The effect of ablation of the olfactory pits on the development of the habenular nuclei in Rana pipiens

Transcription

1 /. Embryol. exp. Morph. Vol 34, 3, pp , Printed in Great Britain The effect of ablation of the olfactory pits on the development of the habenular nuclei in Rana pipiens By BYRON K. NORRIS 1 AND VICTOR B. EICHLER 1 From the Department of Biology, Wichita State University, Kansas SUMMARY The habenular nuclei in the diencephalon of the frog, Rana pipiens, are asymmetrical structures: two discrete cell groups develop on the left side (as medial and lateral nuclei), while a single nucleus is formed on the right side. Experimental animals were subjected to bilateral removal of the olfactory pits at an early embryonic stage, and were maintained with normal control animals until metamorphosis was complete. The length, relative volume and cell number for each of the three nuclei were determined in the control and experimental animals at regular intervals during larval development. In the control animals, the left medial nucleus developed similarly to the right nucleus spatially and temporally; however, the left lateral nucleus was significantly different in its development in the three parameters measured. In the experimental animals the left medial and the right habenular nuclei were alone affected by the removal of the olfactory pits. The results provide experimental evidence that the right and left medial, but not the left lateral, habenular nuclei are centers receiving afferent olfactory fibers. INTRODUCTION The habenular nuclei in the brain of vertebrates are small clusters of cells located in the anterior dorsal diencephalon, at the level of the epiphysis. The nuclei appear as elongated oval structures with compact neural cell bodies surrounding a relatively cell-free interior. In amniote vertebrates these groups of nerve cell bodies are symmetrically located on either side of the third ventricle; however, many anamniotes exhibit a left-right asymmetry. The first report of the existence of this asymmetry appears to have been by Goronowitsch (1833), who described the asymmetry in the habenula of the sturgeon. In this species of fish, the right nucleus was found to be strikingly larger than that of the left side. Gaupp (1896) published his findings on the asymmetrical nature of the frog's habenulae, and showed that on the right side there is a single nucleus, while the left side has a large medial nucleus and a much smaller lateral nucleus. Within the next five years several reports appeared indicating that a left-right asymmetry existed also in the habenular nuclei of a variety of cyclostomes and bony fishes. 1 Authors' address: Department of Biology, Wichita State University, Wichita, Kansas 6728, U.S.A. Requests for reprints should be sent to Dr Eichler.

2 658 B. K. NORRIS AND V. B. EICHLER However, the findings of an anatomical asymmetry in the vertebrate brain were generally ignored during the first half of the present century, for it was generally accepted that the vertebrate brain was composed of mirror image halves. The existence of asymmetries in the brain of amphibians, reptiles, birds and mammals was even specifically denied during this time in books authored by such eminent investigators as Kappers, Huber & Crosby (1936) and Beccari (1943). Beginning in the early 195s, at the time Frontera (1952) published his very detailed account of the anuran diencephalon, there had been little attention paid to the interesting asymmetry present, in its development or its fiber connections. But in the last five years, several investigators have noted in independent studies (Braitenberg & Kemali, 197; Morgan, O'Donnell & Oliver, 1971; Eichler & Norris, 1974) that in the frog brain there are indeed two distinct cell groups on the left side, while in the corresponding position on the right side there is only a single group of cells. The present communication reports our findings relative to two interesting, but unanswered questions. We have asked, first, what is the pattern of development of this asymmetry in anuran amphibians? That is, what comparisons can be made temporally and spatially in the normal development of the cell group on the right side with each of the two cell groups on the left side? Secondly, we asked what the nature of the neural input is to these nuclei. We wondered if functionally, do both of the nuclei on the left side have the same neural input as that on the right side and, if not, which of the left nuclei is functionally analogous to the right one? MATERIALS AND METHODS Eggs were obtained from adult Rana pipiens females by induced ovulation and subsequent fertilization in the laboratory following the method of Rugh (1934). Five days post-fertilization the olfactory pits of the experimental frog embryos were surgically extirpated using fine glass needles and hair loops, under a dissecting microscope. Approximately one hundred and fifty embryos were operated on in full-strength Holtfreter's solution, and they were kept in this solution for 2-3 h following the operation in order to promote wound healing. Groups of ten embryos were kept in glass finger bowls containing 1 % Holtfreter's solution for 1 or 2 post-operative days and then were transferred to plastic dish pans (3 x 33 x 15 cm) which held two liters aerated tap water. All animals were maintained in uncrowded condition at room temperature and were exposed to approximately equal numbers of light and dark hours. On the eleventh day washed, canned spinach was placed into the pans so food was available ad libitum. Food and water were changed daily. Ten control and ten experimental animals were sacrificed at each of the following development stages of Taylor & Kollros (1946): stage I, V, X, XV,

3 Development of frog habenular nuclei 659 XX and XXV, which correspond roughly to ages of 1, 2, 4, 6, 8 and 1 days, respectively. At stage XXV the animals have completed metamorphosis: all four limbs have emerged, the tail is completely resorbed, and the animal is ready to emerge from the water onto land. The heads of the younger animals (stages I and V) were removed from the body and placed in Bouin's fixative for 2 days. The heads of the older larvae were trimmed by removing the lower jaw, and only the dorsal part of the head was placed in the fixative. The tissues were dehydrated in alcohols, cleared in methyl salicylate followed by xylene, and embedded in paraffin. Serial sections, cut at 1 /.im, were stained with Delafield's hematoxylin and counterstained with eosin. The length of each habenular nucleus was determined by counting the number of sections containing each one, and this number was multiplied by the section thickness. The relative volume of each nucleus was determined by the method of 'paired comparisons', where an image of each section of the right, left medial, and left lateral habenular nucleus was projected at a magnification of x 165, the outline of the projected image was carefully traced onto bond paper, and each tracing (representing one 1 /m\ thick section through one of the habenular nuclei) was then cut out. The combined weights of the tracings representing each of the three nuclei at the various developmental stages, when compared, gave a measure of the relative volumes of the respective structures. The total number of cells in each nucleus were determined by counting the cells in ten representative non-adjacent sections in every animal except the two youngest stages, where only the nuclei in five non-adjacent sections were counted. The cells around the perimeter and contained within the lumen of each nucleus were counted at a magnification of x 1 using a hand tally counter. The average of cells contained in ten sections was multiplied by the total number of sections in the specific nucleus to give an estimation of the total cell population. A correction for cell estimates was determined following the method of Ebbesson & Tang (1965). The statistical comparison of the length, relative volume, and total number of cells present in each of the three habenular nuclei of the control and experimental animals is presented in Table 1. RESULTS A. Length of habenular nuclei Fig. 1 compares the length of the right, left medial and left lateral nuclei. In Fig. 1A it can be seen that the left lateral nucleus develops later than the left medial nucleus; it is not present at either stages I or V. Also, the left medial nucleus is at all stages significantly longer than the left lateral nucleus. There is a dramatic increase in the length of the control and experimental left medial nucleus during stages I through XV. At stages XV through XX there is 42 E M B 34

4 66 B. K. NORRIS AND V. B. EICHLER Larval stage Table 1. Comparisons of length, relative volume, and number of cells in Length /tm Right c e medial e lateral e Relative volume Right c e medial e lateral e Cell number Right c e medial e lateral e each of the habenular nuclei of control and experimental animals^ I V X XV * XX * -9-7* * c, control animals; e, experimental animals. * P < 5, Student's /-test; ** P < 1, Student's Mest. f Each value represents the mean score for ten animals. Left B Right XXV f ** * ** ** Control ** ** Experimental V X XV XX XXV V X XV XX XXV Fig. 1. Length of the habenular nuclei of control and experimental animals; A, left medial and left lateral nuclei; B, right nucleus.

5 Development of frog habenular nuclei 661 a leveling off of the rate of development of the experimental left medial nucleus compared to the control animals increase. At stages XX-XXV there is a significant decrease in the length of the experimental left medial nucleus while the length of the control medial nucleus continues to increase. In Fig. 1A the lengths of the left lateral nuclei in experimental and control animals are also compared. Animals in both experimental and control groups exhibit no left lateral nucleus at stages I or V. At all stages examined thereafter, the left lateral nucleus was found to be present. Its development, however, is significantly reduced in comparison to the medial nucleus of the left side. The length of the left lateral nucleus of the experimental animals is not significantly different from the length of the left lateral nucleus of the control animals at any of the stages examined. The length of the control and experimental left lateral nucleus increases between stages V and XV. At stages XV through XXV the length of the left lateral nucleus in larvae of both the experimental and control groups neither increases nor decreases significantly as is observed in the left medial nucleus. In Fig. IB the length of the right nucleus in the experimental and control animals is compared. The length of the control and experimental right nucleus greatly increases between stages I through XV. Between stages XV to XX there is a leveling off of the rate of development of the experimental right nucleus in terms of length as compared to the continued increase in length of the control right nucleus. Between stages XX and XXV in the experimental group of larvae there is a significant decrease in the length of the right nucleus comparable to that found in the left medial nucleus of the same animals, while the right nucleus in the control animals increases in length throughout development similar to the medial nucleus on the left side of these animals. A similarity clearly exists in the development of the left medial and right nuclei of larvae in both the control and experimental groups. Also, only the left medial and the right nuclei are affected by the ablation of the olfactory pits as shown by the decrease in the length of these cell groups in later developmental stages. The development of the lateral nucleus begins later, it develops slower and remains smaller than the other two habenular nuclei; it also is not significantly affected by extirpation of the olfactory pits at any of the larval stages studied. These features clearly set the left lateral nucleus apart from the left medial and the right nucleus. B. Relative volume of habenular nuclei The relative volumes of each of the three nuclei were determined at the six selected larval stages in both the experimental and control groups. The results are compared in Fig. 2 A for the left medial and the left lateral nuclei. The relative volumes of the left medial nuclei of animals in the control and experimental groups show a great increase between stages I through XV. The relative volume of the left medial nucleus is less at all stages examined in the 42-2

6 662 B. K. NORRIS AND V. B. EICHLER experimental animals when compared to the unoperated ones, but the difference is only of statistical significance at stages XX and XXV, the same stages where the decrease in the length was observed. The relative volume of the left lateral nucleus of the animals in the experimental and control groups is not significantly different at any larval stage examined, but at every stage examined the lateral nucleus is significantly smaller than the medial nucleus. Right V X XV XX XXV V X XV XX XXV Fig. 2. Relative volume of the habenular nuclei of control and experimental animals: A, left medial and left lateral nuclei; B, right nucleus. Fig. 2B compares the relative volumes of the right nucleus in control and experimental animals. The relative volume of this nucleus increases in both groups between stages I and XV. The volume difference in this nucleus between the two groups becomes significant by larval stage XV. This reduction in volume reflects the decrease in the length observed in the right nucleus of the experimental animals. The development of the left medial and the right nucleus in larvae of both the experimental and control groups in terms of volume, shown in Fig. 2, indicates that the left medial and the right nuclei are not significantly different, while a significant difference in this parameter is noted between the left lateral and right nuclei through all of the larval period. C. Number of cells in the habenular nuclei In Fig. 3 the total cell population of all three nuclei are shown. Fig. 3 A compares the total cell population for the two left nuclei in both the experimental and control animals. The left medial nucleus contains significantly more cells at all stages examined than does the left lateral nucleus in both the control and

7 Development of frog habenular nuclei 663 experimental groups. The number of cells contained in the left medial nucleus increases between stages I through XV in animals in both the experimental and control groups. There is less increase in the number of cells contained in the left medial nucleus of the experimental animals between stages XV and XX, while this nucleus in control animals continues to increase in cell number. The left medial nucleus of the experimental animals has undergone a significant decrease in the cell number by stage XX compared to the number of cells in the same nucleus of control animals. 2 1 Left Control Experimental B 2 1 Right Control 5 Medial 5 ; 1 I 1 I 5 i V X XV XX XXV 1 V X XV XX XXV Fig. 3. Total number of cells in the habenular nuclei of control and experimental animals: A, left medial and left lateral nuclei; B, right nucleus. There is no significant difference in the number of cells populating the left lateral habenular nucleus in the operated animals when compared to the unoperated control animals at any of the larval stages studied. At each stage examined, however, there were significantly fewer cells in this nucleus than in its medial companion on this side. Fig. 3B shows the total cell population of the right nucleus, and reflects the changes found in the length and relative volume. There is a great increase in the number of cells contained in this nucleus between stages I and XV in both the experimental and control animals. Between stages XV and XX in the experimental animals there is less increase in the number of cells while in the control animals the cell numbers continue to increase. Between stages XX and XXV there is a significant decrease in the total number of cells observed in the right nucleus of the experimental animals when compared to the increase of the cell number shown in the control animals. The number of cells at each larval stage

8 664 B. K. NORRIS AND V. B. EICHLER examined in both control and experimental animals are clearly similar in the right and left medial nuclei and clearly dissimilar in either of these when compared to the left lateral nucleus in the same animals. DISCUSSION The most complete descriptions of the neural connections of the habenular nuclei in amphibians are given by Herrick (1948) and Frontera (1952), and both authors indicate that the major bundles of afferent fibers enter the habenula from areas under strong olfactory influence. Experimental proof of this association has not been shown in amphibians, although it has been demonstrated in mammals (Rausch & Long, 1971). To test experimentally whether the habenula complex of amphibians receives olfactory sensory information, and to determine which of the three prominent nuclei in this region are involved, the olfactory pits were ablated on young embryos at an age before the olfactory nerves had contacted the brain. It is well known that the removal of peripheral sensory end-organs results in a hypoplasia in the corresponding neural centers (see Hughes, 1968, for a review). If any of the habenular nuclei receive olfactory information via the nasal pits, a decrease in size and/or cell number in the corresponding neural centers would be expected. The experimental nature of the present study allows an extension and clarification of the functional nature of the frog habenular nuclei beyond that of recent studies from other laboratories (Braitenberg & Kemali, 197; Morgan et al. 1971). It also presents an interesting discrepancy in that the two centers reported by these authors to be homologous on the basis of morphological appearance appear not to be the homologous pair in terms of developmental criteria nor analogous as revealed by their functional connections. Using the Bodian protargol method, Braitenberg & Kemali (197) report that a fine net of agrophilic fibers appears to course irregularly within the right and left lateral habenular nuclei, but are absent in the left medial nucleus. We have confirmed this observation in our laboratory (Fig. 4). However, it is clear from the results of the present study, as will be discussed below, that it is the left medial nucleus, and clearly not the left lateral nucleus, which develops spatially and temporally in a manner similar to the right habenular nucleus. Moreover, the left medial nucleus also is affected in the same way, to the same degree, and at the same developmental age, as the right nucleus following embryonic removal of the olfactory pits. The left lateral nucleus is not significantly affected in size nor in cell number as a result of the experimental manipulations of this investigation. Unfortunately, the data available at the present time do not allow a satisfactory resolution to this discrepancy of homologies based on morphological appearance on the one hand, and homologies based on developmental sequence and functional connections on the other. In our material we have found that the left lateral nucleus develops at approxi-

9 x < ^JH * Fig. 4. Transverse section through the diencephalon of an adult Rana pipiens frog. Fibrous nature of right {Hr) and left lateral (HI) nuclei may be compared with the non-fibrous appearance of the left medial (Hm) habenular nucleus. Epiphysis (E) is indicated. Marker = 25 /*m. b I" I ON

10 666 B. K. NORRIS AND V. B. EICHLER mately larval stage V, which contrasts with the finding of Morgan et al. (1971) who describes this cell group as a secondary development which does not form completely as a separate nucleus until after metamorphosis. This difference may reflect the different species of Rana frog used in the two experiments. In Rana pipiens we find no left lateral nucleus generally observable in animals of either the control or experimental groups before stage V; however, in two control animals the left lateral nucleus was present to a small degree (approximately 3 jiim in length in each animal) by this early larval stage. This cell group could thus be considered a secondary development to the medial nucleus, which is regularly present as early as stage I, but both are present throughout most of the larval development of this species. The left lateral nucleus was present in all our animals at stage X, the next older stage examined. The reduction in the length, relative volume and total cell number observed in the right and left medial habenular nuclei of the operated animals would indicate some important nerve connection between the peripheral sensory endorgan and the neural center. The hypoplasia noted indicates that the right and left medial nuclei are centers receiving afferent olfactory fibers. Because the experimental left lateral nucleus did not demonstrate any such reduction in the three parameters measured, it appears that the left lateral nucleus is not a center receiving afferent olfactory fibers. It is thus concluded that the right habenular nucleus and the left medial habenular nucleus of the frog brain are homologous and analogous structures based on their developmental pattern and functional responses. The lateral habenular nucleus on the left side of the frog brain is unlike these other two diencephalic nuclei in both development and functional connections. We have no satisfactory answer to explain why the two nuclei which are affected by the embryonic extirpation of the olfactory pits do not show a significant response until near the end of the larval period. It has been suggested (Norris, 1974) that the olfactory sense becomes of increased importance as the adult stage is approached. If the semi-terrestrial frog depends more on an acute olfactory sense than the aquatic tadpole, this would be a possible explanation, but such data is not available at present. Risser (1914), in fact, has reported that tadpoles of the toad react more strongly to olfactory stimulation than adults, and Noble (1954) supports this view for amphibians in general. On the other hand, it may be related in some way to the general maturation of the central nervous system in middle to late prometamorphic stages in response to the development of a functioning hypothalamo-hypophyseal system as metamorphic climax approaches (Etkin, 1965, 1966). The latter explanation has recently been shown (Eichler & Gray, 1975) to be valid for the prometamorphic changes in developmental rate attending continuous illumination or darkness in larval frogs of the same species. The authors wish to thank Patricia Taylor for her skilled laboratory assistance.

11 Development of frog habenular nuclei 667 REFERENCES BECCARI, N. (1943). Neurologia Comparta. Firenze: Sansoni Edizioni Scientifiche. BRAITENBERG, V. & KEMALI, M. (197). Exceptions to bilateral symmetry in the epithalmus of lower vertebrates. J. comp. Neurol. 138, EBBESSON, S. O. E. & TANG, D. (1965). A method for estimating the number of cells in histological studies. /. R. microsc. Soc. 84, EICHLER, V. B. & NORRIS, B. K. (1974). Normal development of an asymmetrical area of the frog brain, and its dependency on olfactory connections. Am. Zool. 14, EICHLER, V. B. & GRAY, L. S., JR. (1975). The influence of environmental lighting on the growth and prometamorphic development of larval frogs. (In preparation.) ETKIN, W. (1965). The phenomena of amphibian metamorphosis. IV. The development of the median eminence. /. Morph. 116, ETKIN, W. (1966). Hypothalamic sensitivity to thyroid feedback in the tadpole. Neuroendocrinology 1, FRONTERA, J. G. (1952). A study of the anuran diencephalon. /. comp. Neurol. 96, GAUPP, E. (1896). Ecker's and Wiedersheim's Anatomie des Frosches. Abt. 2. Braunschweig. GORONOWITSCH, N. (1833). Das gehirn und die Kopfnerven von Cyclothons acclinidens. Morph. Jb. 12, HERRICK, C. J. (1948). The Brain of the Tiger Salamander. Chicago: University of Chicago Press. HUGHES, A. F. W. (1968). Aspects of Neural Ontogeny. London: Logo Press Ltd. KAPPERS, C. U. A., HUBER, G. C. & CROSBY, E. (1936). The Comparative Anatomy of the Nervous System of Vertebrates Including Man. New York: The Macmillan Co. MORGAN, M. J., O'DONNELL, J. M. & OLIVER, R. F. (1971). Development of left-right asymmetry in the habenular nuclei of Rana temporaria. J. comp. Neurol. 149, NOBLE, G. K. (1954). The Biology of the Amphibia. New York: Dover Publ., Inc. NORRIS, B. K. (1974). The effect of ablation of the olfactory pits on the development of the habenular nuclei in Rana pipiens. M.S. dissertation, Wichita State University. RAUSCH, L. J. & LONG, C. J. (1971). Habenular nuclei: a crucial link between the olfactory and motor systems. Brain Res. 29, RISSER, J. (1914). Olfactor reactions in amphibians. J. exp. Zool. 16, RUGH, R. (1939). Induced ovulation and artificial fertilization in the frog. Biol. Bull. mar. biol. Lab., Woods Hole 66, TAYLOR, A. C. & KOLLROS, J. J. (1946). Stages in normal development of Rana pipiens larvae. Anat. Rec. 94, (Received 28 April 1975)