- Email: [email protected]
The neurons and their postnatal development in the basilar pontine nuclei of the rat
Brain Research
Bulletin,
Vol. 5, pp. 271-283. Printed in the U.S.A.
The Neurons and Their Postnatal Development in the Basilar Pontine Nuclei of the Rat CATHERINE Depurtments
of Physiology”
E. ADAMS,* JOHN G. PARNAVELAS,? GREGORY AND DONALD J. WOODWARD*? and Cell Biology,
i The University
Received
of Texas Health
A. MIHAILOFFt
Science
Center,
Dallas,
TX 75235
17 March 1980
AND D. J. WOODWARD. 7%~ neurwu and Iheir postnatut development of basilar pontine neurons was investigated utilizing a variety of Golgi techniques in female Sprague-Dawley rats ranging in age from 0 (birth) to 120 days. Basilar pontine neurons in 120-day old animals demonstrated many of the characteristic morphological features observed in adult animals. The perikarya of these neurons exhibited a variety of shapes, gave rise to from 2-7 dendrites, and ranged in size from 8-45 pm. Dendritic patterns were generally divisible into four groups. The most common pattern was a multipolar arrangement of relatively long dendrites, the surfaces of which were studded with a variety of protrusions. The next two most common patterns each exhibited a sparse complement of dendritic appendages but differed in other respects. One was characterized by stout, gradually tapering dendrites arising from large somata while the other possessed perikarya of intermediate sizes and long, thin dendrites of uniform caliber. The final and least common pattern was a bipolar configuration comprised of infrequently branching dendrites originating from small, ovoid or spindle-shaped cell bodies. Surface appendages were present only in small numbers on such neurons. Axons of adult basilar pontine neurons were found to arise from either the somata or a proximal dendrite and appeared on occasion to give rise to collaterals within the pontine gray. At birth, the perikarya of basilar pontine cells were small and irregular in shape. Dendrites and axons were thin, short and displayed bulbous growth cones on their terminal segments and along their lengths. By the end of the first postnatal week, the dendrites had become more uniform in diameter, while the cell bodies had increased considerably in size. Surface appendages on somata and dendrites first appeared 10-12 days after birth, differing from the adult in their greater density and wider distribution. The end of the second postnatal week was marked by a major dendritic growth spurt which continued until Day 16. At that time, most basilar pontine neurons attained adult dendritic patterns. A gradual disappearance of proximal dendritic appendages coupled with the persistence of distal surface appendages was also noted during the second postnatal week. This process was completed by postnatal Day 24 when the basilar pontine neurons appeared to attain adult morphology. ADAMS, C. E., J. G. PARNAVELAS,
developmenf
in rhe hasilarpontine
Basilar pontine nuclei Development Rat
G. A. MIHAILOFF
nuclei of the rat. BRAIN RES. BULL. 5(3)277-283, 1980.-The
Cerebro-cerebellar
systems
THE maturation of motor control in the rat is a gradual process which occurs for the most part during the first three
weeks of postnatal life [ 19,361. The understanding of the evolution of this process is contingent upon knowing the state of maturation of the major motor centers during this postnatal period. Previous investigations [4, 5, 6, 7, 8, 16, 31, 32, 331 have described various aspects of the development of the cerebral and cerebellar cortices of the rat. An important adjunct to these developmental studies would be a description of the maturation of the basilar pons which provides a major link between the cerebral cortex and cerebellum [ 1, 2, 3, 10, 11, 12, 13, 23, 24, 381. The aim of the present study is to describe the morphological development of basilar pontine neurons in the rat as a first step in understanding the maturation of the basilar pons and of the cortico-ponto-cerebellar system. METHOD
Female Sprague-Dawley albino rats of ages 0 (newborn), 2,4,6,8, 10, 12, 14, 16, 18,20,24 and 120 days were used in
Copyright 0 1980 ANKHO
Corticopontine
system
Pontocerebellar
system
this developmental study. All rats were sacrificed under ether anesthesia and the brains quickly removed from the skull and placed either in Golgi-Cox (entire brains) or Golgi-Kopsch (blocks containing only the pons) solutions. Following impregnation, 120 pm coronal sections were cut and processed respectively by the Van der Loos [37] moditication of the Golgi-Cox technique or the Golgi-Kopsch method [ 141. The findings reported below were based on camera lucida drawings of a few hundred cells and detailed observations of several hundred other cells. RESULTS
Neurons in the Pontine Nuclei of Adult Ruts Golgi impregnation of the adult basilar pons revealed a heterogeneous population of neuronal cell types (Fig. 1). The perikarya of pontine neurons vary considerably both in shape and size, ranging approximately from 10-45 pm in length and 8-30 pm in width, and appear to be distributed randomly throughout the different pontine nuclear regions. Pontine neurons also display a highly variable dendritic mor-
International Inc .-0361-9230/80/030277-07$01.20/O
278
FIG. 2. Cell a and b illustrate the appearance
of basilar pontine
neurons on the day of birth (Day 0). The small perikaryal sizes, the
FIG. 1. Camera lucida drawings of cells illustrating the heterogeneous neuronal population comprising the basiiar pans of the adult rat. The most common neuronal type is represented by cell d which displays a multipolar dendritic pattern, relatively long dendrites, and a variety of surface appendages. Cell a is an example of a less frequently encountered cell type which is characterized by thick. gradually tapering dendrites nearly devoid of surface spines or protrusions and a large cell body. Another subgroup of relatively spine-free pontine neurons is represented by cell b. These neurons differ from the previously described subgroup in that their somata are usually of an intermediate size and the dendrites are long, thin, and of a more uniform caliber. Cell c illustrates the least frequently impregnated neuron type in the basilar pons which is characterized by a small cell body and only two to three dendrites arranged in a bipolar pattern. The arrow indicates the axon of this neuron which branches distally.
phology and in coronal sections appear as multipolar (up to seven primary dendrites) or oligopoIar (primarily fusiform and bipolar) cells. Their dendritic shape appears to conform to four broad patterns, but the criteria distinguishing them are not rigid or sufftciently consistent to allow description of separate and distinct cell types. The most common pattern is one displayed by the majority of cells throughout the basilar pons which have relatively long dendrites exhibiting numerous spines and protrusions and radiating in all directions from the cell body (Fig. 1,d). The next two most common varieties, which are commonly seen in the regions of the cerebral peduncles, are characterized by cells with a sparse complement ofdendritic spines and protrusions. One group is distinguished by large somata and stout primary dendrites which gradually tapered d&ally (Fig. I,a), while the other variety exhibits somata of intermediate size and long, thin dendrites of uniform caliber (Fig. I,b). Some of these neurons project dendrites into the cerebral peduncle while
irregular somatic and dendritic surfaces as well as the presence of growth cone-like enlargements on dendritic tips and at branch points are charactetistic of growing neurons. Axons (arrow, cell b) also appear immature on the day of birth, being very fine and beaded. By postnatal Day 4 (cells c and d), the somata of pontine neurons increase dramatically in size. The dendrites and axons (arrows) also increase in both length and diameter, but still retain the irregular surfaces characteristic of growing neurons. Growth cones, especially evident in cell d, are still abundant.
others extend dendritic branches into surrounding areas. All three subgroups of pontine neurons described above have dendrites which branch frequently and span distances which vary from approximately 50 to a few hundred micrometers. Common to all these neurons is the presence of dendritic appendages which range in shape from short, stubby stalks to long highly-branched and intertwined protuberances which sometimes form a claw-like arrangement and whose density and disposition varies considerably amongst pontine neurons. The final and least common dendritic pattern is a bipolar configuration exhibited by cells which are infrequently impregnated. These cells have smaller, ovoid OI spindle-shaped perikarya, with an oligopolar or bipolar (majority) dendritic pattern in which individual dendrites branch only rarely and disptay a small number of appendages (Fig. 1,c). The axons of adult pontine cells are found to arise either from the cell body or from one of the primary dendrites and are generally not impregnated beyond the initial segment. However. a few were traced for a considerable distance and occasionally were found to give rise to axon collaterals within the pontine gray.
At birth and display 7.a). These ous growth
(Day 0). basilar pontine neurons appear immature features indicative of growth (Fig. 2.a and b: Fig. include irregular perikaryal surfaces and numercone-like enlargements along the length of most
ADAMS ET AL.
279
FIG. 3. By postnatal Day 6 (cells a and b), the dendrites of basilar pontine neurons have increased in length and have become more uniform in diameter. The presence of numerous growth cone-like enlargements at the distal ends of dendrites indicates that both the multipolar (cell a) and bipolar (cell b) subgroups of basilar pontine neurons are still immature. Dendritic appendages become conspicuous for the first time on pontine ceits by postnatal Day 10 (celi c) and increase further in density by twelve days after birth (cetl d). The various appendages were more widely distributed at these ages than was commonly found for adult basilar pontine cells. Arrows point to axons of cells c and d.
FIG. 4. A major increase in dendritic length has occurred for each of the pontine neuronal subtypes by postnatal Day 14. The bipolar cell type (cell a) closely resembles its adult counterpart, except for the much greater density of surface appendages present at this stage of development. Cells b and c, representing the two subclasses of relatively aspinous basilar pontine neurons, also appear to have a greater compiement and distribution of spines than seen in the adult. Growth cone-like enlargements and beading are still apparent in the more distal portions of dendrites (cell d). Arrows point to axons of cells b, c and d.
dendrites and axons, particularly at branch points and terminal dendritic segments. These growth cone-like enlargements commonly give rise to distinct filopodia at this early stage of postnatal development. The size and shape of the cell perikarya is variable with the majority of cells exhibiting a somatic length of approximately 15-20 pm. At birth, the preponderance of impregnated neurons are multipolar, with many dendrites giving rise to secondary and some tertiary branches, but a few bipolar neurons are also present throughout the pontine nuclei. The axons of most neurons, identified by their relatively thin and smooth appearance as well as a characteristic bend near the origin from the scma or a proximal dendrite, could only be traced for short distances from the perikaryon and some display growth cone-like enlargements similar to those characterizing the dendrites of these neurons. Several morphological changes become apparent in pontine neurons by the fourth postnatal day (Fig. 2,c and d). Cell perikarya appear to have undergone a marked increase in size, some displaying lengths up to 45 pm. Somatic shapes are varied and no apparent relationship exists between cell shape and position within the pontine gray at this stage of postnatal development. The dendrites, which range in appearance from thick to tine and beaded, are longer and more
branched at this stage than at Day 0, and growth cone-like enl~gements are still conspicuous on cell perikarya, dendrites and axons. At the beginning of the second postnatal week (Fig. 3,a and b; Fig. 7, b and c), the most notable change in basilar pontine neurons is the greater uniformity of dendritic and axonal diameters although some beading and growth conelike structures can stiI1 be seen on the distal ends of some processes. The most striking morphological feature of developing neurons at Days 10 and 12 (Fig. 3,c and d) of postnatal life is the precipitous increase in the number of dendritic appendages. Growth cone-like enlargements are still present, becoming more sparse in the 12 day old animals. A noticeable increase in dendritic branching, length, and diameter occurs between postnatal Days 12 and 16. Figure 4 illustrates postnatal Day 14 which marks the midway point of this dendritic growth spurt. The bipolar cells (Fig. 4,a) are more advanced in their dendritic maturation, followed in succession by the relatively aspinous neurons (Fig. 4,b and c) and then by the multipolar neuronai subgroup (Fig, 4,d). At postnatal Day 16, dendritic growth appears to be complete in that dendritic dimensions and branching patterns are within adult values (Fig. 5). A marked decrease in the
i
50vm
f
dendritic fields (length and branching pattern) of the multipolar (cell c), aspinous (cells b and a). and bipolar (cell d) cell FIG. 5. The
types at Day 16 resemble those of their adult counterparts. The number ofgrowth cones is also markedly reduced. Spine density and distribution are still greater for all pontine neuron types at this age than found in the adult rat basilar pons. Arrow points to axon of cell d.
number of growth cone-like enlargements also occurs at this time. Dendritic appendages on most neurons appear to have been lost proximal to the first dendritic bifurcation although some somatic protuberances are still present (Fig. 5,b and d). Distally, however, spiny protrusions and appendages are still nume~us (Fig. 5,a and c). An interesting feature present in the Golgi preparations at Day 16 is the high frequency of impregnated axons, many of which are seen to course for several hundred micrometers. sometimes giving rise to axon collaterals. Finally, the only notable morphological feature of development occurring between Day 16 and postnatal Day 24, when basilar pontine cells appear to reach maturity. is the continuation of the process in which dend&tic protrusions decrease in number proximally (Fig. 6,a and b) and persist or even increase in number (Fig.6,c) distal to the first dendritic branch point.
The present study shows that neurons in the basilar pons of the adult rat fall into two distinctive classes. The great majority ofcells are rnuiti~~r with hotly variable de~dritic morphoIogies, resembling closely the projection neurons of the pontine gray described by other investigators for a variety of mammals: Ramon y Cajal [34t_neonatal mouse and
i.
FIG. 6. Basilar pontine neurons in the mathave become rn~~~~logitally mature by postnatal Day 24. &ndriiic appendages have undergone a gradual recession from proximal regions and appear to be retained beyond initial dendritic branch points in all cell types while having become less dense overall on those neurons which belong to the relatively aspinous neuronal subtypes (cells a and b). The variety of surface appendages common to the adult multipolar cell type are well i~~ust~t~ in cell c. Ax&s of some basiktr pontine cells (arrow, cell a) appear to have acquired a my&r sheath in that the diameter of the axon increases markedly just distal to the characteristic bend.
human infant; Cooper and Fox iIS]-adult monkey; Mihailo~ and King [21’J--adult opossum; ~ihaiio~ if iii. 125]--adult rat. The axons of these neurons presumably enter the cerebellum via the brachium pontis, aithvugh the evidence used in designating this large population of cells as projection neurons is for the most part indirect. A marked simil~ty exists between the rno~hol~ of these celis and other projection neurons in the brain, e.g., the thalamocortical projection cells in the mammzdian thalamus 117, 18, 28, 30, 351. Basilar pontine neurons comprising a second category are scarce, have relativdy small and elongated perikaryn (up to 20 pm in length), are oligopoiar or bipolar (majority) and branch i~equently. These cells are strikingly sin&r to the interneurons found in the basilar pans of adult monkey tl5] adult rat [25] and to the intemeurons present in the thalamic nuclei of the rat 19. 17, 291 and other mammals [ 15,171. The morphogenetic changes described in this study OCcurred within the first twenty-four days of life and, with the exception of three stages, occurred gradually. The first noticeable change occurred during the first four days of postnatal life when the neuronal perikarya displayed signs of rapid growth. The second conspicuous change in the morphology of cells was observed between Days JO and 18. During this time, the ap~n~ges which were scattered OR the dendritic surfaces of the pontine neurons initially. ROWap_ pear to be eliminated proximally and retained or perhap, increased in number distal to the first dendritic branch point.
NEURONAL
A Day
281
POSTNATAL DEVELOPMENT
C Day 8
0
50wn
FIG. 7. Photomontages of rat basilar pontine neurons at various postnatal ages. Day 0 (birth)-the irregular somatic surface and large number of dendritic growth cone-like enlargements illustrate the immature state of this neuron (A). Dav 6-dendritic growth cone-like enlargements (arrows) are still evident on this bipolar basilar pontine neuron, giving the dendritic surface anirregular appearance. The soma, on the other hand, appears more mature due to the absence of any surface irregularities (B). Day a-the perikarya of basilar pontine neurons have increased considerably in size by postnatal Day 8 and the dendrites have become more uniform in diameter (C). Duy 20-basilar pontine neurons are approaching maturity by this time, the final dendritic growth spurt having been completed by Day 16. Many pontine neurons at this age, as evidenced in this photomicrograph, exhibit proximal dendritic segments which are relatively free of surface protrusions (D).
282
AJIAMS t.7 Al,
This dist~bution persisted in the adult rat 1251 and may be correlated with experimental electron microscopic findings which demonstrate that the major pontine afferent projection, the cortico-pontine system, terminates primarily on distal dendrites and their spines [20, 22, 271. The third striking change was observed between Days 12 and 16, at which time a marked increase in dendritie b~nchjng and length occurred. Perhaps the proiiferation of appendages between Days 10 and 12 and the marked increase in dendritic complexity between postnatal Days 12 and 16 is related to synaptogenesis in the pontine nuclei. However, information on synapse formation is not presently available for this b~instem region and, with the exception of the afferents
from the motor-sensory cortex [ 191,little is known concerning the development of the tierent and effentnt systems. Knawledge of the maturation of the intrinsic and extrinsic connections of the pontine nuclei will facilitate understanding of the development of cerebra-ecrebellar interaction and motor control in general.
We acknowledge the financial support by U.S.P.H.S. grant (EY 02964-011 to J.G.P., U.S.P.H.S. grant INS 12644)to G.A.M., NSF grant BNS-77-01174 to D.J.W and an award from the Biological Humanjcs ~~~u~ati~)n,
REFERENCES I. Allen, G. I. and N. Tsukahara. Cerebrocerebellar communication systems. P/q&f. Rev. 54: 957-1006, 1974. 3. .Allen, G. I., H. Kam and T. Ckhima. The mode of synaptic linkgage in the cerebra-ponto-cerebetlar pathway of the cat. I. Responses in the bra&urn pontis. E.rpl Brclin Rrs. 24: l-14. 1975. 3. Allen, G. I., H. Kern, T. Oshima and K. Toyama. The mode of synaptic linkage in the cerebra-panto-cerebeellar pathway af the cat. Il. Responses of single cells in the pontine nuciei. k:.$ Bruin Rcs. 25: 15-36, 1975. 4. Altman, J. Autoradiographic and histoiogical studies of . postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rate. .1. cornp. .Ne~ml. 136: 269-294, 1969. 5. Altman, J. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the t~nsitionai molecular layer. J. crmp. Neuroi. 145: 353-398, 1972. 6. Altman, J. Postnatal development of the cerehellar cortex in the rat. II. Phases in the maturation of Purkinje cells of the molecular layer. J. cwnp. Ne~rd. 145: 399-464. 1972. 7. Altman, J. Postnatal development of the cerebellar cortex in the rat. III. Matu~tion of the components of the granular layer. .I. c’omp. Ncpurtrf. 145: 465-5 14, 1972. 8. Berry, M. Development of the cerebral neocortex of the rat. In:
New York: Academic press, 19j4, pp. 7-67. 9. Brauer, K. and U. Shober. Qualitative und quantitative Ultersuchun8en am Corpus geni~ulatum laterale (Cgl) der Laborratte. 1. Zur Struktur des Cgl unter bescmderer Beticksichtyung der Go@-Architektanik. J. Hirt~/ksh. 14: 389-398, 1973. 10. Brodal, P. The corticopontine projection in the rhesus mankey. Origin and principles of organization. Brain 101: 251-283, 1978. II. B&al, P. The pontocerebellar projection in the rhesus monkey. An ex~~mental study with retr~de axonal transpon of HRP. ~~~f~r~~.~(,i~,~I~~ 4: 193-208, 1979. 12. Bume. R. A.. G. A. Mihailoff and D. J. Woodward. Visual corticopontine input to the paraflocculus: A combined autoradiographic and horseradish peroxidase study. Brain RCF. 143: 139-146, 1978. 13. Burne, R. A.. M. A. Eriksson, J. A. Saint-Cyr and D. J. Woodward. The o~ni~ati~n of the ~ntine projection to lateral cerebellar areas in the rat: dual zones in the pons. Bruiti RPS. 139: 340-347, 1978. 14. Colonnier, M. The tangential organization of the visual cortex. J. Amt. 98: 327-344, 1964. 15. Cooper, M. H. and C. A. Fox. The basilar pontine gray in the adult monkev fMucura ~ulurfi~}: A Golgi study. J. ~~r,rn~. Ncrir&. 168: i4S-134, 1976. 16. Deza, I,. and E. Eidelberg. Development of cortical electrical activity in the rat. &.‘p/ Nrurol. 17: 425438, 1%7. 17. Grossman. A., A. R. Lieberman and K. E. Webster. A Golpi study of the rat darsal lateral geniculate nucleus. J. c‘r~~nf~. Nfwd.
150: 441-466,
1973.
18 Guiiiery, R. W. A study of Go&i preparations from the dorsal lateral Keniculate nucleus of the adult cat. J. r0my. Nfvff-1 12s 21-50. i966. 19 Hicks. S. P. and J. D’Amato. Motor-sensory cortexcorticospinal system and develdping locomotion and placing in rats. Am. .I. Anur. 143: l-42, 19’75. 20 Hollander. H., P. Brodal and F. Walberg. Electron microscopic observations on the structure of tlte pontine nuclei and mode of termination of c~~ico~ntine fibers. E.q~l Brtrirr Rr. 7: 9-5-l IO, 1969. 21 Mihailoff. G. A. and J. S. King. The basilar pontine gray of the opossum: A correlated light an& electron microscopic analysis. J. tonrp. N:rurol. 159: 521-522, 1973. 22 Mihailoff. G. A. Two types of degenerating axon terminals . .-. . in. the basilar pontine nuclei of the opossum fo~o~i~ cerebral cortical lesions. Nrun~ci. Lrbr~. S: 219-224. 1978. 23 Mihailoff, G. A., R. A. Bume and D. J. Woodward. Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: An autoradiographic study. Bruirr Hes. 145: 347-354, 1978. 24 Mihailoff. G. A.. G. F. Martin and M. A. Linauts. The mn-- I--.. tocerebellar system in the opmxrn, an HRP study. Brain Rt*tfuc, Evcttftr., 1980, in press. 25 Mihai!off, G. A.. C. B. McArdle and C. F,. Adams. The cytoarchitecture, cytology and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies. .I. c’onrp. Nf~urot., 1980, in press. 26 Mihailoff, G. A. and C. B. MeArdle. The cytnarchitecture, cytology and synaptic orgaaizatian of the basilar potine nuclei in the rat. Ii. Electron microscopic studies. .I. cctnrp. Ncrrrt~l., /1980, in press. 27. Mihailoff. G. A., C. B. Watt and R. A. Bume. Evidence demonstrating that both the corticopontine and cerebellopontine systems are composed of two separate neuronal elements. An electron microscopic and horseradish peroxidase study in the rat. J. wmp. Neurot.. 1980, in press. 2X. Morest. D: K. The neuronal ~~~~ture of the medial geniculate bodv of the cat. J. Ancrr. 98: 611-630. 1964. 29. Mounty: E. J.. J. G. Pamavelrts and A. R. Lieberman. Theneurons and their postnatal development in the ventral lateral geniculate nucleus of the rat. Anat. Emhryol. 151: 35-51, 1977. 30. Pamavelas,‘J. G., E. J. Mounty. R. Bradford and A. R. Lieberman. The postnatal develo~e~t of neurons in the dorsal lateral genicufate nucl@~s of the rat: A Golgi study. J. c’orn~. Nrrm*i, 171: 481-500, 1977. 31. Pamavelas, J. G.. R. Bradford. L. 3. Mounty and A. R. Lieberman. The development of non-pyramidal neurons in the visual cortex of the rat, Arrur. Emhrytt. 155: t-14, 1978. 32 Pure, D. G. and D. J. Woodward. Maturation of evoked climhing fiber input to rat ecrebellar Purkinje cells (I.). !?~pi &ii/i Rcs. 28: 85-100, 1977. 33. Pure, D. G. and D. J. Woodward. Maturatmn 01 WoKed mossy fiber input to rat cerebellar Purkinje cells. E.rpl B&n Rr.$. 2R: 427-441. 1977. ,I..
__“.
NEURONAL
34. Ramon I’Homme
POSTNATAL
DEVELOPMENT
S. Histologic due Susteme Nerveux de et des Vartebres. Vol. I. Paris: A. Maloine, 1909, pp.
y Cajal,
960-978. 35. Scheibel, M. E. and A. B. Scheibel. Patterns of organization of specific and non-specific thalamic fields. In: The Thalamus. edited by D. P. Purpura and J. M. D. Yale. New York: Columbia University Press, 1966, pp. 13-46. 36. Tilney, F. Behavior in its relation to the development of the brain. II. Correlation between the development of the brain and behavior in the albino rat from embryonic states to maturity. Bull. Neural. Inst. 3: 252-358, 1933.
283 37. Van der Loos, H. Dendro-denditische
verbindingen in de schors der grote hersenen. Thesis, H. Stam, Haarlem, 1959. 38. Wiessendanger, R., M. Wiesendanger and D. G. Ruegg. An anatomical investigation of the corticopontine projection in the primate (Macaca fascicularis and Saimini sciurrus). II. The projection from frontal and parietal association areas. Neamscience 4: 747-765, 1979.
Bulletin,
Vol. 5, pp. 271-283. Printed in the U.S.A.
The Neurons and Their Postnatal Development in the Basilar Pontine Nuclei of the Rat CATHERINE Depurtments
of Physiology”
E. ADAMS,* JOHN G. PARNAVELAS,? GREGORY AND DONALD J. WOODWARD*? and Cell Biology,
i The University
Received
of Texas Health
A. MIHAILOFFt
Science
Center,
Dallas,
TX 75235
17 March 1980
AND D. J. WOODWARD. 7%~ neurwu and Iheir postnatut development of basilar pontine neurons was investigated utilizing a variety of Golgi techniques in female Sprague-Dawley rats ranging in age from 0 (birth) to 120 days. Basilar pontine neurons in 120-day old animals demonstrated many of the characteristic morphological features observed in adult animals. The perikarya of these neurons exhibited a variety of shapes, gave rise to from 2-7 dendrites, and ranged in size from 8-45 pm. Dendritic patterns were generally divisible into four groups. The most common pattern was a multipolar arrangement of relatively long dendrites, the surfaces of which were studded with a variety of protrusions. The next two most common patterns each exhibited a sparse complement of dendritic appendages but differed in other respects. One was characterized by stout, gradually tapering dendrites arising from large somata while the other possessed perikarya of intermediate sizes and long, thin dendrites of uniform caliber. The final and least common pattern was a bipolar configuration comprised of infrequently branching dendrites originating from small, ovoid or spindle-shaped cell bodies. Surface appendages were present only in small numbers on such neurons. Axons of adult basilar pontine neurons were found to arise from either the somata or a proximal dendrite and appeared on occasion to give rise to collaterals within the pontine gray. At birth, the perikarya of basilar pontine cells were small and irregular in shape. Dendrites and axons were thin, short and displayed bulbous growth cones on their terminal segments and along their lengths. By the end of the first postnatal week, the dendrites had become more uniform in diameter, while the cell bodies had increased considerably in size. Surface appendages on somata and dendrites first appeared 10-12 days after birth, differing from the adult in their greater density and wider distribution. The end of the second postnatal week was marked by a major dendritic growth spurt which continued until Day 16. At that time, most basilar pontine neurons attained adult dendritic patterns. A gradual disappearance of proximal dendritic appendages coupled with the persistence of distal surface appendages was also noted during the second postnatal week. This process was completed by postnatal Day 24 when the basilar pontine neurons appeared to attain adult morphology. ADAMS, C. E., J. G. PARNAVELAS,
developmenf
in rhe hasilarpontine
Basilar pontine nuclei Development Rat
G. A. MIHAILOFF
nuclei of the rat. BRAIN RES. BULL. 5(3)277-283, 1980.-The
Cerebro-cerebellar
systems
THE maturation of motor control in the rat is a gradual process which occurs for the most part during the first three
weeks of postnatal life [ 19,361. The understanding of the evolution of this process is contingent upon knowing the state of maturation of the major motor centers during this postnatal period. Previous investigations [4, 5, 6, 7, 8, 16, 31, 32, 331 have described various aspects of the development of the cerebral and cerebellar cortices of the rat. An important adjunct to these developmental studies would be a description of the maturation of the basilar pons which provides a major link between the cerebral cortex and cerebellum [ 1, 2, 3, 10, 11, 12, 13, 23, 24, 381. The aim of the present study is to describe the morphological development of basilar pontine neurons in the rat as a first step in understanding the maturation of the basilar pons and of the cortico-ponto-cerebellar system. METHOD
Female Sprague-Dawley albino rats of ages 0 (newborn), 2,4,6,8, 10, 12, 14, 16, 18,20,24 and 120 days were used in
Copyright 0 1980 ANKHO
Corticopontine
system
Pontocerebellar
system
this developmental study. All rats were sacrificed under ether anesthesia and the brains quickly removed from the skull and placed either in Golgi-Cox (entire brains) or Golgi-Kopsch (blocks containing only the pons) solutions. Following impregnation, 120 pm coronal sections were cut and processed respectively by the Van der Loos [37] moditication of the Golgi-Cox technique or the Golgi-Kopsch method [ 141. The findings reported below were based on camera lucida drawings of a few hundred cells and detailed observations of several hundred other cells. RESULTS
Neurons in the Pontine Nuclei of Adult Ruts Golgi impregnation of the adult basilar pons revealed a heterogeneous population of neuronal cell types (Fig. 1). The perikarya of pontine neurons vary considerably both in shape and size, ranging approximately from 10-45 pm in length and 8-30 pm in width, and appear to be distributed randomly throughout the different pontine nuclear regions. Pontine neurons also display a highly variable dendritic mor-
International Inc .-0361-9230/80/030277-07$01.20/O
278
FIG. 2. Cell a and b illustrate the appearance
of basilar pontine
neurons on the day of birth (Day 0). The small perikaryal sizes, the
FIG. 1. Camera lucida drawings of cells illustrating the heterogeneous neuronal population comprising the basiiar pans of the adult rat. The most common neuronal type is represented by cell d which displays a multipolar dendritic pattern, relatively long dendrites, and a variety of surface appendages. Cell a is an example of a less frequently encountered cell type which is characterized by thick. gradually tapering dendrites nearly devoid of surface spines or protrusions and a large cell body. Another subgroup of relatively spine-free pontine neurons is represented by cell b. These neurons differ from the previously described subgroup in that their somata are usually of an intermediate size and the dendrites are long, thin, and of a more uniform caliber. Cell c illustrates the least frequently impregnated neuron type in the basilar pons which is characterized by a small cell body and only two to three dendrites arranged in a bipolar pattern. The arrow indicates the axon of this neuron which branches distally.
phology and in coronal sections appear as multipolar (up to seven primary dendrites) or oligopoIar (primarily fusiform and bipolar) cells. Their dendritic shape appears to conform to four broad patterns, but the criteria distinguishing them are not rigid or sufftciently consistent to allow description of separate and distinct cell types. The most common pattern is one displayed by the majority of cells throughout the basilar pons which have relatively long dendrites exhibiting numerous spines and protrusions and radiating in all directions from the cell body (Fig. 1,d). The next two most common varieties, which are commonly seen in the regions of the cerebral peduncles, are characterized by cells with a sparse complement ofdendritic spines and protrusions. One group is distinguished by large somata and stout primary dendrites which gradually tapered d&ally (Fig. I,a), while the other variety exhibits somata of intermediate size and long, thin dendrites of uniform caliber (Fig. I,b). Some of these neurons project dendrites into the cerebral peduncle while
irregular somatic and dendritic surfaces as well as the presence of growth cone-like enlargements on dendritic tips and at branch points are charactetistic of growing neurons. Axons (arrow, cell b) also appear immature on the day of birth, being very fine and beaded. By postnatal Day 4 (cells c and d), the somata of pontine neurons increase dramatically in size. The dendrites and axons (arrows) also increase in both length and diameter, but still retain the irregular surfaces characteristic of growing neurons. Growth cones, especially evident in cell d, are still abundant.
others extend dendritic branches into surrounding areas. All three subgroups of pontine neurons described above have dendrites which branch frequently and span distances which vary from approximately 50 to a few hundred micrometers. Common to all these neurons is the presence of dendritic appendages which range in shape from short, stubby stalks to long highly-branched and intertwined protuberances which sometimes form a claw-like arrangement and whose density and disposition varies considerably amongst pontine neurons. The final and least common dendritic pattern is a bipolar configuration exhibited by cells which are infrequently impregnated. These cells have smaller, ovoid OI spindle-shaped perikarya, with an oligopolar or bipolar (majority) dendritic pattern in which individual dendrites branch only rarely and disptay a small number of appendages (Fig. 1,c). The axons of adult pontine cells are found to arise either from the cell body or from one of the primary dendrites and are generally not impregnated beyond the initial segment. However. a few were traced for a considerable distance and occasionally were found to give rise to axon collaterals within the pontine gray.
At birth and display 7.a). These ous growth
(Day 0). basilar pontine neurons appear immature features indicative of growth (Fig. 2.a and b: Fig. include irregular perikaryal surfaces and numercone-like enlargements along the length of most
ADAMS ET AL.
279
FIG. 3. By postnatal Day 6 (cells a and b), the dendrites of basilar pontine neurons have increased in length and have become more uniform in diameter. The presence of numerous growth cone-like enlargements at the distal ends of dendrites indicates that both the multipolar (cell a) and bipolar (cell b) subgroups of basilar pontine neurons are still immature. Dendritic appendages become conspicuous for the first time on pontine ceits by postnatal Day 10 (celi c) and increase further in density by twelve days after birth (cetl d). The various appendages were more widely distributed at these ages than was commonly found for adult basilar pontine cells. Arrows point to axons of cells c and d.
FIG. 4. A major increase in dendritic length has occurred for each of the pontine neuronal subtypes by postnatal Day 14. The bipolar cell type (cell a) closely resembles its adult counterpart, except for the much greater density of surface appendages present at this stage of development. Cells b and c, representing the two subclasses of relatively aspinous basilar pontine neurons, also appear to have a greater compiement and distribution of spines than seen in the adult. Growth cone-like enlargements and beading are still apparent in the more distal portions of dendrites (cell d). Arrows point to axons of cells b, c and d.
dendrites and axons, particularly at branch points and terminal dendritic segments. These growth cone-like enlargements commonly give rise to distinct filopodia at this early stage of postnatal development. The size and shape of the cell perikarya is variable with the majority of cells exhibiting a somatic length of approximately 15-20 pm. At birth, the preponderance of impregnated neurons are multipolar, with many dendrites giving rise to secondary and some tertiary branches, but a few bipolar neurons are also present throughout the pontine nuclei. The axons of most neurons, identified by their relatively thin and smooth appearance as well as a characteristic bend near the origin from the scma or a proximal dendrite, could only be traced for short distances from the perikaryon and some display growth cone-like enlargements similar to those characterizing the dendrites of these neurons. Several morphological changes become apparent in pontine neurons by the fourth postnatal day (Fig. 2,c and d). Cell perikarya appear to have undergone a marked increase in size, some displaying lengths up to 45 pm. Somatic shapes are varied and no apparent relationship exists between cell shape and position within the pontine gray at this stage of postnatal development. The dendrites, which range in appearance from thick to tine and beaded, are longer and more
branched at this stage than at Day 0, and growth cone-like enl~gements are still conspicuous on cell perikarya, dendrites and axons. At the beginning of the second postnatal week (Fig. 3,a and b; Fig. 7, b and c), the most notable change in basilar pontine neurons is the greater uniformity of dendritic and axonal diameters although some beading and growth conelike structures can stiI1 be seen on the distal ends of some processes. The most striking morphological feature of developing neurons at Days 10 and 12 (Fig. 3,c and d) of postnatal life is the precipitous increase in the number of dendritic appendages. Growth cone-like enlargements are still present, becoming more sparse in the 12 day old animals. A noticeable increase in dendritic branching, length, and diameter occurs between postnatal Days 12 and 16. Figure 4 illustrates postnatal Day 14 which marks the midway point of this dendritic growth spurt. The bipolar cells (Fig. 4,a) are more advanced in their dendritic maturation, followed in succession by the relatively aspinous neurons (Fig. 4,b and c) and then by the multipolar neuronai subgroup (Fig, 4,d). At postnatal Day 16, dendritic growth appears to be complete in that dendritic dimensions and branching patterns are within adult values (Fig. 5). A marked decrease in the
i
50vm
f
dendritic fields (length and branching pattern) of the multipolar (cell c), aspinous (cells b and a). and bipolar (cell d) cell FIG. 5. The
types at Day 16 resemble those of their adult counterparts. The number ofgrowth cones is also markedly reduced. Spine density and distribution are still greater for all pontine neuron types at this age than found in the adult rat basilar pons. Arrow points to axon of cell d.
number of growth cone-like enlargements also occurs at this time. Dendritic appendages on most neurons appear to have been lost proximal to the first dendritic bifurcation although some somatic protuberances are still present (Fig. 5,b and d). Distally, however, spiny protrusions and appendages are still nume~us (Fig. 5,a and c). An interesting feature present in the Golgi preparations at Day 16 is the high frequency of impregnated axons, many of which are seen to course for several hundred micrometers. sometimes giving rise to axon collaterals. Finally, the only notable morphological feature of development occurring between Day 16 and postnatal Day 24, when basilar pontine cells appear to reach maturity. is the continuation of the process in which dend&tic protrusions decrease in number proximally (Fig. 6,a and b) and persist or even increase in number (Fig.6,c) distal to the first dendritic branch point.
The present study shows that neurons in the basilar pons of the adult rat fall into two distinctive classes. The great majority ofcells are rnuiti~~r with hotly variable de~dritic morphoIogies, resembling closely the projection neurons of the pontine gray described by other investigators for a variety of mammals: Ramon y Cajal [34t_neonatal mouse and
i.
FIG. 6. Basilar pontine neurons in the mathave become rn~~~~logitally mature by postnatal Day 24. &ndriiic appendages have undergone a gradual recession from proximal regions and appear to be retained beyond initial dendritic branch points in all cell types while having become less dense overall on those neurons which belong to the relatively aspinous neuronal subtypes (cells a and b). The variety of surface appendages common to the adult multipolar cell type are well i~~ust~t~ in cell c. Ax&s of some basiktr pontine cells (arrow, cell a) appear to have acquired a my&r sheath in that the diameter of the axon increases markedly just distal to the characteristic bend.
human infant; Cooper and Fox iIS]-adult monkey; Mihailo~ and King [21’J--adult opossum; ~ihaiio~ if iii. 125]--adult rat. The axons of these neurons presumably enter the cerebellum via the brachium pontis, aithvugh the evidence used in designating this large population of cells as projection neurons is for the most part indirect. A marked simil~ty exists between the rno~hol~ of these celis and other projection neurons in the brain, e.g., the thalamocortical projection cells in the mammzdian thalamus 117, 18, 28, 30, 351. Basilar pontine neurons comprising a second category are scarce, have relativdy small and elongated perikaryn (up to 20 pm in length), are oligopoiar or bipolar (majority) and branch i~equently. These cells are strikingly sin&r to the interneurons found in the basilar pans of adult monkey tl5] adult rat [25] and to the intemeurons present in the thalamic nuclei of the rat 19. 17, 291 and other mammals [ 15,171. The morphogenetic changes described in this study OCcurred within the first twenty-four days of life and, with the exception of three stages, occurred gradually. The first noticeable change occurred during the first four days of postnatal life when the neuronal perikarya displayed signs of rapid growth. The second conspicuous change in the morphology of cells was observed between Days JO and 18. During this time, the ap~n~ges which were scattered OR the dendritic surfaces of the pontine neurons initially. ROWap_ pear to be eliminated proximally and retained or perhap, increased in number distal to the first dendritic branch point.
NEURONAL
A Day
281
POSTNATAL DEVELOPMENT
C Day 8
0
50wn
FIG. 7. Photomontages of rat basilar pontine neurons at various postnatal ages. Day 0 (birth)-the irregular somatic surface and large number of dendritic growth cone-like enlargements illustrate the immature state of this neuron (A). Dav 6-dendritic growth cone-like enlargements (arrows) are still evident on this bipolar basilar pontine neuron, giving the dendritic surface anirregular appearance. The soma, on the other hand, appears more mature due to the absence of any surface irregularities (B). Day a-the perikarya of basilar pontine neurons have increased considerably in size by postnatal Day 8 and the dendrites have become more uniform in diameter (C). Duy 20-basilar pontine neurons are approaching maturity by this time, the final dendritic growth spurt having been completed by Day 16. Many pontine neurons at this age, as evidenced in this photomicrograph, exhibit proximal dendritic segments which are relatively free of surface protrusions (D).
282
AJIAMS t.7 Al,
This dist~bution persisted in the adult rat 1251 and may be correlated with experimental electron microscopic findings which demonstrate that the major pontine afferent projection, the cortico-pontine system, terminates primarily on distal dendrites and their spines [20, 22, 271. The third striking change was observed between Days 12 and 16, at which time a marked increase in dendritie b~nchjng and length occurred. Perhaps the proiiferation of appendages between Days 10 and 12 and the marked increase in dendritic complexity between postnatal Days 12 and 16 is related to synaptogenesis in the pontine nuclei. However, information on synapse formation is not presently available for this b~instem region and, with the exception of the afferents
from the motor-sensory cortex [ 191,little is known concerning the development of the tierent and effentnt systems. Knawledge of the maturation of the intrinsic and extrinsic connections of the pontine nuclei will facilitate understanding of the development of cerebra-ecrebellar interaction and motor control in general.
We acknowledge the financial support by U.S.P.H.S. grant (EY 02964-011 to J.G.P., U.S.P.H.S. grant INS 12644)to G.A.M., NSF grant BNS-77-01174 to D.J.W and an award from the Biological Humanjcs ~~~u~ati~)n,
REFERENCES I. Allen, G. I. and N. Tsukahara. Cerebrocerebellar communication systems. P/q&f. Rev. 54: 957-1006, 1974. 3. .Allen, G. I., H. Kam and T. Ckhima. The mode of synaptic linkgage in the cerebra-ponto-cerebetlar pathway of the cat. I. Responses in the bra&urn pontis. E.rpl Brclin Rrs. 24: l-14. 1975. 3. Allen, G. I., H. Kern, T. Oshima and K. Toyama. The mode of synaptic linkage in the cerebra-panto-cerebeellar pathway af the cat. Il. Responses of single cells in the pontine nuciei. k:.$ Bruin Rcs. 25: 15-36, 1975. 4. Altman, J. Autoradiographic and histoiogical studies of . postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rate. .1. cornp. .Ne~ml. 136: 269-294, 1969. 5. Altman, J. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the t~nsitionai molecular layer. J. crmp. Neuroi. 145: 353-398, 1972. 6. Altman, J. Postnatal development of the cerehellar cortex in the rat. II. Phases in the maturation of Purkinje cells of the molecular layer. J. cwnp. Ne~rd. 145: 399-464. 1972. 7. Altman, J. Postnatal development of the cerebellar cortex in the rat. III. Matu~tion of the components of the granular layer. .I. c’omp. Ncpurtrf. 145: 465-5 14, 1972. 8. Berry, M. Development of the cerebral neocortex of the rat. In:
New York: Academic press, 19j4, pp. 7-67. 9. Brauer, K. and U. Shober. Qualitative und quantitative Ultersuchun8en am Corpus geni~ulatum laterale (Cgl) der Laborratte. 1. Zur Struktur des Cgl unter bescmderer Beticksichtyung der Go@-Architektanik. J. Hirt~/ksh. 14: 389-398, 1973. 10. Brodal, P. The corticopontine projection in the rhesus mankey. Origin and principles of organization. Brain 101: 251-283, 1978. II. B&al, P. The pontocerebellar projection in the rhesus monkey. An ex~~mental study with retr~de axonal transpon of HRP. ~~~f~r~~.~(,i~,~I~~ 4: 193-208, 1979. 12. Bume. R. A.. G. A. Mihailoff and D. J. Woodward. Visual corticopontine input to the paraflocculus: A combined autoradiographic and horseradish peroxidase study. Brain RCF. 143: 139-146, 1978. 13. Burne, R. A.. M. A. Eriksson, J. A. Saint-Cyr and D. J. Woodward. The o~ni~ati~n of the ~ntine projection to lateral cerebellar areas in the rat: dual zones in the pons. Bruiti RPS. 139: 340-347, 1978. 14. Colonnier, M. The tangential organization of the visual cortex. J. Amt. 98: 327-344, 1964. 15. Cooper, M. H. and C. A. Fox. The basilar pontine gray in the adult monkev fMucura ~ulurfi~}: A Golgi study. J. ~~r,rn~. Ncrir&. 168: i4S-134, 1976. 16. Deza, I,. and E. Eidelberg. Development of cortical electrical activity in the rat. &.‘p/ Nrurol. 17: 425438, 1%7. 17. Grossman. A., A. R. Lieberman and K. E. Webster. A Golpi study of the rat darsal lateral geniculate nucleus. J. c‘r~~nf~. Nfwd.
150: 441-466,
1973.
18 Guiiiery, R. W. A study of Go&i preparations from the dorsal lateral Keniculate nucleus of the adult cat. J. r0my. Nfvff-1 12s 21-50. i966. 19 Hicks. S. P. and J. D’Amato. Motor-sensory cortexcorticospinal system and develdping locomotion and placing in rats. Am. .I. Anur. 143: l-42, 19’75. 20 Hollander. H., P. Brodal and F. Walberg. Electron microscopic observations on the structure of tlte pontine nuclei and mode of termination of c~~ico~ntine fibers. E.q~l Brtrirr Rr. 7: 9-5-l IO, 1969. 21 Mihailoff. G. A. and J. S. King. The basilar pontine gray of the opossum: A correlated light an& electron microscopic analysis. J. tonrp. N:rurol. 159: 521-522, 1973. 22 Mihailoff. G. A. Two types of degenerating axon terminals . .-. . in. the basilar pontine nuclei of the opossum fo~o~i~ cerebral cortical lesions. Nrun~ci. Lrbr~. S: 219-224. 1978. 23 Mihailoff, G. A., R. A. Bume and D. J. Woodward. Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: An autoradiographic study. Bruirr Hes. 145: 347-354, 1978. 24 Mihailoff. G. A.. G. F. Martin and M. A. Linauts. The mn-- I--.. tocerebellar system in the opmxrn, an HRP study. Brain Rt*tfuc, Evcttftr., 1980, in press. 25 Mihai!off, G. A.. C. B. McArdle and C. F,. Adams. The cytoarchitecture, cytology and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies. .I. c’onrp. Nf~urot., 1980, in press. 26 Mihailoff, G. A. and C. B. MeArdle. The cytnarchitecture, cytology and synaptic orgaaizatian of the basilar potine nuclei in the rat. Ii. Electron microscopic studies. .I. cctnrp. Ncrrrt~l., /1980, in press. 27. Mihailoff. G. A., C. B. Watt and R. A. Bume. Evidence demonstrating that both the corticopontine and cerebellopontine systems are composed of two separate neuronal elements. An electron microscopic and horseradish peroxidase study in the rat. J. wmp. Neurot.. 1980, in press. 2X. Morest. D: K. The neuronal ~~~~ture of the medial geniculate bodv of the cat. J. Ancrr. 98: 611-630. 1964. 29. Mounty: E. J.. J. G. Pamavelrts and A. R. Lieberman. Theneurons and their postnatal development in the ventral lateral geniculate nucleus of the rat. Anat. Emhryol. 151: 35-51, 1977. 30. Pamavelas,‘J. G., E. J. Mounty. R. Bradford and A. R. Lieberman. The postnatal develo~e~t of neurons in the dorsal lateral genicufate nucl@~s of the rat: A Golgi study. J. c’orn~. Nrrm*i, 171: 481-500, 1977. 31. Pamavelas, J. G.. R. Bradford. L. 3. Mounty and A. R. Lieberman. The development of non-pyramidal neurons in the visual cortex of the rat, Arrur. Emhrytt. 155: t-14, 1978. 32 Pure, D. G. and D. J. Woodward. Maturation of evoked climhing fiber input to rat ecrebellar Purkinje cells (I.). !?~pi &ii/i Rcs. 28: 85-100, 1977. 33. Pure, D. G. and D. J. Woodward. Maturatmn 01 WoKed mossy fiber input to rat cerebellar Purkinje cells. E.rpl B&n Rr.$. 2R: 427-441. 1977. ,I..
__“.
NEURONAL
34. Ramon I’Homme
POSTNATAL
DEVELOPMENT
S. Histologic due Susteme Nerveux de et des Vartebres. Vol. I. Paris: A. Maloine, 1909, pp.
y Cajal,
960-978. 35. Scheibel, M. E. and A. B. Scheibel. Patterns of organization of specific and non-specific thalamic fields. In: The Thalamus. edited by D. P. Purpura and J. M. D. Yale. New York: Columbia University Press, 1966, pp. 13-46. 36. Tilney, F. Behavior in its relation to the development of the brain. II. Correlation between the development of the brain and behavior in the albino rat from embryonic states to maturity. Bull. Neural. Inst. 3: 252-358, 1933.
283 37. Van der Loos, H. Dendro-denditische
verbindingen in de schors der grote hersenen. Thesis, H. Stam, Haarlem, 1959. 38. Wiessendanger, R., M. Wiesendanger and D. G. Ruegg. An anatomical investigation of the corticopontine projection in the primate (Macaca fascicularis and Saimini sciurrus). II. The projection from frontal and parietal association areas. Neamscience 4: 747-765, 1979.