morphogenesis of the trigeminal mesencephalic nucleus in ... · from the departments of anatomy and...

/ . Embryol. exp. Morph. Vol. 31, 1, pp. 99-121, 1974 9 9

Printed in Great Britain

Morphogenesis of the trigeminalmesencephalic nucleus in the hamster: cytogenesis

and neurone death

By KEITH E. ALLEY1

From the Departments of Anatomy and Oral Anatomy,Colleges of Medicine and Dentistry, University of Illinois,

Chicago, Illinois

SUMMARY

The chronology of cellular development in the trigeminal mesencephalic nucleus of thehamster has been studied by light and electron microscopy. Major developmental featuresare compressed into a two-week period that spans pre- and postnatal maturation. Specialemphasis is focused on the sequence of developmental events, with the following pointsanalyzed in this report: cell proliferation, cytological and axonal development, cell death,and cluster formation.

These centrally located primary sensory neurones seem to arise from a separate germinalpopulation found in the alar plate of the midbrain and pons. The neurones are remarkable fortheir precocious formation and characteristic appearance. In hamster embryos they are firstdetected when the neural tube is only a couple of cell layers thick. Prenatally, the neuronesgrow rapidly and have doubled in diameter by birth. Axons form shortly after the cells areformed. This is marked by a sudden upsurge in cytoplasmic tubules and filaments in one endof the soma. Postnatally, cellular growth continues at a reduced rate. However, shortly afterbirth many small spines protrude from the cell body, thus increasing the surface area availablefor nutritive exchange. Myelination of the axon commences 6 days after birth.

Cell counts on a series of pre- and postnatal animals detected a considerable overproductionof neurones in the mesencephalic nucleus. Prior to birth there is a rapid elimination of almost50 % of these cells. Degenerating cells were identified in both light and electron microscopes.The temporal relationship between peripheral innervation, cell death and synaptogenesissuggests a developmental mechanism that provides some flexibility in the formation of syn-aptic connexions in the mesencephalic nucleus.

Clusters of 2-4 tightly packed neurones are a characteristic feature of the developing andadult mesencephalic nucleus. Specialized junctions connect adjacent neurones and are thoughtto provide the basis for electrotonic coupling within the nucleus. Macula adhaerens plaqueswere present in both pre- and postnatal hamsters. However, close appositions were onlyidentified postnatally.

1 Author's address: Division of Neurobiology and Department of Oral Biology, Universityof Iowa, Iowa City, Iowa 52242, U.S.A.

7-2

100 K. E. ALLEY

INTRODUCTION

During the past century the structure of the mesencephalic nucleus of thetrigeminal nerve has been intensively studied in a wide variety of vertebratespecies. From these investigations it has become evident that this nucleus hasmany features that are not characteristic of the primary sensory neuroneslocated in either dorsal root or cranial ganglia. Notable among these specialaspects are their central location (Weinberg, 1928), the presence of synapticendings on the soma (Hinrichsen & Larramendi, 1968, 1970; Alley, 1973), andthe possible existence of electrotonic coupling between small clusters of theseneurones (Hinrichsen & Larramendi, 1968, 1970; Baker & Llinas, 1971).

Functionally the mesencephalic nucleus is believed to mediate proprioceptiveinput from the jaw apparatus (Corbin & Harrison, 1940; Jerge, 1963). Peri-pheral processes of these neurones terminate in annulospiral and flower sprayendings in the spindle receptors of the masticatory muscles (Szentagothai, 1948;Kawamura, Funakoshi, Tsukamoto & Takata, 1959; Jerge, 1963), and in ten-sion receptors located in the periodontal ligament of the teeth (Corbin &Harrison, 1940; Jerge, 1963; Kidokoro, Kubota, Shuto, Sumino, 1968a, b).

Although both the structure and function of the adult mesencephalic nucleusare well documented, little is known about the development of these neurones.Their large size, lack of dendrites, and characteristic location in the midbrainand pons make them ideal subjects for both quantitative analyses of neuronalnumber during ontogenesis and electron microscopic observations of cellularmaturation. Moreover, the dramatic shift of feeding behavior from the ratherstereotyped movements of suckling to the more intricate activity of chewingsuggests a greater role for proprioceptive feedback during mastication. Inpostnatal hamsters the timing of these events seems to be correlated with thematuration of the mesencephalic nucleus.

The purpose of this analysis is twofold: first, it outlines the chronology ofcytogenesis, emphasizing cytoplasmic growth and reorganization, the develop-ment of somatic spines, and the formation of the axon. Secondly, the role ofcell death during morphogenesis of the mammalian mesencephalic nucleus isevaluated by cell counts and histological observation, and the timing of neuronaldeath is compared with the onset of synaptogenesis. It was hoped that informa-tion on the developmental process might provide additional insight into thecentral location of this nucleus.

A previous report of cell death in the mammalian mesencephalic nucleuswas first published in abstract form (Alley & Du Brul, 1972).

MATERIALS AND METHODS

Golden hamsters (Mesocricetus auratus) were procured from Con OlsonAnimal Supply of Madison, Wisconsin. All animals were housed in a constant

Morphogenesis of the mesencephalic nucleus 101

temperature, 12-h light-dark environment. A rigorous schedule of timed matingswas employed and at appropriate intervals from 9 days post coitus (p.c.) tobirth the pregnant females were anesthetized with Nembutal and the fetal pupssurgically removed. Postnatal pups were also anesthetized with Nembutal andcollected at 24 h intervals until 15 days of age. Twenty-five-day postnatal (p.n.)and adult animals were also used. A total of 93 specimens was gathered foranalysis in the light and electron microscopes.

Brains of litter-mates were prepared according to the protocol of Nissl,Cajal reduced silver, and Golgi (Valverde, 1970) techniques. Material to bestained with cresylviolet was embedded in paraffin and serially sectioned ineither frontal or sagittal planes. These brains were used to calibrate neuronalsize and to determine cell number during maturation. Further aspects ofsomatic and axon developments were examined on the Golgi and reduced silverpreparations. However, considerable difficulty was encountered in impregnatingneurones of the mesencephalic nucleus in fetuses younger than 12 days p.c.

Fixation for electron microscopy was accomplished either by direct im-mersion or by vascular perfusion. The fixative consisted of a combination ofaldehydes: 2-5% paraformaldehyde, 2-5% glutaraldehyde, 0-001% calciumchloride, and 0-1 M cacodylate buffer at a final pH of 7-4 (Karnovsky,1965).

Early fetuses were immersed in cold fixative immediately after severing theumbilicus, while older fetuses and all postnatal animals were fixed by vascularperfusion. Each brain was kept overnight in cold fixative. The following daysmall pieces of tissue were dissected from the caudal region of the nucleus in thepons and postfixed in 1 % osmium in 0-1 M cacodylate for two hours. Materialwas dehydrated in graded acetone and embedded in Epon-Araldite. Each piecewas oriented so that the sections would be cut in a coronal plane.

Mallory stained sections, 1 /im thick, were used to localize the mesencephalicnucleus prior to thin sectioning. These thin sections were also of considerableaid in analyzing cell morphology during development. Furthermore, semi-serialseries of 1 jtim plastic sections through the extent of the mesencephalic nucleuswere prepared for the youngest embryos studied.

Thin sections were cut on a Porter-Blum MT-2 ultratome, double stainedwith uranyl acetate and lead citrate and viewed on a Hitachi 7-S electronmicroscope.

Serial-sectioned brain stems from 29 animals were used to count the numberof neurones in the developing mesencephalic nucleus. Cell counts were doneaccording to the selected-section method (Konigsmark, 1970). Every othersection was carefully reviewed and the neurones of the mesencephalic nucleuscounted. Only those cells containing a definite nucleus and nucleolus wereincluded in the data. Final estimates of the size of the neuronal population weredetermined by multiplying the actual cell count by the interval.

Certain difficulties were encountered in identifying accurately the earliest

102 K. E. ALLEY

Fig. 1. Alar plate of the midbrain in 9J days p.c. hamster embryo. Large arrowsindicate the mesencephalic nucleus sandwiched between the mantle layer (top)and germinal epithelium. Note the mitoses located near the nucleus (Small arrows).x800.

neurones of the mesencephalic nucleus in the paraffin sections. From 9\ daysp.c. to 10 days p.c. cytoplasmic outlines were indistinct and the intensely stainedgerminal cells tended to mask their appearance. Later in the gestation periodcare was taken to account for other groups of neurones in the immediate vicinity.Particular attention was paid to the neurones of the locus coeruleus.

Additional quantitative procedures used in the investigation will be describedin the appropriate sections.

OBSERVATIONS

Initial appearance of the mesencephalic nucleus

The mesencephalic nucleus is conspicuous by its precocious appearance inthe alar plate of the midbrain and pons at 9 \ days p.c. At this stage the neuraltube is characterized by a mitotically active ventricular zone and a thin inter-mediate zone. The early cells of the mesencephalic nucleus are sandwichedbetween these two layers (Fig. 1). On the 1 fim. sections these neurones werereadily distinguished from the adjacent germinal cells by differences in nuclearand cytoplasmic architecture.

Although the precise primordia of these neurones could not be identified withcertainty, it is interesting to note that in embryos from 9\ days p.c. to \2\ daysp.c. a few mitotic profiles were encountered at some distance from the ventricu-

Morphogenesis of the mesencephalic nucleus

10-5d.p.c. "o

11 d.p.c.

103

15

15

15

15

15

7 10 13 16 19 22 25/im 7 10 13 16 19 22 25 28/im 7 10 13 16 19 22 25 28/mi

9d.p.n. 15 11 d.p.n 13d.p.n.

7 10 13 16 19 22 25 28//m 7 10 13 16 19 22 25 28 31 //m 1 10 13 16 19 22 25 28 31 34 37 /mi

15 d.p.n.

7 10 13 16 19 22 25 28 31 34 37 40/mi

15 25 d.p.n.

7 10 13 16 19 22 25 28 31 34 37 40 //in

15 Adult

7 10 13 16 19 22 25 28 31 34 3740 43 46 //m

Fig. 2. Histograms of cell diameter in the developing mesencephalic nucleus.Abscissa - neuronal diameter; ordinate - percentage of cells, d.p.c, Days postcoitus; d.p.n., days post natal.

lar surface in close proximity to the cells of the mesencephalic nucleus (Figs. 1,5).Even though additional evidence will be required to pin down the origin ofthese neurones, the close proximity of the mitotic cells suggests that themesencephalic nucleus may arise from an independent primordia within theneural tube.

Somatic growth and maturation

The timing and rate of cell growth and development were evaluated for bothpre- and postnatal neurones. Indices of growth are portrayed as histograms ofcell diameter (Fig. 2) and as estimates of the surface area and volume of the

104 K. E. ALLEY

Table 1. Quantitation of somatic growth during development of the mesencephalicnucleus

Age(days)

10 p.c.10* p.c.11 p.c.12p.c.13 p.c.14 p.c.

Newborn1 p.n.2 p.n.3 p.n.5 p.n.7 p.n.9 p.n.11 p.n.13 p.n.15 p.n.25 p.n.Adult

Av. diameterof soma(/tm)

8-410-611-411 -915-615-817-5.18-519121021-221-420-823-325026-827-7341

Surface areaof soma(/*m2)

22235340844576478496210751145138514111438138417041962225524093651

Cytoplasmicvolume of soma

(/<m3)

31062377885819852030275733153580483649905128418781808567101761124320569

soma (Table 1). All neuronal measurements were done with a filar micrometer onthe Nissl-stained paraffin sections. The size recorded was the largest diameter ofeach profile. At least 100 neurones along the length of the nucleus were measuredat each stage. Data from either frontal or sagittal planes gave comparabledistributions of cell size. It was assumed that this was indicative of a spheroidalshape for their soma. However, it might also suggest that the neurones lack apreferential axis of orientation.

Before birth the cells expand very rapidly. At 10 days p.c. each cell averagesless than 9 /an in diameter. Six days later the diameter has doubled as a resultof a tenfold volumetric increase. Postnatally the cell body grows at a muchreduced rate. However, during this period many other factors of importance tothe final maturation of these neurones take place.

Simultaneously with cellular growth there are marked changes in the organiza-tion of the cytoplasm which are apparent in both light and electron microscopes.Early neurones, 9\ days p.c, are irregularly round, oval, or elongated but fewcytoplasmic processes are present. The large nucleus is eccentrically positionedin the soma and nucleoli are usually flattened against the nuclear envelope. Theremainder of the karyoplasm is granular in appearance and more electron densethan in the adult (Fig. 3). Many free ribosomes or small polysomal clusters areevenly distributed in the cytoplasm. In addition, a few strips of granularendoplasmic reticulum are present, but distinct Nissl bodies were not found.


FIGURES 3-5

Fig. 3. Electron micrograph of an immature neurone in the mesencephalic nucleus,9 | days p.c. x 15000.Fig. 4. Mesencephalic neurone and process 10J- days p.c. x 8000.Fig. 5. A cluster of three neurones in the mesencephalic nucleus of an 11 days p.c.fetus. Note the mitotic cell (asterisk) surrounded by these neurones, x 4300.

K. E. ALLEY

FIGURES 6, 7

Fig. 6. Two somatic spines forming palisades adjacent to a small synapse, x 26000.Fig. 7. An early spine configuration, similar to Fig. 6, in a 5 days p.n. hamster pup.x 26000.

Mitochondria are also distributed throughout the cytoplasm, while stacks ofGolgi cisternae and vesicles are confined to the perinuclear area. Very fewfilaments or tubules could be identified at this stage.

By 10^ days p.c. cell processes are frequently found in the neighborhood ofthe mesencephalic nucleus (Fig. 4). The eccentric orientation of the nucleusconfines the cytoplasm to one pole of the cell, and the cell processes arise fromthis side of the soma. Ribosomes exist free, as rosettes or polysomal chains, butthere is also an increase in the number of membrane-bound particles. The mostremarkable change is the tremendous upsurge in the number of neurotubules andfilaments at one pole of the neurone.

A major re-ordering of the cytoplasmic organelles begins about 12 days p.c.(Fig. 5). Most prominent is the increasing basophilia of the peripheral cyto-plasm as a result of the condensation of ribosomes (Fig. 5).

Postnatally, free and membrane-bound ribosomes coalesce into distinctNissl granules that form a peripheral basophilic ring. Small patches of Golgiapparatus also migrate into this region, and by 4 days p.n. the cytoplasmicfeatures of these neurones are generally complete.

Somatic spines

Small thorns protrude from the neurones of the adult mesencephalic nucleus(Fig. 6). Hinrichsen (1968) suggested that these may be vestiges of the largerprocesses sometimes observed during cellular development. However, otherobservations on the cerebellum have implicated spine formation with synapto-


Table 2. Quantitative features of somatic spines on mesencephalic neurones

Age(daysp.n.)

57

101225

Adult

Perimetersurveyed

(/im)

16721152755

1014965

1727

Spinescounted

18212885

10676

118

Density(spines/100 /*m)

10-911111-310-57-9

6-8

Diameter*of spine

base (/*m)

0-280-260-270-260-26

0-27

* Average measurement of 20

Perimeter*of spine

(jim)

2-442-512-242-302-32

2-35

spines.

Calculatednumber ofspines/cell

540615690720740

920

Perimeterincreasedue to

spines (%)

2119201816

14

genesis (Larramendi, 1969). In order to determine if either of these was applic-able to the mesencephalic nucleus a developmental analysis of these spines wasundertaken.

Prenatally the cell membrane is smooth and devoid of any outpocketings(Fig. 3, 4, 5). Shortly after birth the cell outline becomes more undulating andtrue spines can be recognized 3 or 4 days later, and by 4 or 5 days p.n. the cellsurface forms a smooth boundary punctuated by definite projections (Fig. 7).The density of spines increases at 5, 7, and 10 days p.n. when the actual surfacedensity is greater than in the adult (Table 2). Subsequently, the number ofspines per 100/tm of perimeter decreases slightly at 15 days p.n., and by 25days p.n. the density is approximately equivalent to the adult figure of 6-8spines per 100 /an of perimeter. This sort of density analysis does not take intoaccount the continued expansion of the surface membrane, thus a decrease insurface density could reflect either the gradual growth of the cell surface or theregression of some spines during maturation. Estimates of the number of thornsprojecting from an average neurone were determined for the mesencephalicnucleus at 5, 7, 10, 12, 25 days p.n. and in the adult (Table 2). The methodemployed was previously used to analyze synaptogenesis in this nucleus and isdescribed in detail elsewhere (Alley, 1973). Briefly, the calculations include thefollowing. First, the percentage of the cell perimeter giving off spines wasdetermined from random cell profiles (spine density x base diameter of spine4- 100). Subsequently, this percentage was used to compute the actual area ofthe surface emitting spines (total surface x percentage of perimeter covered).Finally, the number of spines per soma was estimated by dividing this portionof the total surface area by the average cross-sectional area of the spine base(spinous surface area •*• cross-sectional area of base). According to thesecalculations, even though spine density decreases dramatically in the periodfrom 15 days p.n. to adulthood, the actual number of projections continues togrow (Table 2). Additional computations that account for the surface added by

108 K. E. ALLEY

Fig. 8. Schematic representation of the adult mesencephalic nucleus portraying theextended length of the axon from the soma to its bifurcation. Taken from Cajal(1911).

the spines indicate that the contribution they make to the total area of the somadeclines with age (Table 2).

Although the timing of spine formation and synaptogenesis coincide, theyare only rarely associated in direct synaptic contact. This situation is truefor both adult and developing hamsters. However, it should be noted thatterminals frequently lie near the base of a spine, but the adhesion is on thesoma (Figs. 6, 7).

Development of neuronal polarity

Cajal (1929) and Tennyson (1965) have outlined the morphologic transforma-tions that occur during cellular maturation in spinal and cranial ganglia. Thesecells begin as simple apolar neurones, pass through a phase of bipolarity, andeventually form a stalk from which the central and peripheral processes aredirected.

Neurones of the mesencephalic nucleus emit a single, large process that passesinto the mesencephalic root. Cajal (1911) indicated that in contrast to adult


Fig. 9. Golgi section and drawing of somatic and axonal structure in themesencephalic nucleus 14̂ - days p.c. x400.

ganglionic neurones, this axon does not bifurcate in the region of the cell body(Fig. 8), but passes into the pons where many collaterals are given off in theregion of the trigeminal motor nucleus. These provide the basis for the mono-synaptic activation of the motoneurones (Szentagothai, 1948; Jerge, 1963;Kidokoro et aJ. 1968 a).

As a reflexion of this adult configuration young neurones in the mesencephalicnucleus appear to bypass the true bipolar phases of development. Instead, eachapolar cell may sprout one or more exploratory processes. Eventually one ofthese becomes dominant while the others appear to be redundant and areusually lost later in development. These findings account for the earlier reportsof a decreasing number of multipolar neurones found with advancing matura-tion of these cells (Pearson, 1949 a, b).

In the hamster this metamorphosis takes place between 9-5 and 14 days p.c.Neurites first appear at 9-5 or 10 days p.c. when cells with multiple short pro-jections are present. By 12 days p.c. the polarity of most neurones is established,with the axon emanating from the pole of the cell opposite the nucleus. Theseaxons have been traced for long distances in 13-, 14- and 15-day fetuses with noevidence of bifurcation (Fig. 9).

After birth the axon increases greatly in diameter as the axoplasm matures.Myelin wrappings first appear around the fibers of the mesencephalic root at6 days p.n.

110 K. E. ALLEY

Table 3. Cell counts in the developing mesencephalic nucleus of the hamster

Neuronal NeuronalAge count count

(days) (right side) (left side) Total

AdultAdult25 p.n.25 p.n.15 p.n.13 p.n.11 p.n.9 p.n.8 p.n.7 p.n.6 p.n.5 p.n.4 p.n.3 p.n.2 p.n.1 p.n.1 p.n.

NewbornNewborn15 p.c.14 p.c.13 p.c.13 p.c.

121 p.c.12J- P.c.12 p.c.

Ill p.c.11 p.c.10 p.c.

7949848509047049168408209509848048328368529747948648448961024116411281190145414221448159214041166

86892082491873891680880086094682484084889410728028468568841056118612841134150814341596142414361076

16621904167418221442183216481620191019301628167216841746204615961710170017902080235024122324296228563044301628402242

Cell death in the developing mesencephalic nucleus

Preliminary data from a small series of pure-bred dogs suggested that manymore neurones are produced than ultimately reach maturity. Since these initialobservations were confined to a limited number of animals, additional quantita-tive data were gained from the more extensive series of developing hamsters.Furthermore, it was thought that the period of cell death would be compactedinto a short span which might provide ample opportunity to observe thedegenerative sequence.

The first age for which reliable quantitative data are available is 10 days p.c.At this stage a bilateral total of 2242 neurones was counted. The definablepopulation increases over the next 36 h, reaching an ultimate of over 3000neurones at 12 days p.c, which is almost twice the adult number (Table 3).During the final days before birth a precipitous decline in cell number occurs. Bybirth the nucleus is almost numerically equivalent to the adult. A comprehensive


38 -

30

o

=s 14

u

- 1 — •

/ / I I I I I

II 13 15

Prenatal

I ' ' ' I I I I I I I

3 5 7 9 11 13 15 25 Adult

Postnatal

Fig. 10. Graphic representation of the unilateral cell total in the mesencephalicnucleus during maturation.

listing of the cell counts is presented in tabular and graphic form in Table 3 andFig. 10.

A search for degenerating neurones was carried out on both pre- and post-natal animals. A few degenerating cells were first identified histologically at 13days p.c. Although only a limited number of dying neurones was observed atany stage, they were most numerous just before birth (Fig. 11). In addition, acouple of instances of dying cells were noted in postnatal animals.

In the light microscope moribund cells were characterized by both nuclearand cytoplasmic changes. The nucleus becomes irregular in shape and stainsmore intensely. The cytoplasm exhibits some swelling and premature peripheralpacking of its ribosomes. Later it becomes very basophilic and disrupted.

Attempts to localize degenerating neurones with the electron microscopewere only partially successful. A couple of examples of early nuclear andcytoplasmic changes were recorded (Fig. 12). The nucleus is more electron denseand its boundary irregularly involuted. The cytoplasm is full of ribosomes.

Although the initial phases of cell degeneration occur rapidly, later eventsmust proceed at a more leisurely pace. Postnatal animals show an accumulationof cellular debris around the viable neurones. The most usual appearance is alarge, round, electron-opaque core surrounded by a granular halo. Frequentlythese particles had been engulfed by glial processes (Fig. 13).

Neuronal clusters

Tightly grouped^aggregates of neurones are a normal occurrence along thelength of the mesencephalic nucleus. Recently, Hinrichsen & Larramendi(1968,1970) identified specialized zona adhaerens and zona occludens junctions

112 K. E. ALLEY

•^3- ^STO ^T»i

«• '•'It%5«fc

* •

JV! u*«

>̂ '

?• 1

w:M

Fig. 1J. Degenerating neurones (arrows) intermingled with normal cells in themesencephalic nucleus at 14^ days p.c. MR - mesencephalic root, LC - locuscoeruleus. x 190.

between these closely applied cells in the mouse. Similar junctions were identi-fied in the adult hamster (Fig. 16, 17).

Individual clusters in developing hamsters have been examined under theelectron microscope in order to determine the time of formation of thesespecializations. At 9j, 10^ and 11 days p.c. cell membranes of adjacent neuronesmay parallel each other for a considerable distance without any definite con-tacts. Small macula adhaerens junctions were identified 12̂ - days p.c. when singleor multiple adhesions dot the surface (Fig. 14). Similar kinds of appositionswere also found in newborn and postnatal animals of 5, 7, 12, 15 and 25 daysp.n. (Fig. 15). The overall length of junctions is longer in older animals. Obser-vations on the formation and development of the zona occludens junctions werenot clear. A suitable plane of section through these junctions is only rarelyencountered.


FIGURES 12, 13

Fig. 12. Low power electron micrograph of early cellular degenerative changes ina newborn hamster, x 3500.Fig. 13. Terminal stages of cellular degeneration are represented by glial phagocytosisof cellular debris, x 9000.

DISCUSSION

Morphogenesis of the trigeminal mesencephalic nucleus in the hamsteroccurs during a 2-week period that spans pre- and postnatal development. Theoutstanding events of ontogeny are schematically illustrated in Fig. 18. Asillustrated these include: A, the formation and initial cytodifferentiation ofneurones; B, development of cellular polarity and cell loss; C, formation ofintranuclear contacts; D, outpocketing of somatic spines; E, synaptogenesis;and F, myelination of the mesencephalic root. In the following sections the firstfour of these topics will be analyzed in terms of associated developmentalmechanisms.

Neuronalformation and cytological maturation

It has been assumed that neurones of the mesencephalic nucleus developfrom neural crest elements that are either retained in or migrate into the neuraltube after closure (Johnston, 1909; Herrick, 1948; Piatt, 1945; Rogers & Cowan,1973). Indirect evidence presented here also suggests that these cells arise froma germinal population other than the ventricular zone of the alar plate. Mitoticcells were frequently found some distance from the luminal surface in closeproximity to the mesencephalic nucleus. In addition, their presence correspondsto the time when the number of neurones in the mesencephalic nucleus isincreasing.

8 E M B 31

K. E. ALLEY

FIGURES 14-17

Fig. 14. Two small macula adhaerens junctions (arrows) at the interface of clusteredneurones in the mesencephalic nucleus 12^ days p.c. x 60000.Fig. 15. Multiple attachment plaques anchor two neurones together in 5-day p.n.hamster, x 26000.Fig. 16. High power electron micrograph of zona adhaerens junction in the adultmesencephalic nucleus, x 140000.Fig. 17. Region of close apposition between adjacent neurones in the adult nucleus,x 90000.

By 9j days p.c. some neurones of the mesencephalic nucleus are apparentin the alar plate of the midbrain and pons. Their conversion from germinalcells into immature apolar neurones is characterized by cytoplasmic changesidentical to those described for other systems (Lyser, 1964; Tennyson, 1965;Fisher & Jacobson, 1970). Most notable of these are the rapid increase incytoplasmic volume and the accumulation of ribosomes and membranous

Morphogenesis of the mesencephalic nucleus

A B

115

Fig. 18. Schematic summation of cellular development in the mesencephalic nucleus.

organelles. Neurotubules and filaments are usually sparse and confined to theperinuclear cytoplasm. A number of authors have implicated proliferation ofneurotubules with the initiation of axonal development (Tennyson, 1965;Lyser, 1968; Fisher & Jacobson, 1970). In the mesencephalic nucleus prolifera-tion of neurotubules and the formation of the neurites take place soon afterthe initial morpho-differentiation of the apolar neurones. By 10̂ - days p.c.cell processes are abundant, and in those instances when continuity betweensoma and neurite could be determined, a rich meshwork of tubules and fila-ments was present in the soma.

Neurones in the mesencephalic nucleus do not pass through a true phase ofbipolarity. Instead, each cell sends out a number of processes. Most are veryshort and penetrate only a short distance from the cell body, giving the cell amultipolar appearance (Pearson, 1949 a, b; Hinrichsen & Larramendi, 1969).Later in development many of the shorter neurites are lost, leaving one mainprocess to enter the mesencephalic root. Around 13 or 14 days p.c. most neur-ones have attained their adult configuration. However, a few neurones maintainthese short processes even into adulthood.

As previously noted, Hinrichsen (1968) suggested that the adult somaticspines might represent vestiges of the lost neurites. However, two facts argueagainst this interpretation. First, there is a time gap of almost one week betweenthe loss of neurites and the onset of spine formation, and during the interim thecell surface is smooth. Second, the actual number of spines is far in excess ofthe number of early processes.

Perhaps a more adequate explanation of spine function can be gleaned from

116 K. E. ALLEY

an analysis of cell growth. It has been well established that the perikaryon isthe metabolic hub of the neurone. The enzyme systems for the production ofcytoplasmic proteins are concentrated in the cell body. Materials are manu-factured here and shipped to the far reaches of the axon (Weiss & Hiscoe, 1948;Droz & Leblond, 1963). Thus it seems crucial that a sufficient surface must beavailable for nutrient exchange. Since the surface area of the soma increasesas a square function while the volume expands as a cube function of the celldiameter, perhaps the tremendous increase of cytoplasmic and axoplasmicvolume during development may outstrip the surface available for exchange.The accumulation of spines provides a significant increase in surface areaboth during postnatal maturation and in the adult. Although their total effectdwindles with time, they still provide a substantial (14 %) increase of somaticmembrane in the adult.

In other systems the presence of spines has been extensively used as anindex of the presence of synapses. Particular attention has been devoted to thePurkinje cell (Hillman, 1969; Larramendi, 1969) and the pyramidal cells ofthe cerebral cortex (Pappas & Purpura, 1961; Jacobson, 1967; Peters &Kaiserman-Abramof, 1970). In contrast to these dendritic spines, somal thornsof the mesencephalic neurones show no special affinity for synaptic endings.

A deeper understanding of spine function has been provided by analyzingthe development of the climbing fiber-Purkinje cell association (Larramendi &Victor, 1966; Kornguth, Anderson & Scott, 1968). Climbing fibers first contactspine-like processes on the soma. As these fibers migrate over the soma and onto the main dendrites the spines regress. Possibly the transitory spines providea guidance system for the wandering climbing fibers. The close timing of spineformation and synaptogenesis in the mesencephalic nucleus initially suggestedthat a similar mechanism may be operant here. However, closer inspection ofthese two events did not substantiate this notion. From their first appearance thespines are totally independent of the developing synapses.

Neuronal death as a morphogenetic mechanism

Genetically planned patterns of cell degeneration are an important morpho-genetic mechanism in vertebrate and invertebrate development (Gliicksmann,1951; Lockshin & Williams, 1965; Saunders, 1966; Saunders & Fallon, 1966).Definite localized arrays of moribund cells occur at selected instances through-out ontogenesis. They help to determine the final size and mold the shape oforgans and organ complexes.

Cell death has also been implicated as a morphogenetic mechanism in thedeveloping nervous system. Dying cells free the early neural tube from the over-lying ectoderm (Gliicksmann, 1951), help shape the optic cup (Silver, 1972),and determine the final size of various neural centers in the spinal cord andbrain stem (Hughes, 1961; Prestige, 1965, 1967a, b; Cowan & Wenger, 1967;Rogers & Cowan, 1973). These authors have indicated that the normal matura-


tion of central-peripheral connectivity is not based on a 1 to 1 relationship, butin fact many more young neurones are produced than eventually connect withthe periphery. Ultimately, the size of a mature neuronal pool is determined by anintricate balance between cellular proliferation and degeneration; and the fateof each neurone is directly dependent on its ability to establish effective contactsin the periphery. Those unable to do so cannot maintain themselves, and quicklyregress. To date, quantitative analyses on the magnitude of this spontaneousregression have concentrated on submammalian vertebrates. Hughes (1961)and Prestige (1965, 1967#, b) counted viable and dying cells in the ventral hornand spinal ganglia of Xenopus laevis and found that there is a continuousproduction and turnover of neurones in these larvae. From a total populationof 10000 neurones only about 1200 reach maturity. In other words, 9 out of 10cells end up in death. Similar investigations of chick trochlear motoneurone(Cowan & Wenger, 1967) and mesencephalic neurones (Rogers & Cowan,1973) have depicted a less severe reduction, averaging 50 and 70 % respectively.

Overproduction of neurones in the mesencephalic nucleus of the hamsterindicates that neuronal death may also play an important role in the ontogenesisof mammalian brains. Although the degree of cell loss in the hamster is muchless exhaustive than in the amphibian spinal cord, it is in general accord withthe decreases noted in the chick. These differences are at least partially accountedfor by the apparent lack of continuous neuronal production and turnover inthe nervous system of higher vertebrates. In contrast, the proliferative and cell-death phases of development occur sequentially. By 12 days p.c. the mesen-cephalic nucleus is numerically complete. These quantitative data along with theapparent lack of degenerating cells prior to this time suggest that no newneurones are added once cell death commences. Perhaps this is an adaptivemodification to limit unnecessary cell wastage, and thus increases the efficiencyof neurogenesis in the bigger-brained higher vertebrates.

Experimental investigations of limb development in submammalian vertebrateshave begun to unravel the complex central-peripheral relationships that occurduring neurogenesis (Hamburger & Levi-Montalcini, 1949; Prestige, 1961 a,b;Cowan, Martin & Wenger, 1968). In essence, three periods are evident andthese correspond to an ever-increasing reliance of the nerve cell on the periphery:

1. A proliferative, pre-axonal stage when the neurone is relatively free fromperipheral influence,

2. A saturation period, as the over-abundance of cells send their axons intothe target organ, and

3. A consolidative phase when definite connexions are formed and the sur-vival of selected neurones is guaranteed by virtue of these peripheral contacts.

To date no information is available on either the growth of axons from themesencephalic root or the ontogenesis of muscle spindles in the jaw muscles.However, the chronology of developmental events within the mesencephalicnucleus suggests that a similar time table may be applicable. The temporal

118 K. E. ALLEY

relationship of axon development and cell death hints that many cell processesreach the jaw muscles about 12 days p.c.

Of added importance is the timing between cell death and the onset ofsynaptogenesis. It would seem to be a futile exercise to form synapses on neuronesthat will not survive. In the mesencephalic nucleus cellular degeneration isgenerally finished at birth, while the early events indicative of synaptogenesiswere first found four days later (Alley, 1973). This assures that synaptic termi-nals will contact only neurones that have already made connexions in the peri-phery and are sure to survive. Aside from the added economy of this sequence,it also introduces a certain level of plasticity into the development of this system.For example, any modification of the periphery, such as an increase or decreasein the muscle mass, will modify the level of cellular degeneration in the mesen-cephalic nucleus (Piatt, 1946; Rogers & Cowan, 1973). If more muscle is availableto the ingrowing nerve fibers one would expect fewer neurones to die. Further-more, the effect of peripheral modification would not be limited to the primarysensory neurones alone. Changes in neuronal number would also effect thesynaptic relationships of those fibers making contact with the cells.

The importance of this sequence and the flexibility it imparts can be bestexplained in terms of adaptive evolutionary change, since any modification ofthe periphery must be accompanied by concomitant alterations of the associatedneural subsystems. Du Brul (1960) has shown that cell number in the adultmesencephalic nucleus of a wide variety of vertebrates is correlated with thesize and complexity of the jaw muscles. In a later report (Du Brul, 1967) heproposed that the target site for evolutionary modification is in the periphery,and that adaptive changes occurring here somehow impress themselves on theappropriate neural centers. Evidence presented here on the chronologicalsequence of cellular proliferation, axon formation, cell death and synapto-genesis provides a plausible developmental mechanism whereby peripheralchanges can alter the size and synaptic patterns in the relevant neural centers.

Intranuclear cell contacts in the mesencephalic nucleus

Cellular interactions are of fundamental importance in the developing as wellas the mature nervous system. One aspect of membrane adhesions receivingmuch attention is their role in morphogenesis. Tight junction and low-resistancecoupling have been found in a number of embryonic systems (Sheridan, 1968;Trelstad, Revel & Hay, 1966). Loewenstein (1967) and Furshpan & Potter (1968)have implicated these junctions in the passage of regulatory molecules directlybetween cells, which could coordinate the growth and differentiation of cellgroups. Pannese (1968) found macula adhaerens and occludens junctionsbetween early primary sensory neurones in the dorsal root ganglia of chicks.However, these appositions are only temporary in nature and are later priedapart by the infiltration of glial cells. No trace of these junctions is left in theadult fowl. Identical macula adhaerens junctions were first identified between

Morphogenesis of the mesencephalic nucleus 119adjacent mesencephalic neurones at 12 days p.c. From this time on they werefrequently found. The earliest adhesions are small, single junctions. Later indevelopment the occurrence of multiple densities becomes more prevalent andsome of these may extend 7 fim. or more in length. Sheffield & Fischman (1970)have suggested that these junctions may serve to stabilize intercellular relation-ships in rapidly growing systems. However, their increasing size with age andpersistence in the adult seem to indicate that they do more than simply anchormesencephalic neurones together. Maybe they encircle patches of the cellsurface, isolating these from the surrounding extracellular space and creating aspecialized intercellular environment. This area could be conducive to theformation of low-resistance electrotonic synapses. A search for zonula occlu-dens junctions in the prenatal hamster was unsuccessful. This may reflect theirsmall size or labile nature, making identification extremely tedious.

It is interesting to speculate that the membrane specializations could serve adual role in the ontogeny of the mesencephalic nucleus. Initially they may besimilar to those observed in other embryonic systems, serving to coordinatematuration of the nucleus. However, in contrast to spinal ganglia neurones thejunctions are not separated by glia. These persist into adulthood and providethe basis for electrical coupling in the adult nucleus (Hinrichsen & Larramendi,1968, 1970; Baker & Llinas, 1971).

The author is grateful to Drs E. L. Du Brul and L. M. H. Larramendi for their continuousadvice and encouragement.

This paper is based on a thesis submitted in partial fulfillment of the requirements for thedegree of Doctor of Philosophy in Anatomy, June 1972. The work was supported by USPHStraining service grant GM-00643.

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{Received 4 April 1973)

morphogenesis of the trigeminal mesencephalic nucleus in ... · from the departments of anatomy and...

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