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The influence of VIIth and VIIIth cranial nerve roots upon the differentiation of Mauthner's cell in Ambystoma
DEVELOPMENTAL
The
BIOLOGY
Influence upon
19,
608-616
of Vllth
the
(1969)
and
VIIIth
Differentiation
Cranial
of Mauthner’s
Nerve Cell
Roots in
Ambystoma’ JEAN PIATT Department
of Anatomy,
of Medicine,
School Philadelphia,
Pennsylvania
Accepted
February
University 19104
of Pennsylvania,
14, 1969
INTRODUCTION
The cells of Mauthner (I\IC) are a pair of giant neurons situated opposite one another in the medulla oblongata of fishes and tailed amphibians. In Ambystoma MC lies between the nucleus motorius tegmenti and the reticular formation at the precise level of entrance of the VIIIth cranial nerve roots (vestibular). The roots of the VIIth lateral-line nerves also enter the medulla at this level but slightly anteriorly (20-50 ,A at stage 46). The large lateral dendrite of NC ramifies extensively throughout the entire field of both VIIth and VIIIth roots, as well as both dorsal and ventral lateral-line tracts of the Xth nerve. The precise and unfailing regularity with which RIG is associated with VIIth and VIIIth nerve roots, particularly the latter, both anatomically and functionally, suggeststhe possibility that differentiation and/or maintenance of the cell may depend to some extent upon the presence of these root fibers during early neurogenesis. The present study is an extension of previous investigations along this line of thought. Experiments done some years ago provide ample evidence that the presence of neither vestibular nor lateral-line roots is always essential for the differentiation of I\IC in Ambystoma (Piatt, 1943, 1947, 1950). Incidental observations by others (Greene, 1924; Detwiler, 1933; Oppenheimer, 1941) and the experiments of Stefanelli (1947a,b) support this conclusion. On the other hand, these same experiments have demonstrated that in roughly one-third of all casesin which vestibular roots are missing, MC is also absent. The effect of lateral-line roots, both VIIth and Xth, upon the differentiation of the cell is lessclear. Further1 This Institutes
research was supported
by
Grant
of Health. 608
1 ROl-NB08112-01
from
the
National
DIFFERENTIATION
OF
MAUTHNER’S
CELL
609
more, a supernumerary MC will occasionally develop in close conjunction with ectopic vestibular root fibers and, to a lesser extent, with ectopic lateral-line roots. It is evident, therefore, that although the presence of vestibular and lateral-line roots is not an indispensable condition for the differentiation and growth of AIC, these roots may in some way influence development of this neuron. In the author’s investigations cited above the data were accumulated from animals operated upon at all ages of embryonic development from stages 21 to 34. Furthermore, many of the embryos had been greatly retarded in their development by prolonged hypothermia prior to removal of the otic vesicle whereas others had not. The data from all groups, however, were lumped together without regard to age at operation or temperature conditions prior to operation. The experiments reported in this paper were designed, therefore, to eliminate the variable of morphological age and, also, to assess the possible effect of differences in chronological age produced by temperature differentials prior to operation. In addition, a much larger number of animals was studied. In all operations it had been intended to eliminate only the vestibular roots, but for some as yet unexplained reason the VIIth lateral-line roots likewise failed to develop in a number of cases, even though VIIth lateral-line ganglia cells were invariably present. Consequently, it has been possible to assess to a greater degree than before the influence of VIIth lateral-line roots upon the differentiation of JIG. The data fall roughly into three categories: (1) influence of vestibular roots upon differentiation of MC, (2) influence of VIIth lateral-line roots upon differentiation of i\IC, (3) influence of preoperative temperature differentials upon the results of categories one and two. PROCEDURE The otic vesicle (primordium of ear and VIIIth ganglion) was extirpated from the right side of Ambystoma maculatum embryos at stage 27. The preauditory ectodermal placode (primordium of VIIth lateralline ganglia) was not excised, but, as already stated, the operation in some way suppressed VIIth lateral-line root development in many cases.All animals were fixed at stage 46. The entire head of each was serially sectioned and impregnated with silver on the slide. Animals were studied for: (1) presence or absence of MC on operated side, (2) presence or absence of vestibular and lateral-line roots. A total of 428 animals I\-as examined. Temperature controls of developing embryos prior to operation were
610
PIATT
carried out as follows. I,arge clutches of eggs were selected and divided into groups of approximately 45 embryos each as soon as the eggs reached the laboratory. Groups from the same clutch were placed in low-temperature incubators kept at different temperatures. Each group was maintained continuously at its respective temperature until stage 27 was reached. Groups were identified by clutch as well as temperature so that possible differences between clutches could be recorded. The temperatures employed were: 5”, S”, lo”, 14”, 18°C. Temperature comparisons have been made chiefly from among the 5”, S”, and 14’ groups. RESULTS
Vestibular root fibers were absent in every one of the 425 animals examined. The VIIIth ganglion was also absent in all but 2 cases. In these 2 cases,only 5 or 6 cells in each case could be positively identified as belonging to the VIIIth ganglion. Mauthner’s cell was absent on the side of operation in 119 animals or 27.8 % of all cases. In 55 of those cases in which MC was missing (46%), absence of the VIIIth roots was the only deviation from the normal condition on the operated side. In 64 casesthe VIIth lateral-line roots were also absent. It should be stated here that in no case were the Xth lateral-line roots absent, and, as would be expected, MC on the non-operated side was always present. These results, therefore, afford conclusive proof that VIIIth root fibers are an important and frequently decisive factor in effecting the differentiation of MC. Since MC developed, despite the absenceof vestibular roots, in almost 75% of the cases,VIIIth root fibers are obviously not the only factor involved. The VIIth lateral-line roots were absent in 173 cases, approximately 40% of the animals. This was an unexpected result. Both dorsolateral and ventrolateral lateral-line ganglia were always present although the number of cells was often reduced in one or the other. Only an extremely small portion of the VIIth lateral-line primordium could have been removed with the extirpation of the ear vesicle, and the presence of lateral-line ganglia in all casesbears this out. Because the VIIIth root was absent in all animals, the data do not indicate whether the VIIth lateral-line roots by themselves have a significant influence on the differentiation of MC but only the effectiveness of this influence when acting in combination with the VIIIth root. It should be borne in mind, therefore, that when the significance of the VIIth lateral-line roots in effecting differentiation of MC is discussed,it refers to the additive effect over and beyond that of the VIIIth root.
DIFFERENTIATION
OF
MAUTHNER’S
611
CELL
When the VIIth lateral-line roots were present, MC differentiated in 200 cases and was absent in 55 cases. When the VIIth roots were missing, I\1C differentiated in 109 cases and was absent in 64 cases. Application of the Chi-square test to these data gives a Chi-square value of 11.461, which is significant at less than the 0.1% level. It is highly improbable, therefore, that these results are due to chance. The presence of VIIth lateral-line fibers (even in the absence of VIIIth root) contributes in some way to the differentiation of .\IC, and the absence of these roots (plus the absence of VIIIth roots) affects adversely differentiation of the cell. The results recorded above with regard to the importance of vestibular and lateral-line roots in effecting the differentiation of MC have included all the data from the entire series, without regard to temperature differentials prior to operation. Table 1 shows the number of cases, together with the percentages, in which MC was absent within each temperature series. The table also indicates the developmental stages at which the groups from different clutches of eggs were placed at their respective temperatures and the hours elapsing before operation. If the stages at which temperature differentials were initially applied are disregarded, there is no apparently significant or close correlation between absence of MC and temperature treatment. Thus, in the three 8” groups, each from a different clutch, 3IC is absent 42.5 %, 28.9 %, and 25.6 %, respectively. T,4BLE
1
SUMMAEY OF THE RIGXTLT~ OF THE I~JFFERISNTIATION THE ABSENCE OF T’IIIth Roow AND J’IIth Cases
40 40 39 40 38 38 38 36 39 40 40
Temperature seriesa I--5” I-8” I-10” I-14” 11-5” 11-8” 11-14” 111-5” 111-B” I\‘-14” 11--w
Stages separated
OF MAUTHNER'S CELL LATERAL-LINE ROOTS
Hours at constant temperature
9 9 9 9 9 9 9 5 5 3 3
792 384 285 192 790 382 190 912 480 216 130
428 a Roman
MC Cases
%
10 17 13 8 6 11 5 13 10 14 12
25.0 42.5 33.3 20.0 15.7 28.9 13.1 36.1 25.0 35.0 30.0
119 numerals
indicate
different
clutches
of eggs.
absent
IN
612
PIATT
Likewise, in the three 14” groups the cell is absent 20.0%, 13.1%, and 35.0%, respectively. If, on the other hand, only those groups that were separated at stage 9 are considered (clutches I and II), there is a better correlation between temperature and absence of AIC. The four groups from clutch I peak at So, with regard to absence of MC. The same is true of the groups from clutch II. Although the absolute number of cases in which MC is absent is consistently greater in the groups of clutch I than in those of clutch II, the relative proportion of cases in which the cell is absent is much the same between equivalent groups. For instance, the percentage of cases in which MC is absent in groups 5’, 8” and 14” in clutch I is 25.0, 42.5, 20.0, respectively, and in clutch II it is 15.7, 28.9, 13.1, respectively. The proportion 25.0:42.5:20.0: :15.7:28.9: 13.1 is remarkably close to the arithmetical expectations. The absolute differences among equivalent groups of these two clutches, therefore, are consistently comparable. Whether the absolute differences indicate genetical differences between the two clutches is, of course, a matter of speculation. It is suggestive, however. The temperature groups of clutches III and IV were initially separated at earlier stages of development than those of clutches I and II. Direct comparison, therefore, between the former and the latter groups is not strictly valid because of this age difference. Equivalent groups of clutches I and II, however, may be considered as single samples for statistical analysis because the treatment was identical in each. The combined 8’ groups of clutches I and II present a total of 78 animals in which MC was absent in 28 cases and present in 50 cases. Similarly, the 14” groups present a total of 78 animals in which JIG was absent in 13 cases and present in 65 cases. The Chi-square value for these data is 6.484, which shows the results to be significant at slightly above the 1% level. It is probable, therefore, that the greater suppression of MC in the 8” groups of clutches I and II as compared to the 14” groups represents a real difference in results. On the other hand, Chi-square values are very low when the 5” groups of clutches I and II are compared with either the 8” or 14” groups. If the above conclusions are valid, the influence of VIIth and VIIIth roots upon the differentiation of MC does not necessarily increase in linear progression with chronological age (lower temperatures) but is greater at 8°C than at temperatures either above or below this. DISCUSSION
Mauthner’s cell is closely related anatomically to a number of intramedullary structures and tracts in addition to its relationship to VIIth
DlFFERENTIATION
OF
MAUTHNER’S
CELL
613
and VIIIth cranial nerve roots, and its functional connections are many and varied. Differentiation of the cell, therefore, is undoubtedly dependent upon other factors than the presence of VIIIth and/or VIIth lateral-line roots. It is not surprising that JIG still develops in approximately 75 97,of the cases in the absence of these root fibers. The factors influencing the differentiation of 1\lC are apparently in delicate balance during development and suppression of one or several of these influences is sufficient in many cases to upset this balance, causing suppression of the cell itself. It is interesting to note in this cormection that almost without exception the differentiation of MC is an “all-or-none” phenomenon. When the cell does differentiate in the absence of vestibular root fibers, it is as large and extensive as on the normal side. Actual measurements have shown this to be the case in Ambystonza (I’iatt, 1950). However, this may not be true for the Amphibia as a whole since in Runa Pesetsky (1960) found that unilateral extirpation of the “acustico lateralis rudiments” resulted in a significantly smaller nucleus in JIG on the side of operation. The nature of the influence exerted by vestibular or lateral-line roots on the differentiation of the cell is quite obscure. Vestibular fibers do not begin to enter the medulla until stage 32 or slightly later (Coghill, 1916), so whatever effect they may have carmot be operative before this time in development. With ordinary histological techniques, ;\IC carmot be definitely identified until about stages 37 or 38. It is possible, therefore, that it is in this period of development between stages 32 and 38 that vestibular root fibers exert their influence upon the differentiation of the cell. There is some evidence, but of a most indecisive character, that -\IC is “determined” as early as the gastrula (Holtfreter, 1931) or medullarg plate (l’iatt, 194.5) stages or by stages 21-23 at the latest (Detwiler, 1944). If JIG, then, is continuously affected by early developmental relations, it is possible that ingrowing root fibers of vestibular or lateral-line nerves afford a final impetus to a series of events which in themselves arc not always decisive. The present study has demonstrated that suppression of L\IC occurs more frequently in the absence of both VIIth and VIIIth roots than in the absence of VIIIth fibers alone. This would imply that the sequential steps in the differentiation of >IC from neuroblast to neuron have a cumulative effect. The manner in which this final influence is implemented, however, is not known. The data derived from comparing the different temperature groups with regard to relative frequency of i\IC suppression are inconclusive. They suggest, however, that there may be a causal connection between
614
PIATT
the speed of embryonic development and its effect upon the sequential factors influencing the differentiation of the cell. It should be made particularly clear that neither hypo- nor hyperthermia by itself affects in any discernible way the development of the cell; the cell is always present on the unoperated side. If the difference between the 8“ and 14” groups separated at stage 9 is not chance variation, then slower development at 8” in some way affects the normal timing of MC differentiation so that VIIIth or VIIth nerve roots are more essential for the completion of the differentiation process than in the 14” groups which developed faster. The fact that no direct linear relationship obtained between length of developmental time and suppression of MC does not alter this proposition. Ogawa (1961) has shown that whereas actin formation is increasingly suppressed with lowering of temperature in the development of salamander embryos, myosin production is strikingly suppressed at 18°C and is clearly promoted at either higher or lower temperatures. Voitkevish (1962) has likewise shown that in anuran development different physiological processes are influenced in opposite directions by heat and cold. It is interesting to mention, too, that the differentiation of irradiated ear grafts is markedly more affected at a continuous temperature of 8°C than at either 14” or 20” (Piatt, 1968). Further speculation is fruitless at this point, however, and more research is needed before valid conclusions may be drawn. SUMMARY
The primordium of the VIIIth cranial ganglion (otic vesicle) was extirpated on the right side of 428 embryos of Ambystoma lnaculatu?n at stage 27. hlauthner’s cell (MC) develops in close association with the VIIIth nerve roots and to a lesser degree with VIIth lateral-line roots. The primary objective of the experiments was to record from a large seriesof animals the number of casesin which MC failed to differentiate in the absence of VIIIth root fibers. In addition, all embryos were separated (stages 3, 5, 9) into various groups and maintained continuously at different constant temperatures (5”, 8”, lo”, 14”, 18°C) until operation. The various temperature groups were then compared among themselves for any constant or significant differences with regard to the suppression of MC on the operated side. Animals were examined at stage 46. The VIIIth root fibers were completely absent in every case on the operated side. Although the VIIth lateral-line ganglia were present in all animals, root fibers failed to develop in 173 cases.This afforded an
DIFFERENTIATION
OF
MAUTHNER’S
CELL
615
opportunity to study the influence of VIIth lateral-line roots upon the differentiation of AJC, as well as the influence of VIIIth roots. Mauthner’s cell was absent in 119 cases (27.8 %). In 55 of these cases only VIIIth root fibers were absent; in 64 cases both VIIIth and VIIth roots were missing. The importance of VIIIth root fibers in influencing the differentiation of NC, therefore, is obvious since in 46 % of those cases in which 1\C failed to develop absence of the VIIIth root was the only deviation from normal. The presence or absence of MC correlated with the presence or absence of VIIth lateral-line roots gives a relatively high Chi-square value of 11.461. The temperature series data are inconclusive in demonstrating a critical or optimal thermal effect upon the sequential steps affecting the differentiation of MC. If the time in early development when temperature differentials were applied is ignored, there are no consistent or significant differences among the temperature groups with regard to the suppressionof ?rlC in the absenceof the VIIth-VIIIth root complex. On the other hand, if only those animals are considered that were separated at stage 9 (the majority of the animals), then there is a low degree of correlation (significance slightly above the 1% level) between greater suppression of MC in the S” groups as opposed to lessersuppression in the 14” groups. These results on temperature influence are in need of further experimentation and analysis. REFERENCES COGHILL, G. E. (1916). Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. II. The afferent system of the head of Amblysloma. J. Comp. iVeuro1. 26, 247-340. DETWILER, S. R. (1933). Further experiments upon the extirpation of Mauthner’s neurones in amphibian embryos (dmblystoma me.zicanunr). J. Ezpfl. 2001. 64, 415-431. DETWILER, S. R. (1944). Restitution of the medulla following unilateral excision in the embryo. J. Exptl. 2001. 96, 129-142. GRKENE, W. F. (1924). The effect of the removal of the embryonic ear vesicle upon amphibian larvae. Doctor’s thesis, Yale University, New Haven, Connecticut. Unpublished. HOLTFRI~GTER, J. (1931). Potenzpriifungen am Amphibienkeim mit Hilfe der Isolationsmethode. Verhandl. Deut. Ges. Zool. (Zool. Bnz.), Suppl. 6, 15&165. OGAWA, Y. (1961). Influence of temperature on the synthesis of contractile proteins in Triturus embryo. dnn. Rept. Natl. Inst. Genet. 12, l-2. OPPENHEIMER, J. RI. (1941). The anatomical relationships of abnormally located Mauthner’s cells in Fundulus embryos. 1. Comp. Neural. 74, 131-167. PESETSKY, I. (1960). hIaintenance and regression of Mauthner’s neuron in larval Rana pipiens. Anat. Record 136, 257.
616 PIATT, J. (1943).
PIATT
The course and decussation of ectopic Mauthner’s fibers in Amblystoma punctatum. J. Comp. Neural. 79, 165-183. PIATT, J. (1945). Origin of the mesencephalic V root cells in Amblystoma. J. Comp. Neural. 83, 35-53. PIATT, J. (1947). A study of the factors controlling the differentiation of Mauthner’s cell in Amblystoma. J. Comp. Neural. 86, 199-235. PIATT, J. (1950). Further studies on the differentiation and growth of Mauthner’s cell in Amblystoma. J. Exptl. 2001. 113, 379-395. PIATT, J. (1968). Dissociability of developmental processes in the morphogenesis of the ear in Amblystoma, as revealed by x-ray damage. Anat. Record 160, 143151. STEFANELLI, A. (1947a). La determinazione istologica ed il differenziamento della cellula nervosa (di Mauthner) indagati co1 metodo degli espianti. Ric. Morfologia 22, l-15. STEFANELLI, A. (1947b). I fenomeni della determinazione della rigenerazione e de1 differenziamento de1 sistema nervoso. Atti. Accad. Nazi. Lincei, Mem. Classe Sci. Fis. Mat. Nat. Ser. III, 1, 27-114. VOITKEVICH, A. A. (1962). The effect of temperature on the function of the preoptic nuclei in the amphibian hypophysis. Bull. Exptl. Biol. Med. (USSR) (English Trans.) 63, 599-602.
The
BIOLOGY
Influence upon
19,
608-616
of Vllth
the
(1969)
and
VIIIth
Differentiation
Cranial
of Mauthner’s
Nerve Cell
Roots in
Ambystoma’ JEAN PIATT Department
of Anatomy,
of Medicine,
School Philadelphia,
Pennsylvania
Accepted
February
University 19104
of Pennsylvania,
14, 1969
INTRODUCTION
The cells of Mauthner (I\IC) are a pair of giant neurons situated opposite one another in the medulla oblongata of fishes and tailed amphibians. In Ambystoma MC lies between the nucleus motorius tegmenti and the reticular formation at the precise level of entrance of the VIIIth cranial nerve roots (vestibular). The roots of the VIIth lateral-line nerves also enter the medulla at this level but slightly anteriorly (20-50 ,A at stage 46). The large lateral dendrite of NC ramifies extensively throughout the entire field of both VIIth and VIIIth roots, as well as both dorsal and ventral lateral-line tracts of the Xth nerve. The precise and unfailing regularity with which RIG is associated with VIIth and VIIIth nerve roots, particularly the latter, both anatomically and functionally, suggeststhe possibility that differentiation and/or maintenance of the cell may depend to some extent upon the presence of these root fibers during early neurogenesis. The present study is an extension of previous investigations along this line of thought. Experiments done some years ago provide ample evidence that the presence of neither vestibular nor lateral-line roots is always essential for the differentiation of I\IC in Ambystoma (Piatt, 1943, 1947, 1950). Incidental observations by others (Greene, 1924; Detwiler, 1933; Oppenheimer, 1941) and the experiments of Stefanelli (1947a,b) support this conclusion. On the other hand, these same experiments have demonstrated that in roughly one-third of all casesin which vestibular roots are missing, MC is also absent. The effect of lateral-line roots, both VIIth and Xth, upon the differentiation of the cell is lessclear. Further1 This Institutes
research was supported
by
Grant
of Health. 608
1 ROl-NB08112-01
from
the
National
DIFFERENTIATION
OF
MAUTHNER’S
CELL
609
more, a supernumerary MC will occasionally develop in close conjunction with ectopic vestibular root fibers and, to a lesser extent, with ectopic lateral-line roots. It is evident, therefore, that although the presence of vestibular and lateral-line roots is not an indispensable condition for the differentiation and growth of AIC, these roots may in some way influence development of this neuron. In the author’s investigations cited above the data were accumulated from animals operated upon at all ages of embryonic development from stages 21 to 34. Furthermore, many of the embryos had been greatly retarded in their development by prolonged hypothermia prior to removal of the otic vesicle whereas others had not. The data from all groups, however, were lumped together without regard to age at operation or temperature conditions prior to operation. The experiments reported in this paper were designed, therefore, to eliminate the variable of morphological age and, also, to assess the possible effect of differences in chronological age produced by temperature differentials prior to operation. In addition, a much larger number of animals was studied. In all operations it had been intended to eliminate only the vestibular roots, but for some as yet unexplained reason the VIIth lateral-line roots likewise failed to develop in a number of cases, even though VIIth lateral-line ganglia cells were invariably present. Consequently, it has been possible to assess to a greater degree than before the influence of VIIth lateral-line roots upon the differentiation of JIG. The data fall roughly into three categories: (1) influence of vestibular roots upon differentiation of MC, (2) influence of VIIth lateral-line roots upon differentiation of i\IC, (3) influence of preoperative temperature differentials upon the results of categories one and two. PROCEDURE The otic vesicle (primordium of ear and VIIIth ganglion) was extirpated from the right side of Ambystoma maculatum embryos at stage 27. The preauditory ectodermal placode (primordium of VIIth lateralline ganglia) was not excised, but, as already stated, the operation in some way suppressed VIIth lateral-line root development in many cases.All animals were fixed at stage 46. The entire head of each was serially sectioned and impregnated with silver on the slide. Animals were studied for: (1) presence or absence of MC on operated side, (2) presence or absence of vestibular and lateral-line roots. A total of 428 animals I\-as examined. Temperature controls of developing embryos prior to operation were
610
PIATT
carried out as follows. I,arge clutches of eggs were selected and divided into groups of approximately 45 embryos each as soon as the eggs reached the laboratory. Groups from the same clutch were placed in low-temperature incubators kept at different temperatures. Each group was maintained continuously at its respective temperature until stage 27 was reached. Groups were identified by clutch as well as temperature so that possible differences between clutches could be recorded. The temperatures employed were: 5”, S”, lo”, 14”, 18°C. Temperature comparisons have been made chiefly from among the 5”, S”, and 14’ groups. RESULTS
Vestibular root fibers were absent in every one of the 425 animals examined. The VIIIth ganglion was also absent in all but 2 cases. In these 2 cases,only 5 or 6 cells in each case could be positively identified as belonging to the VIIIth ganglion. Mauthner’s cell was absent on the side of operation in 119 animals or 27.8 % of all cases. In 55 of those cases in which MC was missing (46%), absence of the VIIIth roots was the only deviation from the normal condition on the operated side. In 64 casesthe VIIth lateral-line roots were also absent. It should be stated here that in no case were the Xth lateral-line roots absent, and, as would be expected, MC on the non-operated side was always present. These results, therefore, afford conclusive proof that VIIIth root fibers are an important and frequently decisive factor in effecting the differentiation of MC. Since MC developed, despite the absenceof vestibular roots, in almost 75% of the cases,VIIIth root fibers are obviously not the only factor involved. The VIIth lateral-line roots were absent in 173 cases, approximately 40% of the animals. This was an unexpected result. Both dorsolateral and ventrolateral lateral-line ganglia were always present although the number of cells was often reduced in one or the other. Only an extremely small portion of the VIIth lateral-line primordium could have been removed with the extirpation of the ear vesicle, and the presence of lateral-line ganglia in all casesbears this out. Because the VIIIth root was absent in all animals, the data do not indicate whether the VIIth lateral-line roots by themselves have a significant influence on the differentiation of MC but only the effectiveness of this influence when acting in combination with the VIIIth root. It should be borne in mind, therefore, that when the significance of the VIIth lateral-line roots in effecting differentiation of MC is discussed,it refers to the additive effect over and beyond that of the VIIIth root.
DIFFERENTIATION
OF
MAUTHNER’S
611
CELL
When the VIIth lateral-line roots were present, MC differentiated in 200 cases and was absent in 55 cases. When the VIIth roots were missing, I\1C differentiated in 109 cases and was absent in 64 cases. Application of the Chi-square test to these data gives a Chi-square value of 11.461, which is significant at less than the 0.1% level. It is highly improbable, therefore, that these results are due to chance. The presence of VIIth lateral-line fibers (even in the absence of VIIIth root) contributes in some way to the differentiation of .\IC, and the absence of these roots (plus the absence of VIIIth roots) affects adversely differentiation of the cell. The results recorded above with regard to the importance of vestibular and lateral-line roots in effecting the differentiation of MC have included all the data from the entire series, without regard to temperature differentials prior to operation. Table 1 shows the number of cases, together with the percentages, in which MC was absent within each temperature series. The table also indicates the developmental stages at which the groups from different clutches of eggs were placed at their respective temperatures and the hours elapsing before operation. If the stages at which temperature differentials were initially applied are disregarded, there is no apparently significant or close correlation between absence of MC and temperature treatment. Thus, in the three 8” groups, each from a different clutch, 3IC is absent 42.5 %, 28.9 %, and 25.6 %, respectively. T,4BLE
1
SUMMAEY OF THE RIGXTLT~ OF THE I~JFFERISNTIATION THE ABSENCE OF T’IIIth Roow AND J’IIth Cases
40 40 39 40 38 38 38 36 39 40 40
Temperature seriesa I--5” I-8” I-10” I-14” 11-5” 11-8” 11-14” 111-5” 111-B” I\‘-14” 11--w
Stages separated
OF MAUTHNER'S CELL LATERAL-LINE ROOTS
Hours at constant temperature
9 9 9 9 9 9 9 5 5 3 3
792 384 285 192 790 382 190 912 480 216 130
428 a Roman
MC Cases
%
10 17 13 8 6 11 5 13 10 14 12
25.0 42.5 33.3 20.0 15.7 28.9 13.1 36.1 25.0 35.0 30.0
119 numerals
indicate
different
clutches
of eggs.
absent
IN
612
PIATT
Likewise, in the three 14” groups the cell is absent 20.0%, 13.1%, and 35.0%, respectively. If, on the other hand, only those groups that were separated at stage 9 are considered (clutches I and II), there is a better correlation between temperature and absence of AIC. The four groups from clutch I peak at So, with regard to absence of MC. The same is true of the groups from clutch II. Although the absolute number of cases in which MC is absent is consistently greater in the groups of clutch I than in those of clutch II, the relative proportion of cases in which the cell is absent is much the same between equivalent groups. For instance, the percentage of cases in which MC is absent in groups 5’, 8” and 14” in clutch I is 25.0, 42.5, 20.0, respectively, and in clutch II it is 15.7, 28.9, 13.1, respectively. The proportion 25.0:42.5:20.0: :15.7:28.9: 13.1 is remarkably close to the arithmetical expectations. The absolute differences among equivalent groups of these two clutches, therefore, are consistently comparable. Whether the absolute differences indicate genetical differences between the two clutches is, of course, a matter of speculation. It is suggestive, however. The temperature groups of clutches III and IV were initially separated at earlier stages of development than those of clutches I and II. Direct comparison, therefore, between the former and the latter groups is not strictly valid because of this age difference. Equivalent groups of clutches I and II, however, may be considered as single samples for statistical analysis because the treatment was identical in each. The combined 8’ groups of clutches I and II present a total of 78 animals in which MC was absent in 28 cases and present in 50 cases. Similarly, the 14” groups present a total of 78 animals in which JIG was absent in 13 cases and present in 65 cases. The Chi-square value for these data is 6.484, which shows the results to be significant at slightly above the 1% level. It is probable, therefore, that the greater suppression of MC in the 8” groups of clutches I and II as compared to the 14” groups represents a real difference in results. On the other hand, Chi-square values are very low when the 5” groups of clutches I and II are compared with either the 8” or 14” groups. If the above conclusions are valid, the influence of VIIth and VIIIth roots upon the differentiation of MC does not necessarily increase in linear progression with chronological age (lower temperatures) but is greater at 8°C than at temperatures either above or below this. DISCUSSION
Mauthner’s cell is closely related anatomically to a number of intramedullary structures and tracts in addition to its relationship to VIIth
DlFFERENTIATION
OF
MAUTHNER’S
CELL
613
and VIIIth cranial nerve roots, and its functional connections are many and varied. Differentiation of the cell, therefore, is undoubtedly dependent upon other factors than the presence of VIIIth and/or VIIth lateral-line roots. It is not surprising that JIG still develops in approximately 75 97,of the cases in the absence of these root fibers. The factors influencing the differentiation of 1\lC are apparently in delicate balance during development and suppression of one or several of these influences is sufficient in many cases to upset this balance, causing suppression of the cell itself. It is interesting to note in this cormection that almost without exception the differentiation of MC is an “all-or-none” phenomenon. When the cell does differentiate in the absence of vestibular root fibers, it is as large and extensive as on the normal side. Actual measurements have shown this to be the case in Ambystonza (I’iatt, 1950). However, this may not be true for the Amphibia as a whole since in Runa Pesetsky (1960) found that unilateral extirpation of the “acustico lateralis rudiments” resulted in a significantly smaller nucleus in JIG on the side of operation. The nature of the influence exerted by vestibular or lateral-line roots on the differentiation of the cell is quite obscure. Vestibular fibers do not begin to enter the medulla until stage 32 or slightly later (Coghill, 1916), so whatever effect they may have carmot be operative before this time in development. With ordinary histological techniques, ;\IC carmot be definitely identified until about stages 37 or 38. It is possible, therefore, that it is in this period of development between stages 32 and 38 that vestibular root fibers exert their influence upon the differentiation of the cell. There is some evidence, but of a most indecisive character, that -\IC is “determined” as early as the gastrula (Holtfreter, 1931) or medullarg plate (l’iatt, 194.5) stages or by stages 21-23 at the latest (Detwiler, 1944). If JIG, then, is continuously affected by early developmental relations, it is possible that ingrowing root fibers of vestibular or lateral-line nerves afford a final impetus to a series of events which in themselves arc not always decisive. The present study has demonstrated that suppression of L\IC occurs more frequently in the absence of both VIIth and VIIIth roots than in the absence of VIIIth fibers alone. This would imply that the sequential steps in the differentiation of >IC from neuroblast to neuron have a cumulative effect. The manner in which this final influence is implemented, however, is not known. The data derived from comparing the different temperature groups with regard to relative frequency of i\IC suppression are inconclusive. They suggest, however, that there may be a causal connection between
614
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the speed of embryonic development and its effect upon the sequential factors influencing the differentiation of the cell. It should be made particularly clear that neither hypo- nor hyperthermia by itself affects in any discernible way the development of the cell; the cell is always present on the unoperated side. If the difference between the 8“ and 14” groups separated at stage 9 is not chance variation, then slower development at 8” in some way affects the normal timing of MC differentiation so that VIIIth or VIIth nerve roots are more essential for the completion of the differentiation process than in the 14” groups which developed faster. The fact that no direct linear relationship obtained between length of developmental time and suppression of MC does not alter this proposition. Ogawa (1961) has shown that whereas actin formation is increasingly suppressed with lowering of temperature in the development of salamander embryos, myosin production is strikingly suppressed at 18°C and is clearly promoted at either higher or lower temperatures. Voitkevish (1962) has likewise shown that in anuran development different physiological processes are influenced in opposite directions by heat and cold. It is interesting to mention, too, that the differentiation of irradiated ear grafts is markedly more affected at a continuous temperature of 8°C than at either 14” or 20” (Piatt, 1968). Further speculation is fruitless at this point, however, and more research is needed before valid conclusions may be drawn. SUMMARY
The primordium of the VIIIth cranial ganglion (otic vesicle) was extirpated on the right side of 428 embryos of Ambystoma lnaculatu?n at stage 27. hlauthner’s cell (MC) develops in close association with the VIIIth nerve roots and to a lesser degree with VIIth lateral-line roots. The primary objective of the experiments was to record from a large seriesof animals the number of casesin which MC failed to differentiate in the absence of VIIIth root fibers. In addition, all embryos were separated (stages 3, 5, 9) into various groups and maintained continuously at different constant temperatures (5”, 8”, lo”, 14”, 18°C) until operation. The various temperature groups were then compared among themselves for any constant or significant differences with regard to the suppression of MC on the operated side. Animals were examined at stage 46. The VIIIth root fibers were completely absent in every case on the operated side. Although the VIIth lateral-line ganglia were present in all animals, root fibers failed to develop in 173 cases.This afforded an
DIFFERENTIATION
OF
MAUTHNER’S
CELL
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opportunity to study the influence of VIIth lateral-line roots upon the differentiation of AJC, as well as the influence of VIIIth roots. Mauthner’s cell was absent in 119 cases (27.8 %). In 55 of these cases only VIIIth root fibers were absent; in 64 cases both VIIIth and VIIth roots were missing. The importance of VIIIth root fibers in influencing the differentiation of NC, therefore, is obvious since in 46 % of those cases in which 1\C failed to develop absence of the VIIIth root was the only deviation from normal. The presence or absence of MC correlated with the presence or absence of VIIth lateral-line roots gives a relatively high Chi-square value of 11.461. The temperature series data are inconclusive in demonstrating a critical or optimal thermal effect upon the sequential steps affecting the differentiation of MC. If the time in early development when temperature differentials were applied is ignored, there are no consistent or significant differences among the temperature groups with regard to the suppressionof ?rlC in the absenceof the VIIth-VIIIth root complex. On the other hand, if only those animals are considered that were separated at stage 9 (the majority of the animals), then there is a low degree of correlation (significance slightly above the 1% level) between greater suppression of MC in the S” groups as opposed to lessersuppression in the 14” groups. These results on temperature influence are in need of further experimentation and analysis. REFERENCES COGHILL, G. E. (1916). Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. II. The afferent system of the head of Amblysloma. J. Comp. iVeuro1. 26, 247-340. DETWILER, S. R. (1933). Further experiments upon the extirpation of Mauthner’s neurones in amphibian embryos (dmblystoma me.zicanunr). J. Ezpfl. 2001. 64, 415-431. DETWILER, S. R. (1944). Restitution of the medulla following unilateral excision in the embryo. J. Exptl. 2001. 96, 129-142. GRKENE, W. F. (1924). The effect of the removal of the embryonic ear vesicle upon amphibian larvae. Doctor’s thesis, Yale University, New Haven, Connecticut. Unpublished. HOLTFRI~GTER, J. (1931). Potenzpriifungen am Amphibienkeim mit Hilfe der Isolationsmethode. Verhandl. Deut. Ges. Zool. (Zool. Bnz.), Suppl. 6, 15&165. OGAWA, Y. (1961). Influence of temperature on the synthesis of contractile proteins in Triturus embryo. dnn. Rept. Natl. Inst. Genet. 12, l-2. OPPENHEIMER, J. RI. (1941). The anatomical relationships of abnormally located Mauthner’s cells in Fundulus embryos. 1. Comp. Neural. 74, 131-167. PESETSKY, I. (1960). hIaintenance and regression of Mauthner’s neuron in larval Rana pipiens. Anat. Record 136, 257.
616 PIATT, J. (1943).
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The course and decussation of ectopic Mauthner’s fibers in Amblystoma punctatum. J. Comp. Neural. 79, 165-183. PIATT, J. (1945). Origin of the mesencephalic V root cells in Amblystoma. J. Comp. Neural. 83, 35-53. PIATT, J. (1947). A study of the factors controlling the differentiation of Mauthner’s cell in Amblystoma. J. Comp. Neural. 86, 199-235. PIATT, J. (1950). Further studies on the differentiation and growth of Mauthner’s cell in Amblystoma. J. Exptl. 2001. 113, 379-395. PIATT, J. (1968). Dissociability of developmental processes in the morphogenesis of the ear in Amblystoma, as revealed by x-ray damage. Anat. Record 160, 143151. STEFANELLI, A. (1947a). La determinazione istologica ed il differenziamento della cellula nervosa (di Mauthner) indagati co1 metodo degli espianti. Ric. Morfologia 22, l-15. STEFANELLI, A. (1947b). I fenomeni della determinazione della rigenerazione e de1 differenziamento de1 sistema nervoso. Atti. Accad. Nazi. Lincei, Mem. Classe Sci. Fis. Mat. Nat. Ser. III, 1, 27-114. VOITKEVICH, A. A. (1962). The effect of temperature on the function of the preoptic nuclei in the amphibian hypophysis. Bull. Exptl. Biol. Med. (USSR) (English Trans.) 63, 599-602.