small dish partially filled with moist sterile sand and stationed in the observation incubator. In all these operations we practised surgical cleanliness.
The observation incubator, provided with electric thermostat, adequate and controlled illumination, moist chambers and fan, was a bacteriological one, placed on its side with glass door uppermost. Through this door the observations were made. The temperatures remained constant to within less than 0·1° C. during the time required for any particular group of readings.
In our first experiments, we applied a few drops of adjusted Ringer-Locke solution, but found evaporation more efficiently controlled by means of the moist chambers and a watch crystal partially covering the hole in the shell. A small thermometer carefully checked against a large standard, and placed with its bulb just in contact with the vitelline area, recorded temperatures at a maximum distance of 1 cm. from the heart. This thermometer was completely enclosed in the incubator and, though inadequate for absolute accuracy, was reliable in measuring differences proportional to the changes in temperature undergone by the heart itself. A few runs with a thermocouple proved that the added difficulties of this method were not compensated for by any appreciable advantages.
The first reading was not taken until the egg had been in the observation incubator hours. For each subsequent change of 1° C. we allowed 1 hour for stabilisation. Embryos exhibiting irregularities during the first hours or later were discarded. In some instances these irregularities were inexplicable; in others traceable to haemorrhage or transcendent temperatures.
The heart, or in some cases the pulse in the vitelline artery, was observed directly or by means of a binocular microscope. At the higher temperatures 100, at the lower, 50 complete cycles were counted for one reading. Time was measured with a stop-watch graduated to tenths of a second. Three or more readings spaced 3 minutes apart were taken at every stable temperature. A complete record usually required 12 hours. In certain instances, the embryos survived for 48, 72 and even 96 hours, and made possible successive runs.
As the main stem of the vagus innervates the heart on the fifth and sixth days of incubation (Lillie, 1919), our study began with three-day eggs. On this material we determined firstly the thermal limits within which our mechanism is not irreversibly changed. Individuals, of course, vary in their tolerance of extremes. Some after exposure to 45° or 20° C. later yield records at certain intermediate temperatures, identical with the first readings ; others may become irregular at 26° and 42° C. In view of these preliminary findings, observations on early and also later stages of development were confined between 24° and 44·5° C. No one embryo however was observed in a single run over this entire range.
Certain results on pre-vagus chicks are assembled in Fig. 1. Starting at or near 37·5° C. the direction of the observations is indicated by arrows. In two instances, curves 1 and 6, the ascending and descending series of points are plotted together and are to be compared respectively with curves 2 and 7—the former giving only descending, the latter only ascending points. The data for 1 and 2 are derived from three, those for 6 and 7 from four individuals. The several rates are too nearly identical to require factoring. Curve 6 has a somewhat greater latitude are based on unselected eggs of mongrel origin. In two of these the sequence of observation is indicated by numbers. The increments are clearly of the orders 16,000 and 18,000.
Of the four orders disclosed by pre-vagus chicks, two are found in Limulus and one in Fundulus at corresponding stages. If three-day chicks were to exhibit critical points, some of the missing orders might emerge and thus establish a still closer resemblance to Limulus in which at certain temperatures sudden changes in rate may be followed by changes of increment. Such findings would not be irreconcilable with the data from Fundulus, for here too there occur shifts (Glaser, loc. cit.) which may or may not involve different temperature characteristics.
As indicated in Fig. 2, a break, not too accurately localised, was found in one Rhode Island Red, R.I., where μ = 20,700 changes to μ = 7,100. On the contrary, the mixed stock shows at least three instances ; one with a vertical shift from which the order 14,000 emerges, and another in which μ= 25,500 changes to μ = 8,300. No particular importance can be attached to μ = 5,000, although this value has likewise been observed in Limulus (Crozier and Stier) and is not excluded from Fundulus (Glaser, loc. cit.). The critical points lie between 35·3° and 36·9° C.
With pre-vagus chicks disclosing so large a repertoire of temperature characteristics, innervation can hardly be expected to yield more than changes in the location of critical points and differences in the frequency with which these same increments emerge.
Our post-vagus material is entirely restricted to pure Rhode Island stock and les records of the seventh, eighth, ninth, eleventh, fourteenth and fifteenth days. As in the earlier stages, critical points are rare; only three instances were encountered, but these fall within a temperature zone whose lower limit is 6° C under that of the myogenic period (cp. Fundulus, Glaser, loc. cit.). In two of these instances μ. = 25,000 breaks to μ = 8,500 (Fig. 3). In the third, the order 21,200 shifts to order 8,100.
The eleven curves, Figs. 4 and 5, illustrate runs on ten different individuals. In accordance with expectation, the values of μ are the recurrent ones of Limulus and Fundulus, and, except in frequency, identical with those exhibited during the pre-vagus period of the chick.
It would be interesting now to find a stage of development capable of yielding some new increment. The fifth day was chosen because partial innervation might prove favourable to the emergence of characteristics not so easily disclosed by the earlier or later stages. Only two transitional embryos are available. Above 33° C. both expose low μ.-values; below this temperature however these individuals give unmistakable exhibitions of μ = 25,000 (Fig. 6).
Omitting the least authentic increments, the remainder classify into six well-established orders, Table I.
While frequencies differ, as it was expected they would, three of the orders exposed by the chick have been found in myogenic Limulus and all of them in Fundulus either before or after innervation.
All increments recurrent in one or more of these animals are assembled in Table II.
Thus heart rhythm in these three types fails to reveal at any period of development symptoms of structural changes in its underlying catenary series. Of this chain of processes, six, possibly seven, links may be considered as capable of verification. Murray’s data (loc. cit.) derived from fragments of chick auricle, when corrected for shifts in pace-maker control, yield the orders 8,000, 12,500, 20,000 and 24,000 (Crozier and Stier, loc. cit.) and harmonise very well with our present findings. In so far as they are published and available for criticism, Cohn’s (loc. cit.) apparently contradictory results from the intact heart of the chick are by no means discordant. Far from indicating a glissando of p-values or an inverse relationship between incremental order of magnitude and age, these observations in two instances clearly yield the order 8,000. In two other cases, if we allow for the changes in pace-maker control suggested by the fan-like dispersion of the data, the apparent orders of magnitude that emerge are the familiar ones 8,000, 11,000, 14,000 and 16,000 (Fig. 7).
The identification of specific temperature characteristics with specific chemical processes is an ideal (Crozier, 1924 b). At present we can do little more than attribute the uniformities exposed by different types of heart to corresponding uniformities in the processes that underlie their respective rhythms. Yet the temptation to go somewhat further is not totally unjustified. Values of the order 16,000 are frequently encountered in connection with processes in which oxidation or the rates of gaseous exchange might easily be the controlling factors (Crozier, 1924 c); μ = 11,000 has been related to OH-ion catalysis (Rice, 1923); whereas μ = 18,200 emerges from reactions in which iodine is concerned. (Cp. Hecht’s 1928 findings and comments on the work of Plotnikow, 1907, Conant and Hussey, 1925, and of Rideal and Williams, 1925.)
These analyses rest on the fact that the equation ln, properly applied, gives an accurate description and objective reproduction of the data. For closeness of fit, see Glaser 1924, 1925 and 1929.
The velocity constants and K1 and K0 inversely proportional to the time required per beat at the absolute temperatures T1 and To. R is the gas constant and μ a factor designated by Crozier (1924 a, b) as the temperature characteristic or critical thermal increment and accounting for changes of rate associated with changes of temperature.
In the graphs μequals slope, but physically and in theory it represents the energy required by a given species of molecule when transforming from the chemically inactive to the reactive state.
A biological process is attributable to a series of underlying chemical reactions linked in definite sequence. Ordinarily such catenary systems can proceed no faster than the component with the slowest rate of acceleration. This is the controlling rate, and in a complicated system is the only one of which μ is a measure. Hence constant and recurrent values of μ implicate constant, recurrent ami specific chemical reactions.