A lucite plate method for 3-dimensional reconstruction of neuronal populations

Journal of Neuroscience Methods, 4 (1981) 63--71 © Elsevier/North-Holland Biomedical Press

63

LUCITE PLATE METHOD FOR 3-DIMENSIONAL RECONSTRUCTION OF NEURONAL POPULATIONS A

JAMES R. AUGUSTINE

Department of Anatomy, University of South Carolina, School of Medicine, Columbia, S.C. 29208 (U.S.A.) (Received October l l t h , 1980) (Revised version received November 25th, 1980) (Accepted December 6th, 1980)

Key words: Lucite plate -- 3-dimensional reconstruction -- oculomotor nucleus -- baboon - - horseradish peroxidase A Lucite plate reconstruction method is described which, when combined with HRP histochemistry, provides an excellent means of visualizing, in 3-dimensional fashion, the functional organization of neuronal populations. Color photomicrographs of representative serial sections were made through the baboon oculomotor nucleus. Each color slide was then projected onto a 9 X12 in. Lucite plate and the configuration of the nucleus at each representative level drawn to scale on the plates. These plates were then stacked one in front of the other yielding a see-through, 3-dimensional reconstruction of the entire nucleus. Color transparencies of every sixth HRP-processed section were made and the image of each section projected onto the Lucite plates on which the configuration of the oculomotor nucleus was previously outlined. Using different colored stars to represent the neurons which supply different oculomotor muscles, the number and location of HRP-positive neurons was then plotted. The end result was an anatomically accurate 3-dimensional reconstruction of the entire labeled population of the oculomotor nucleus including the location of subnuclei that can be viewed from any side or at any angle. With adequate reference points these data can be entered into a computer graphics device, then viewed and manipulated in 3-dimensional fashion.

INTRODUCTION Since the introduction of the horseradish peroxidase (HRP) histochemical p r o c e d u r e as a u s e f u l n e u r o a n a t o m i c t e c h n i q u e , n u m e r o u s a p p l i c a t i o n s o f t h i s m e t h o d h a v e b e e n p u b l i s h e d . F o r e x a m p l e , in t h e r a b b i t i t h a s b e e n demonstrated that one can identify hypoglossal neurons which contain HRP reaction product within 10--14 h after injection of the tracer into the tongue musculature (Kristensson, 1975). Thus this method has been frequently used to study the location of nerve cell bodies following intramuscular injection o f t h e t r a c e r ( K r i s t e n s s o n a n d O l s s o n , 1 9 7 1 ; G a c e k , 1 9 7 4 ; M i z u n o e t al., 1 9 7 5 ; S t r i c k e t al., 1 9 7 6 ; R a d p o u r , 1 9 7 7 ; A k a g i , 1 9 7 8 ; K u m e e t al., 1 9 7 8 ; M a t s u d a e t al., 1 9 7 8 ; U e m u r a e t al., 1 9 7 9 ; G l i c k s m a n , 1 9 8 0 ; R a d p o u r a n d

64

Gacek, 1980; Yoshida et al., 1980). As Gacek (1974) noted, neurons of all sizes are clearly labeled and may be differentiated from unlabeled cells. Similarly, all neurons in each functional subgroup are clearly identified using this method. In our study of the functional organization of the o c u l o m o t o r nucleus in the baboon, using horseradish peroxidase histochemistry (Augustine et al., 1980) we encountered the problem of appreciating the differences from author to author in the arrangement of the o c u l o m o t o r subdivisions and of understanding the rostrocaudal and dorsoventral definition of this nucleus. To resolve this problem and to clearly appreciate the anatomic arrangement of the entire o c u l o m o t o r nucleus in the baboon, a 3-dimensional reconstruction of the o c u l o m o t o r nucleus was prepared using clear Lucite plates. The results obtained from intramuscular HRP injections of the oculomotor muscles in a series of baboons were then compiled using this Lucite plate reconstruction method. This marriage of the previously described HRP histochemical technique to the method described here allows one to obtain an anatomically accurate 3-dimensional view of the baboon o c u l o m o t o r nucleus and a clear idea of the relative positions of the functional neuronal populations within the o c u l o m o t o r nucleus. By this simple method and with comparatively little effort and cost one would be able to aquire a 3-dimensional view of any neuronal population in any animal. Reconstructions from serial electron micrographs might also be possible with this method. MA TER I ALS AND METHODS

Color photomicrographs were made of every sixth neutral red stained serial section through the entire b a b o o n o c u l o m o t o r nucleus. Each of the resulting 18 color slides was then projected with the aid of a photographic enlarger onto a 9 × 12 in. clear Lucite plate. Using a black fine-point marker the configuration of the o c u l o m o t o r nucleus at each representative level was drawn to scale on the Lucite plates. Since the sections used in this series were well stained, no difficulty was encountered in delineating the boundaries of the nuclear group. Brain stem nuclear groups such as the o c u l o m o t o r nucleus, to which this m e t h o d has been applied, are characterized morphologically by changes in the number and distribution of their individual cells from one level of the nucleus to the next. The 18 Lucite plates, each representing a 40 pm section through the o c u l o m o t o r nucleus, were then stacked one in front of the other yielding a see-through 3-dimensional reconstruction of the nucleus. For purposes of illustration, only the left o c u l o m o t o r nucleus is outlined on the Lucite plate model shown in Fig. 1A. This simple m e t h o d of utilizing contour information from serial sections provides an unparalleled appreciation of the external shape of the entire o c u l o m o t o r nuclear complex in 3-dimensions, that is anatomically accurate and which can be viewed from any side or at any angle. The location of the midbrain aqueduct and several measured reference points from the aqueduct were depicted on each Lucite

65 C~

A

Ii II!l!

Fig. 1. A: p h o t o g r a p h o f a L u c i t e p l a t e m o d e l s h o w i n g t h e left o c u l o m o t o r n u c l e u s outlined o n 9 L u c i t e plates. F o r t h e p u r p o s e o f clarity o n l y h a l f o f t h e L u c i t e plates are s h o w n . B: p h o t o g r a p h o f t h e L u c i t e p l a t e m o d e l s h o w i n g t h e left o c u l o m o t o r n u c l e u s o u t l i n e d o n 9 L u c i t e plates. T h e n u m b e r a n d d i s t r i b u t i o n o f H R P - t a b e l e d cells r e s u l t i n g f r o m i n j e c t i o n o f t h e c o n t r a l a t e r a l s u p e r i o r r e c t u s m u s c l e are p l o t t e d as b l a c k stars.

66 plate as convenient landmarks to align the sections (plates) and maintain anatomically correct spatial relations between sections. This Lucite plate reconstruction m e t h o d was adapted from that previously employed by Baetens et al. (1979) to demonstrate the t w o types of islet cells in the endocrine pancreas of the rat. The b a b o o n oculomotor nucleus and the Lucite plate reconstruction were then subdivided into rostral, middle, and caudal thirds in accordance with Pearson's (1944) developmental study of the human oculomotor nucleus. There were 36 stained sections and 6 Lucite plates representing each third of the nucleus with a total of 108 sections and 18 plates representing the entire o c u l o m o t o r nucleus. Each plate represented a 40 pm section with a 200 pm interval between plates. Depending upon the level, each section, and therefore each plate of the reconstruction, could be further subdivided in a dorsoventral fashion, that is, from near the midbrain aqueduct (dorsal) toward the interpeduncular fossa (ventral), into a dorsal and a ventral zone as described in the human fetus by Pearson (1944). Interposed between these zones is an intermediate zone. This additional zone was indirectly defined b y Warwick (1953) in his classic study of the representation of the extraocular muscles in the oculom o t o r nuclei of the monkey. Each section through the o c u l o m o t o r nucleus and each of the 18 Lucite plates could be assigned to the rostral, middle, or caudal third of the nucleus and each section (plate) may include dorsal, intermediate, and/or ventral zones. These subdivisions are often apparent in stained preparations of the normal b a b o o n o c u l o m o t o r nucleus, b u t become more distinct following utilization of horseradish peroxidase histochemistry. This subdivision of the o c u l o m o t o r nucleus could easily be represented on the Lucite plates. Thus one of the distinct advantages of this method is the ease with which the Lucite plates can be manipulated such that one can add information to the plates and further visualize in 3-dimensional fashion details of the anatomy of the nucleus under investigation. Secondly, corrections can be made at any time by examination of the original section under low- or high-power magnification and the corrections transferred to the Lucite plates. With this Lucite plate reconstruction at hand, it is possible to give a complete description of the form and spatial relationships of a 3-dimensional structure such as the oculomotor nucleus, even though the information is derived from serial 2-dimensional sections. Once the outlines of the o c u l o m o t o r nucleus through its rostral, middle, and caudal thirds are estabh'shed and drawn to scale on the Lucite plates along with an indication of the zones present at each level, it is possible to plot the number and location of HRP-positive neurons on the Lucite reconstruction. A number of experiments were carried o u t in which individual o c u l o m o t o r muscles were injected with H R P in a series of baboons. Cells in the o c u l o m o t o r nucleus which contained H R P reaction product following injection of the tracer into one of the muscles of the eyeball or into the levator muscle of the upper eyelid were carefully recorded on the Lucite

67 plates, both as to their number and their precise distribution. The technique used is a modification of the photographic m e t h o d described by Howarth and Warwick {1952), but utilizes the Lucite plate m e t h o d previously described. Color photomicrographs were made of every sixth HRP processed section through the ocutomotor nucleus in each animal used in this study. Using these transparencies, the image of each section was then projected onto the Lucite plates on which the configuration of the oculomotor nucleus was previously outlined. Using different colored symbols (stars), the number and location of HRP-positive neurons was then plotted on the Lucite plates, thus providing a permanent record of the location and distribution of HRPpositive neurons on each representative section. A different colored star was used to represent the cells which supply the muscles of the eyeball as well as to represent the cells which supply the levator of the upper eyelid. With these maps in hand, it was possible to compare the results from different animals in which different muscles were injected with the tracer. HRP-posirive neurons which were not heavily labeled often escaped identification at low magnification. This variability in density of reaction product within cells on the same section could easily be compensated for by the use of the original sections examined under high-power magnification. The results could then be charted on the Lucite plates and corrections made at any time on the Lucite model. A composite 3-dimensional reconstruction of the entire labeled population of the oculomotor nucleus on Lucite plates was the end result. It was thus possible to clearly recognize the overall nuclear configuration from rostral to caudal, the zones of the nuclear complex at each representative level and appreciate the position of neuronal populations which supply the individual extraocular muscles. Fig. 1B shows the left oculomotor nucleus as outlined on the Lucite plates. The black stars represent those cells which were found to contain HRP-reaction product following injection of the right superior rectus muscle in the baboon. DISCUSSION A number of methods and techniques have been utilized over the years to obtain a 3-dimensional appreciation of a spatial structure based upon data obtained from serial 2-dimensional sections. Born's (1883) m e t h o d for 3-dimensional reconstruction was based upon the use of wax plates of uniform thickness. Although Born's m e t h o d was one of the earliest techniques described for reconstruction purposes, the procedure is well known in light microscopy and has recently been applied to electron microscopic studies. Werner and Morgenstern (1980) produced a wax model from serial electron micrographs of h u m a n blood platelets using the m e t h o d of Born. Table 1 provides a survey of different materials used by various authors over the past 100 years to obtain a 3-dimensional reconstruction from serial 2-dimensional sections.

68 TABLE 1 MATERIALS USED TO OBTAIN A 3-DIMENSIONAL RECONSTRUCTION FROM SERIAL 2-DIMENSIONAL SECTIONS Year

Author

Medium

1883 1887 1887 1899 1902 1927 1937 1955 1958 1965 1966 1971 1971 1973 1974 1975 1976 1978

Born His Strasser Vosmaer Kerr Lapin Rolshoven Popper and Elias SjSstrand Trujillo-Cen6z Pedler and Tilly J}rgensen Los Knobler and Stempak SjSstrand Emmers and Tasker Gribnau and Lammers Schook and Blom

1979 1980

Baetens et al. Werner and Morgenstern

Wax plates Clay with graphical reconstruction Glass plates Celluloid plates Frosted glass sheets Gelatin plates Cellophane plates Lantern slides Plasticine using electron microscopic sections Balsa wood Polystyrene sheets Acryl plates Microfilm and prints Plexiglas squares Vinyl sheets Cardboard and styrofoam Polystyrene plates Combination light microscopic and electro n microscopic 3-dimensional reconstruction from a single block Lucite plates Wax model from serial electron microscopic sections

Los (1973) noted that plastic representation is absolutely essential for the study and teaching of anatomy and embryology. He advocated the construction of a series of plastic reconstructions in space, of increasing detail, all from the same original material and all of the same tangible and comprehensive format. He termed this method 'reconstructive morphology'. This thought-provoking article by Los (1973) presents some possibilities and some limitations of this methodology. Ware and LoPresti (1975) have recently published a rather thorough review of some methods used for 3-dimensional reconstruction from serial sections including a discussion of graphical models, model building, serial section cinematography and computer methods. This excellent paper is highly recommended to the interested reader. Baetens et al. (1979) utilized immunofluorescent staining of the endocrine pancreas along with a 3-dimensional reconstruction method to show two types of islets of Langerhans. Color photographs from each immunofluorescent stained section were copied to scale onto Lucite sheets with a colored pen used to represent different cell types. The Lucite plate reconstruction method described here was adapted from the method employed by Baetens et al. (1979).

69 The variety of methods noted in Table 1 is evidence of the creativity of m a n y investigators in attempting to further understand problems of a n a t o m y and embryology. No one technique can be designated as the 'best-all-around', principally because the problems under study are so diverse as to require a wide assortment of methods or perhaps the application of two or more methods to a single research endeavor. The Lucite plate m e t h o d described here in combination with horseradish peroxidase histochemistry provides a simple and inexpensive means of studying the problem of nuclear organization. Precise quantitative analysis may be made of the nucleus being studied by employing these methods. With an anatomically accurate appreciation of the clear-cut 3
70

method described here was presented at the meeting of the Southern Society of Anatomists, held October, 1980, in Augusta, Ga., U.S.A. This endeavor was supported in part by a University of South Carolina, School of Medicine, Intramural Grant. REFERENCES Akagi, Y. (1978) The localization of the m o t o r neurons innervating the extraocular muscles in the oculomotor nuclei of the cat and rabbit, using horseradish peroxidase, J. comp. Neurol., 181: 754--762. Augustine, J.R., Des Champs, E.G. and Ferguson, J.G., Jr. (1980) Functional organization of the oculomotor nucleus in the baboon, Amer. J. Anat., submitted for publication. Baetens, D., Malaisse-Lagae, F., Perrelet, A. and Orci, L. (1979) Endocrine pancreas: three-dimensional reconstruction shows two types of islets of Langerhans, Science, 206: 1323--1325. Born, G. (1883 ) Die PlattenmodeUiermethode, Arch. mikrosk. Anat., 22 : 584--599. Emmers, R. and Tasker, R.R. (1975) The Human Somesthetic Thalamus With Maps for Physiological Target Localization During Stereotactic Neurosurgery, Raven Press, New York, pp. 96--105. Gacek, R.R. (1974) Localization of neurons supplying the extraocular muscles in the kitten using horseradish peroxidase, Exp. Neurol., 44: 381--403. Glicksman, M.A. (1980) Localization of motoneurons controlling the extraocular muscles of the rat, Brain Res., 188: 53--62. Gribnau, A.A.M. and Lammers, G.J. {1976) The preparation of graphical and threedimensional reconstructions of the developing central nervous system, Acta morph. neerl, scand., 14: 1--18. His, W. (1887) IJber die Methoden der plastischen Rekonstruktion und fiber deren Bedeutung fiir Anatomie und Entwickelungsgeschichte, Anat. Anz., 2: 382--394. Howarth, P. and Warwick, R. ( I 9 5 2 ) A photographic method of recording the precise distribution of nerve or other cells, J. Med. Lab. Tech., 10: 21--23. J~gensen, M. (1971 ) Three-dimensional reconstruction histology. A short survey of available methods and description of a new technique. Acta path. microbiol, scand. (A), 79: 298--302. Kerr, J.G. (1902) The development of Lepidosiren paradoxa. Part II. Quart. J. microsc. Sci., 45: 1--46. Knobler, R.L. and Stempak, J.G. (1973) Serial section analysis of myelin development in the central nervous system of the albino rat: an electron microscopical study of early axonal ensheathment. In D.H. Ford (Ed.), Neurobiological Aspects of Maturation and Aging, Progress in Brain Research, Vol. 40, Elsevier, Amsterdam, pp. 407--423. Kristensson, K. (1975) Retrograde axonal transport of protein tracers. In W.M. Cowan and M. Cu~nod (Eds.), The Use of Axonal Transport for Studies of Neuronal Connect tivity, Elsevier, Amsterdam, pp. 60--82. Kristensson, K. and Olsson, Y. (1971) Retrograde axonal transport of proteins, Brain Res., 29: 363--365. Kume, M., Uemura, M., Matsuda, K., Matsushima, R. and Mizuno, N. (1978) Topographical representation of peripheral branches of the facial nerve within the facial nucleus: a HRP study in the cat, Neurosci. Lett., 8: 5--8. Lapin, M.L. (1927) Eine neue Methode fiir die Anfertigung durchsichtiger plastischer Rekonstruktionen ('Gelatine-Rekonstruktion'), Z. wissenseh. Mikr., 44: 134--164. Los, J.A. (1971) A new method of three-dimensional reconstruction of microscopical structures based on photographic techniques, Acta morph, neerl, seand., 8: 273--279.

71 Los, J.A. (1973) Reconstructive morphology --possibilities and limitations, Acta morph. neerl, scand., 11: 263--278. Matsuda, K., Uemura, M., Kume, M., Matsushima, R. and Mizuno, N. (1978) Topographical representation of masticatory muscles in the motor trigiminal nucleus in the rabbit: a HRP study, Neurosci. Lett., 8: 1--4. Mizuno, N., Konishi, A. and Sato, M. (1975) Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. comp. Neurol., 164: 105--116. Pearson, A.A. (1944) The oculomotor nucleus in the human fetus, J. comp. Neurol., 80: 47--63. Pedler, C. and Tilly, R. (1966) A new method of serial reconstruction from electron micrographs, J. roy. microsc. Soc., 8 6 : 1 8 9 - - 1 9 7 . Popper, H. and Elias, H. (1955) Histogenesis of hepatic cirrhosis studied by the threedimensional approach, Amer. J. Path., 31 : 405--442. Radpour, S. (1977) Organization of the facial nerve nucleus in the cat, Laryngoscope (St. Louis), 87: 557--574. Radpour, S. and Gacek, R.R. (1980) Facial nerve nucleus in the cat. Further study, Larynogoscope (St. Louis), 90: 685--692. Rolshoven, E. (1937) Rekonstruktionen histologischer Objekte auf durchsichtigen Werkstoffen, Z. wissensch. Mikr., 54: 328--338. Schook, P. and Blom, N. (1978) A three-dimensional reconstruction method preserving light microscopic and transmission electron microscopic information, Acta morph. neerl, scand., 16: 157--170. SjSstrand, F.S. (1958) Ultrastructure of retinal rod synapses of the guinea pig eye as revealed by three-dimensional reconstructions from serial sections, J. ultrastruct. Res., 2: 122--170. SjSstrand, F.S. (1974) A search for the circuitry of directional sensitivity and neural adaptation through three-dimensional analysis of the outer plexiform layer of the rabbit retina, J. ultrastruct. Res., 49: 60--156. Strasser, H. (1887) 0 b e r die Methoden der plastischen Rekonstruktion, Z. wissensch. Mikr., 4: 168--208. Strick, P.L., Burke, R.E., Kanda, K., Kim, C.C. and Walmsley, B. (1976) Differences between alpha and gamma motoneurons labeled with horseradish peroxidase by retrograde transport, Brain Res., 113 : 582--588. Trujillo-CenSz, O. (1965) Some aspects of the structural organization of the intermediate retina of dipterans, J. ultrastruct. Res., 13: 1--33. Uemura, M., Matsuda, K., Kume, M., Takeuchi, Y., Matsushima, R. and Mizuno, N. (1979) Topographical arrangement of hypoglossal motoneurons: an HRP study in the cat, Neurosci. Lett., 13: 99--104. Vosmaer, G.C.J. (1899) Eine einfache Modifikation zur Herstellung von Platten-Diagrammen, Anat. Anz., 16: 269--271. Ware, R.W. and LoPresti, V. (1975) Three-dimensional reconstruction from serial sections, Int. Rev. Cytol., 40: 325--440. Warwick, R. (1953) Representation of the extra-ocular muscles in the oculomotor nuclei of the monkey, J. comp. Neurol., 98: 449--504. Werner, G. and Morgenstern, E. (1980)Three-dimensional reconstruction of human blood platelets using serial sections, Europ. J. Cell Biol., 20: 276--282. Yoshida, Y., Miyazaki, T., Hirano, M., Shin, T., Totoki, T. and Kanaseki, T. (1980) Location of motoneurons supplying the cricopharyngeal muscle in the cat studied by means of the horseradish peroxidase method, Neurosci. Lett., 18: 1--4.