Growth and target finding by axons of the corticospinal tract in prenatal and postnatal rats

GROWTH AND TARGET FINDING BY AXONS OF THE CORTICOSPTNAL TRACT IN PRENATAL AND POSTNATAL RATS D. J. sCHKLiYI:K and E. G. Jn~us James L, O’Leary Division of Experimental Neurology and Neurological Surgery, and McDonnell Center for the Study of Higher Brain Function. Departments of Neurology and Neurologic~~l Surgery. and of Anatomy and Neorob~o~o~y, Washington University School of Medicine. St. Louis. Missouri 631 10. U.S.A. Abstract .-The growth of the corticospinal tract was studied in prenatal and neonatal rats using the anterograde transport of horseradish peroxidase injected into the cerebral cortex as a marker in fightmicroscopic preparations. The findings were compared with electron-microscopic observations on normal material at the same ayes. Labelfed corticofugal axons traverse the dien~~phafon by gestational day 17.5. reach the pontine nuclei by gestational day 19.5, and the caudal limit of the medulla oblongata by gestational day 20.5, just before birth. On the day after birth. labelled corticospinal axons have crossed in the pyramidal decussation and extended into the dorsal columns of the upper cervical spinal cord. C’orticospinai axons reach the thoracic segments by postnatal day 3, the lumbar segments by day 6 and the sacral segments by day 9. The lower end of the spinal cord is reached only after postnatal day 14. Beside the principal carticospinal tract m the dorsal columns. two other smaller corticospinal tracts occupy an intermediate position in the base of the cervical dorsal horn and a lateral position in the lateral white column. The intermediate tract is not found helow cervical levels. Growth cones are seen at the tips of axons in light- and electron-mlcroscoplc material. The first corticospinal axons. less than 03 microns in diameter and grouped in tight fascicles, grow through a dense fabric of astrocptic and other glial processes in which no obvious prc-existing channels could be identified. Growth of corticospinal axons into the dorsal horn adjacent to the main tract is delayed until 2-3 days after the initial arrival of the tract at a given segment. This begins in the cervical segments only after the t~~aIarna~art~~d1fibers have invaded the sensory-motor carfex though the parent pyramidal cells of the tract are still highly immature. The rate of extension of corticospinal axons is not constant. Growth down the dorsal columns is characterized by accelerated growth spurts on postnatal days 4 and 9. Much slower growth characterizes initial outgrowth through the diencep~l~ilon and later ingrowth into the spinal gray matter. There rs approximately a three-fold increase in the numbers of cortlcospirial axons in the cervical segments between postnatal days 5 and 10. Myehnation commences between postnatal days IO and I?. It is concluded that the d~velopn~~l~l of the cortico~pinal tract in the rat displays fcntt~rcs that xc common to other developing paihways in the rat and other specks. Initial outgrowth of cnrticospinai axons 1s mdependent of at&rent innervation. occurring at a time when the pitrent cell bodies ~tre ver? immature. The early growth of corticospinal axons to the licinlty of their targets 1s followed h) u substantial waiting period, comparable to that seen in other systems. before final invasion of the target. The factors responsible for the initiation of the second growth spurt. carrylng axons into the target gray matter are not known. However. the fin;rl invasion of prq matter takes place oniy after the cells of origin of corticospinal XIOI~Shave received a slibst~[ntial a&rent input. The rate of initial growth of corticospinal axons down the dorsal columns i> not constant, but v;tries from region to region. Electronmicroscopy has falled to detect any morphological cvidcnce for factors that might guide or promnte the growth of oorticospinal axons. The majority of eorticospinal axons exclusive of the first ‘patb~nders’ seem to grow as tight fasckles in which individual axons contact only one another.

Previous studies of the development of alkenr and efferent connectivity in the somatic sensory and motor cortex of the rat have revealed features suggesting several phases of axon growth, possibly directed by separate mcchanisms~ Thafamic afferents to the sensorimotor cortex accumulate in the white matter underlying the cortical piate during the 17th through -,~bb~~,~iuri(~~i.~: E. embryonic day; HRP, horseradish peroxidase: P. postnatal day: PCST. principal corticospinal tract; Si. first somatic sensory area.

20th days of gestation but do not invade the overlying cortex until the day of birth (day Zl).“’ A similar waiting period is observed during geniculostriate innervation in the rat I3 and on a more protracted time scale in the thalamic innervation of cat sensorimotor cortex31 and monkey striate corte~.“~~~ The axcms of the corpus callosum connecting the two sensorimotor cortices also follow a similar pattern of early accumuIation and later ingrowth during de~eIopment. but the growth of callosal fibers into the cortex is delayed a few days behind that of the thalamic fibers.““.34 This

1837

IX38

Schreyer

D. J.

and F.. G. Jones

suggests that differential growth signals may exist, but their nature is undetermined. It is possible that the phenomenon of early axon growth towards a target followed by a waiting period after which a new growth spurt carries the axons into the target to form synapses. is a feature common to may neural pathways. The corticospinal tract of the rat is amenable to analysis in these terms because of its length and because it develops over a rather protracted time course. largely after birth.3 A delayed innervation of the spinal gray matter by corticospinal axons has already been reported in the rat“ and hamster.20 It is also of interest to determine whether any correlation exists between the maturation of the corticospinal tract and arrival of innervation in the cortex from which it arises, In the sensorimotor cartex of both the cat and the rat.“‘.“’ there is evidence that the outgrowth of corticospinal axons may occur independently of afferent innervation and even before the elaboration of a substantial dendritic tree by the parent cells. but it is not clear whether cortical innervation leads or follows the invasion of the spinal gray matter by the corticospinal fibers, We have employed the anterograde transport of horseradish peroxidasc to label growing corticospinal axons in the fetal and neonatal rat. We have exploited the extended geometry of the tract in order to provide accurate temporal and morphometric data relating to the questions outlined above. These data arc supported by electron-microscopic observations in which it has also been possible to describe the nature of the ‘substrate’ over which the corticospinai fibers must grow. Some of the results have appeared in preliminary

Postnatal 50”,, point to

coronal

younger) received

suture.

from

three

the

suture

PI

cortex

All

perfusions

postfixed 3O”,,

for

secttons planes

tn

All

Two

phosphate

thionm. traced

with

it camera

peroxidase

transport

(HRP.

corticospinal

Sigma

axons

of the type

2lst

VI)

m prenatal,

rats bred in our laboratory. day of gestation

was

neonatal

Only

were

or postnatal

day 0 (PO). The day upon

embryonic animals

day

either smears

(EO). The

0

to

and adult

Iintrcatcd

from

embryonic

dnd

study.

for

animals,

subjected gestation

to laparotomy under chloral

immersed

in a water bath filled

females

were

saline at 37°C.

The heads of three of four fetuses were in each case identified and sutured made

in the of SOP/,HRP

nal suture

to the uterine

uterus

was injected

at a point

glass

micropipette

t&14

h the mother

were

removed

and

wall.

A small

and in the fetal skull into the cortex

223 mm lateral (tip

diameter

was re-opened fixed

E17.5. E19.5 and E20.5).

by

opening

and 0.05 beneath

microns).

and the injected

transcardial

was

microhter the coro-

to the midline, 100

or

magnification.

tract.

the total

The

length

sections digital

or

of

levels

electron-micro-

analyser.

The

a~ the trme of

br,rrna

were tixcd

by

and 5”,, acetic :tcrd in X0”,, ethalo”,,

>cctioned

neutral

forrnalin.

at 20 rnrcrorr\

embedded

rn the

sagrttal

Other hyde

Irttermutes

and

were perfused

l..S”,, glutaraldehyde or with

a mixture

of 1.5”:,

0.5”,, dimethyl

lcrn in 0.1 M

phosphate

buffer

ber’\c blocks

were then

taken

regions

of the spinal

The

embedded on

the

grid

in a Zeiss

I tnicron

blue and azure

thick

(pH cord

lead

EM9S sections

phosphate

buffer

glutaraldehyde.

7.4). Sagittal ccrtical,

or trans-

thoractc

posttixed

resin

and

cttratc”

They with

and

for eiec-

in thin

electron-mrcroscope. were stained

?“,,

and 0.5”;, acro-

and prepared

were

Spurr’s

with

A“,, paraformalde-

sulfoxidc

from

blocks in

uttlt

in 0.1 M

osmium sections

were

then

Corremethylene

11.”

RESU 1.1 S

is termed

on the 17th, 19th or 20th day of hydrate anesthesia while partially with normal

wrth

photographed

I

experimental

pregnant

on gela-

The

are those at the time of death.

For the study of prenatal

tett-;I-

plane

lative

spermatozoa

of the mothers

rtsrng

rnotrnted

w’erc also prcpar&

in 5”,, formalin

paraffin

the spinal

or horizontal

of axon& .II sc!ected

animals.

nol (Jr by pet-fustcm with

examined

Wistar

day 21 (E21

which

ages given

label

at low

traced

littermatcs

immersion

in

frozen

c~~untet stained

then

cortlcospinal

of the inJected

starned

born on the

used for postnatal

is thus

in vaginal

horseradish

used

those litters

day of birth

were observed

termed

enzyme

lucida

using a Zeiss MOP-3

fixatmn

micron

FIRP

wcrc

wet-e

cord and the number

measured

graphs

sections

sections

of the labelled

the spinal

and

overnight

the chromogen.”

~5

Relevant

phos-

H;L~ removed

plane. or. *rth for

VI I”,,

IM

m (f

tn the sagrttal

reacted

coronal

I: mixture

Fini

slides and one of these wit\

tetroxide. anterograde

were

HRP

perfusion.

m~rner~cd

huffer-.

to the hrarn.

serves of alternate

trn-subbed

iron-mtcroscopy,

The

to 4 h. then

secttons

~,i‘ the

MIIII

ncuraxts

rats

11f 5(t”,,

edpc

out

and

IWO adult

glutaraldehyde

7.41. The entire

henlidine

lumbar

PROCEDURES

carried

anterior

tn a serves of ages

transcardral

were cut tn the tranaver\e

methyl

were

anterior-

and 2.5”,

cord still attached

length

resultrng

at a

(P!

14 h

of 0.2 mtcroliter\ the

30 mm

sucrose

1 :! mm

and

of

syringe

survived

4X h before

were

(pH

phate buffer

ir.1 microliter

to P14. Additionally.

along

and survived

pamformaldehyde.

FXPERIMENTAL

with

Hamrlton

Animals

injections

p:traformaldchyde

in

injected

a I microliter

or 4X h (PS and older)

at perfusion mto

were

through

2 3 mm later-al to the midline

the

(pH 7.4)

form.”

animals

HRP

perfusion

using a After

fetuses (ages

‘Plw ,itr.s (!I ~njec’tions. The in)ect)ons in this study were large and directed at the first somatic sensory (SI) and motor areas.7.3” Pyramidal cells with somata in layer Vb of these areas are the origin of the majority of corticospinal axons.35 Our injections almost always included these areas and adjacent cortical regions in animals sufficiently old for the cytoarchitecture to have become obvious. In younger animals, the injections were large enough presumptively to involve the same areas. The striatum and the hippocampus of the same side were often ikluded in the injection with occasional spread to other basal nudei and the contralateral cortex.

Growth and target findings in prenatal Prrnatul derrlopment. At E17.5, the earliest time examined. many labelled axons can be seen descending from the cortex in the rostra] portions of the internal capsule. However. at this age, no labelled fibers can be seen caudal to the level of the diencephalon. Corticofugal axon tips at E19.5 have grown into the cerebral peduncle and have reached the pontine nuclei. The labelled’axons in the pyramidal tract continue to extend over the pontine nuclei and regain the ventral surface of the caudal pons by E20.5. Thereafter. labelled fibers continue to extend along the ventral surface to the caudal limit of the medulla, but do not begin to decussate till about the time of birth. Rostrally, at the ventral midbrain, the developing cerebral peduncle is now thicker and more densely-labelled. presumably due to the addition of more cortical axons. At this time no obvious labelled axons have departed from the tract as it traverses the pontine nuclei (Fig. 1D) as will happen after birth (Fig. 1E). In our material, E20.5 was the earliest age that we could distinguish expansions at the tips of growing axons, a constant feature of the neonatal material (see below). Derelopment of the pyramidal decussation and innerration qf the cercical segments. We were unable to prepare material for age E21/PO because of the problems of performing surgery at the time of birth. On the day after birth (PI) labelled corticospinal axons have decussated as a series of bundles and extended a few millimeters into the upper cervical spinal cord. After crossing, corticospinal fibers come to lie just dorsolateral to the central canal (Fig. 1). The majority of fibers abruptly bend dorsomedially. enter the ventralmost part of the dorsal columns and, making a third deflection. extend caudally from there. A few fibers do not bend back toward the midline. but continue dorsolaterally and enter lateral and intermediate corticospinal tracts or curve rostrally toward the dorsal column nuclei. At early stages. the decussation often looks less orderly than in the adult with a few fascicles or single fibers charting abnormal courses away from the principal bundles (Fig. IB). These aberrant fibers are not seen after P3. From the first postnatal days. corticospinal fibers are observed descending from the decussation in three groups (Fig. IA). The largest group, by far, is the dense principal corticospina] tract (PCST) subjacent to the ascending sensory pathways at the ventral angle of the dorsal columns. At Pl and P2. the boundaries of this tract are sharp, with no fibers straying from the tightly packed group either across the midline or into the adjacent gray matter (Fig, 2A). Lateral to the PCST are found two other groups of corticospinal axons. The second largest component of the corticospinal projection is a group of loose fascicles running through the base of the dorsal horn deep to the spinal trigeminal nucleus in the upper cervical segments (Figs 1A. IF and 2B). This intermediate group extends caudally at about the same rate as the

and postnatal

rats

1839

leading fibers of the PCST for the first two postnatal days. However, on P2 and P3, during extension through the lower cervical segments, these fibers arch laterally and enter the dorsolateral white matter before the thoracic segments of the spinal cord are reached. Figure 2A shows the configuration of these fibers at P3. No labelled axons are seen descending in the intermediate position below the cervical enlargement in any of our material. A third component of the corticospinal projection consists of a few loosely grouped axons running in the dorsolateral white matter (Figs lA, 1F and 2A-E). This, the smallest group, seems to lag slightly behind the most advanced PCST fibers. Unlike the intermediate group, axons in the lateral position will continue to extend to lower levels of the cord. The lateral group does not appear to be augmented at lower cervical levels, so that it presumably does not receive the intermediate corticospinal axons. There is a two-day delay between the first arrival of axons in the dorsal columns and their subsequent growth into the adjacent gray matter. Though forming a dense tract in the upper cervical segments at Pl and P2, the first axons extend laterally into the neck of the dorsal horn at these levels only at P3. By this time some of the axons of the PCST have already reached thoracic levels. By P4, a thin plexus of fibers extends about 100 microns laterally from the PCST. being more pronounced at upper than lower cervical levels. These fibers continue to extend caudally and laterally across the entire dorsal horn by P5 with a relatively more dense 100~micron wide plexus adjacent to the PCST persisting into adulthood (Fig. 2). After P5 it becomes more difficult to distinguish the intermediate and lateral corticospinal fibers because of the increasingly heavy labelling of PCST outgrowth. In the adult, corticospinal labelling is heaviest at the base and medial third of the dorsal horn and in the intermediate zone. Label is absent from the substantia gelatinosa and from the immediate vicinity of spinal motor neurons. Innrrwtion Q’thr thwack segmenta. Labelled fibers of the PSCT can be seen extending about 2 mm into the upper thoracic segments at P3. The leading fibers continue to extend throughout the thoracic segments at P4 at an accelerated rate (see below). The leading fibers of the PCST begin to look fewer in number and more loosely packed at P4. though they remain confined to the white matter upon initial arrival at a given level. By P5 the PCST has just reached the lumbar enlargement. Corticospinal axons in the lateral columns have grown through the thoracic segments by P6 with no apparent diminution in their numbers. At thi\ age the lateral white columns have grown thicker and lateral corticospina] fibers at cervical and thoracic levels are restricted to the deeper white matter. adjacent to the dorsal horn, Innerwtion of’ lumbar und wcral segment.s. The lumbar enlargement is not quite reached by the most

fibers of the PCST during the accelerated growth on P4. By P6, however, these fibers have begun to extend into the enlargement and hn\c I:‘+ versed the upper 3 or 4 lumbar segments by P-i. At P8, labelled axons are seen at the caudal limit of the enlargement and grow the next day (P9) at 617 accelerated rate. continuing through most of the sacral \pinal cord by P10. Elongation of PCST axons contmuc‘b at a constant rate from here. but. because of the continuing growth of the spinal cord itself. the caud:il segments are not reached until Pi.3 or P13. The innervatioii of tht: lumbar- and sacr:tl g~-a\, matter follows a slightly different pattern than at more rostra1 levels. A rostrocaudal time gradient ib still observed. Growth of fibers into the lumbar pray matter begins on P7 and cxtonds to sacral ic~clc hq P9- 10. However. this l~~gr~~~~t~l IS initially diffu\c. the first appearing fibers &en extending ~~~~~d[)lat~r~iil! across much of the dorsal horn. At a given IcwI. stubadvanced

Fig. l.(A)

Horizontal

aged postnatal principal

((3).

corticospinal

intermediate

section some

aberrant

PZ. LabeHrng

~o~lnt~rstain.

of the pontrne

nuclei

Sagittal

tracts

section

joining

Fig. 2. Horizontal

adult

cortex.

Bar reprcsentb

arising

nuclei

sectlon

showing

pattern.

tract

the

middie ingrowth

indtcate

corticospinal

Fig. 3. tab&led

fibers of the advancing

of an animal

Arrows

indicate

sections

from

indicate

labelled

decussation.

aged Pi.

flame-shaped

an animal

(D) Horizontal

fibers of the principal

>ection

reg~m

:imrnal.

Bar rcprescnts Ieating

astrocyte astrocytic

c

cuneate

CG cs

dorsal gray commissure principal corticospinal

DC

dorsal

column

nuclei

DS

dorsal

median

septum

G

gracile

nucleus

CC

growth

process

fasciculus

cone

tract

ages showing

nucki

of fibers

matter

cortico~pinal liom

identified

f’rom tht

cer\,ical

lowest

tract

point

(C)

reached

of an animal

Thiorun ~~~~lnterst~~i~. Bars

from tltc ccrvicat aged

Near-adjacent

Pi

aged

IT

f-1 Thionin

oi an amma

arroli

indicate

jun~t:on.

in (A

at this age.

Straight

Into

L:i. (V’I The

in (1‘ I.

section

region

cones.

in pans.

reglrm

microns

14) Sagittai

the thoraclc

as growth

indicates

pyranudnl

traiit.

is visible

IDC).

passage: t)i the

growth

tract

in 1.4 E) and X0

50

200 mici 311s. (G)

column-gray

,Ihhre\iatlons

As

represent

column

1. (A). initial

corticospinal

axons

and pl-incipal

the dense plc~.us at the dorsal

hcction

an

the decussat~r)t~ (arrow).

in the dorsal

da!

S mm 113ic‘1 .Ind 25 microns

A

(IT)

bundles

at difkrcnt

Bar

sections.

intermediate

trart

tract.

arrow

~orti~~)5p~n~~l tract.

Sagittal

ILT),

at 1’4 ((‘1 :~nd extensrvc

prmqal

and

at Pt.

at po\tnatal

100 m~ronh

irtrm

aged EZO. (FI frivabion

in an animal

r~sc to termination&

The lateral

prmcipaf tr,.&i‘t IIT).

at P5 fD.

expansions

peduncle

corticospmal

Ingrowth

(B) Horlrontai

aged P3. Open

cerebral

the

section

pyramidal

of fiher

ccr\ical

to form

(C) Saglttal

t B)

axon5

the nuclei

ot” the lateral

the region

of labelled

.4ntcrogradely~labelied

in an adult

gikmg

ami

(CS) beyond

Arrows

the intermediate

junction

rise to the

500 rnicrcmr.

Bundles

and intermediate

tract (arrows)

labelling

aged 1’12. I.ahelling

kom

tract

(Di

invading

in ai) animal

give

iabell~i?g of the *tppf)site

decussation

200 microns.

furrow)

(arrow)

aged PI.

the pyramidal

700 microns.

~~~~lnt~rst~~it~. Bars represent

region

from

Bar represents

(P) without

corticozpinal

sections

in an anlmat

drcussation

at P3 (B), substantial

labelling

indicates

to contralateral

at the mcdullospinal

corticospinal

the gray matter

Some

counterstam

f’rom an animal

the principal

labelled

tracts.

by fibers of the pyramidal

(F) Transverse

corticospinal

eorticospmal

B~tr represents

to the pontinc

microns.

(t.T)

dccussation

decussatian

cord

and lateral

of pyram&l

passing dorsal

spinal

the pyramidal

ones (arrow)

animal

and cervical

from

the pyramIda

(CS). T‘hionin

deoussatlon

rising

is due to spread of lnjcctiori through

tract

Thionin

the pyramldal

fibers

(IT),

corticospmal aged

through

3. Labelled

tract5

Horizontal including

section

day

sequent ingrowth over the next three days increases the density of labelling across the dorsal horn. It is only at later ages (P14) that the relatively dense IWmicron plexus adjacent to the PCST becomes distit;gllish~~b~e. This dense media) regton :hen persists into adulthood. In sacral regions. the delay between arr~~i of PCST fihet-s in the w-hitr matter and thei! initial ramification 111the gi-al matter i\ reduced to one day or IuV7 ,\ \er> small number of lateral corticospinal fibers NC wx at lumbar levels in animals aged P9 to adult. WC halt not observed lateral fiber\ in the sacral spinal cord. iA/lO~ph~~lO~ 1‘ rJifd ~~IOrph~Wft~~~~~ it/ lilC d~'~~lOpit?~] c~r~r~r~pir~trL WN~. In the best expzrmiental cases. near-solid tilling of corticospinal axons with HRP was obtained. Because of the reprodn~ibilit~ of the labelling pattern. its progressive lengthenkg lcith age and its c~~rrelittion with the electron-mrcrnscopic prep-

P5.

h~lrlzontaf

CurM

2irrows

indicates

pvramldal

showing

the leading

SO microns

in iAl and fB?.

in IDi

used on Figure\ GI

glial process

I(‘

Internal

i-r

l~ltermed~ate

1.7

lateral

t)

oligodendrocyte

P

pontine

PT SP

pyramidal spinal

capsule corticospinai

corticospinai

iract

tract

nuclei tract

trig&minal

nuclear

complex

A

‘t

-.

Fig. I IX41

c

Pl P2 P3 P4

AGE

(days)

”

p6 P7 P8 PQ PI0

. .

P13

l%

P14

I

‘. 1

‘

.

r

r

1

2

3

4

5

LENGTH

(cm)

Fig. 4. Graph showing the furthest extension of the principal corticospinal tract (filled circles) and total length of the spinal cord (open squares) at the ages indicated. Both measures are from the caudal limit of the pyramidal decussation. Note greater than average growth rates at P-4 and P&9, -..__-___

_-.-.

_.--__--..-

Fig. 5. I micron-thick plastic transverse sections from high thoracic regions of the spinal cords of animals aged embryonic day 20 (A), postnatal day 0 (B). postnatal day 5 (C). postnatal day 12 (D) and adult postnatal day 35. Unmyelinated corticospinal tract is seen in (Cl. Myelination is commencing in (D) and is complete in (E). Region through which tract must grow is indicated by X in (A) and (B). Note that myelination commences in cuneate (C) before gracife (G) fascicufus. Richardson er aLZz stain. Bars indicate 50 microns in (A-D) and 100 microns in (E). Fig. 6.(A) Sagittal plastic section from the high lumbar region of an animal aged postnatal day 0 showing the gracile fasciculus (G), central gray matter (CG) and the region (X) through which the corticospinal tract will grow. Bar indicates 50 microns. (B) Electron-micrograph from region indicated by X in (A) showing interweaving astroglial processes and possibly some axons of indeterminate origin, x 7000. (C, D) Electron-micrographs from the dorsal columns in the middle thoracic region of an animal aged E20 showing astroghal processes and dark cells of the dorsal median septum (DS). In D there are some axons, presumed to belong to the gracile fasciculus. Transverse sections (C) x 18,000 (D) x 7000. Fig. 7. Electron-micrographs from the deep part of the dorsal columns. (A) Middle thoracic region of an animal aged E20 showing pale astroglial processes and darker radial processes (arrows) of dorsal median and an adjacent septum. x 15,000. (B, C) The high cervical region of animals aged EZO (B) and PO (C) showing fasicles of small profiles (arrows) interpreted as corticospinal axons entering the spinal cord between glial processes (Cl). Both sections: transverse plane. x 18,000. (D) Axons of the gracile fasciculus (below) in the low cervical region of an animal aged P5 are surrounded by a c~~ns~derable extracellular space. x 18,ooO.(E) Longitudinal section from the high cervical region of an animal aged PO. Dorsal root axons at the periphery of the dorsal columns forming synapses (arrows). x t4@.% (F) Shows a growth cone (GC) in the corticospinal tract in the high cervical region of an animal aged P2. Transverse plane. x 35,000. Fig. !&(A-C) Transverse sections of the corticospinal tract in the low cervical regions of animals aged postnatal day 5 showing tightly packed smail axons and growth cones (arrows, CC). In (0 several growth cones are closely associated with astrogtial processes (As) but this is relatively nncon~rn~~n.(D1 Commencing myelination in the corticospinal tract in the high thoracic region of an animal aged P12. Arrows show two large axons associated with astrocytic processes (As). 0: ohgodendrocyte. x 7ooO. (A, D) x 7000, (B) x 23.000. (C) x 18,000. 1844

Growth and target findings in prenatal and postnatal rats we assume that the labelling extends to the axons. Individual axons were fairly straight in all regions of the white matter and were about 0.5 microns in diameter. In some regions, particularly at the decussation of the pyramids and in the intermediate position in the cervical spinal cord. bundles of axons were often tightly fasciculated (Fig. IC). In other regions. especially at the leading front of the PCST and the lateral corticospinal group. individual axons were clearly distinguishable. showing minimal or no contact with their fellows (Figs 3A. 3B and 3D). The major trunk of the PCST. however. was tightly packed in a dense tract. A few glial cells, primarily astrocytes, were seen to be included in the pyramidal tract in paraffin and plastic sections. but we were unable to determine from light-microscopy what degree of interaxonal separation their processes provided. The leading front of the PCST was composed of up to a dozen labelled axons. These extended up to 100 microns further than the next following fibers. Following fibers increased in number gradually, thus providing no obvious basis for a clear division between early and later arriving corticospinal axons (Fig. 3). Characteristic expansions 2 x 5 microns in extent were found at the tips of growing axons. whether in white or gray matter, from E20.5 to PI4 (Fig. 3B). We have tentatively identified them as growth cones. Growth cones were seen at the growing point and at all levels within the tract in the spinal cord as late as Pl4. Their numbers. however. are reduced 45 days after the arrival of the first corticospinal axons at a particular level. The PCST continues to get thicker and more densely labelled through the first two postnatal weeks. As time progresses. it clearly contains more fibers. a fact that is also detectable by electron-microscopy. Thus, the PCST is not constructed by a massive, synchronous outgrowth of cortical axons. but rather by the successive addition of more and more fibers on the heels of the leading group. The distance that the leading fibers of the PCST have extended caudally from the spinomedullary junction was measured in a series of neonatal animals and related to the total length of the spinal cord. No correction was made for shrinkage of the frozen sections during histological preparation. This factor was assumed to be rather small and appeared constant at all ages. The numbers obtained. nevertheless. reflect relative. rather than absolute length of the PCST. During the first two postnatal weeks. considerable lengthening of the spinal cord takes place, but no correction was included for this factor either. Our numbers reflect the total degree of elongation, independent of causal factors, As can be seen from Fig. 4, the overall rate of extension of the front of the PCST is about 2.6 mm, day. The rate, however, is not constant, There are periods of accelerated elongation at P334 and P8 9. followed by periods of growth slower than average.

arations.

tips of the growing

I x49

The significance of these deviations from a Constant growth rate is not understood. The earlier growth spurt takes place during extension through the thoracic segments while the latter spurt occurs during growth through the sacral segments. The scatter of data points for each age increases with increasing age, This probably reflects animal-toanimal variation in overall growth rate after a near synchronous start for the first PCST axons. This increasing scatter may account for the smaller size of the later growth spurt as calculated by our methods.’ The elongation rate at P3-4 is nearly three-fold the overall rate, while the rate at P889 is just over twofold the overall rate. The growth rate of corticofugal axons in the brain was estimated from measurements of prenatal material. Growth between E 17.5 and E 19.5 proceeded at about 1.2 mm/day. This corresponds to growth through the diencephalon and midbrain, and is considerably slower than growth through the spinal cord. However. growth along the vjentral surface of the pons and medulla (El9.520.5) proceeds at about 2.4 mm/day. much closer to the rate of extension through spinal cord. No attempt was made to measure the rate of growth through the pyramidal decussation because of its complex geometry. Growth of PCST axons from the tract into the adjacent spinal gray matter was comparatively slow. PCST axons can extend down the dorsal columns a distance of many times the width of the dorsal horn (up to 0.5 mm) in one day, though. as indicated above, they take more than one day to traverse the dorsal horn mediolaterally.

Phtic~ .wcrions. The spinal cord of fetal and neonatal rats was sampled in transverse and sagittal sections at selected levels and age intervals covering a period from before the entry of corticospinal fibers into the cord to the commencement of myelination in the corticospinal tract, The levels examined in I micron thick plastic sections and in ultrathin sections were: high cervical. low cervical, high thoracic. mid thoracic and upper lumbar. The ages examined ranged from E20 to PI4 with one animal sampled at P35 and others at ages in excess of three months. In plastic sections from older animals. the corticospinal tracts can be detected as two tightly packed aggregations of thinly myelinated axons in the ventral part of the dorsal columns. separated by the dorsal median septum and abutting directly on the dorsal gray commissure. The gracile fasciculus and. at appropriate levels. the cuneate fasciculus can be readily seen lying dorsal and dorsolateral to the corticospinal tract and filling in the remainder of the space between the two dorsal horns (Fig. 5E). There is no clear-cut septum between the corticospinal and the two ascending tracts. They are mainly distinguished by differences in the packing density and myelination of the constituent fibers,

IX50

D. J. Schreyer and E. G. Jones

In immature animals, after the entry of corticospinal axons into the cord but prior to the onset of myehnation (at P12, Fig. 5D), the corticospinal tract can be detected as a condensation of unmyelinated axons in the same relative position as in the adult (Fig. 5C). The early myelination of the cuneate fasciculus (at P3-4) also helps to delimit the corticospinal tract in the cervico-thoracic region. The gracile fasctculus does not commence myehnating until P9910: prior to this time. there is only an indistinct boundary between it and the corticospinal tract. Detection of the corticospinal tract in plastic sections from the four levels sampled at the different ages generally parallels the time scale over which labelled fibers were shown to enter the successive segments in the HRP material. The tract is invariably thinner in the lumber segments than at upper levels but over the time-span sampled, there is a progressive increase in both relative and absolute thickness of the tract at all levels (Fig. 5). Prior to the entry of corticospinal fibers into the cord, the dorsal columns are narrow and homogeneous (Figs 5A and SB). They are composed of the unmyelinated fibers of the presumed gracile and cuneate fasciculi with numerous intervening clearer areas suggestive of neuroglial processes arising from the astrocytes identifiable in the tract. Appearunces prior to urricd of’ corricospintrl frhem Electron-micrographs from all levels at ages prior to the entry of fibers into the cord and, at later ages, from levels below that yet reached by the growing fibers reveal no obvious pre-existing framework over which the fibers must grow. In the early stages. the ventral parts of the dorsal columns have a structure not obviously different from the remainder of the dorsal columns. The columns consist of large, irregularly oriented glial processes with watery cytoplasm intervening between small fascicles of unmyelinated fibers presumably belonging to the gracile and cuneate fasciculi (Fig. 6A-D). There are no large extracellular spaces in the dorsal columns in any of our material. except immediately beneath the subpial basal lamina in the more dorsal parts of the gracile fascicuh (Figs 5A. B; 7D. E). Large extracellular spaces were the rule here until about P12. even though the axons and glial cells in the region appeared well fixed. The fascicles of axons in the dorsal columns before arrival of the corticospinal tracts are oriented rostrocaudally but no preferred orientation can be discerned in the associated glial processes though most appear longitudinal (Figs 6B-D). Other cellular processes are dorsoventrally aligned and often extend through the dorsal columns from the central gray matter to the pial surface (Figs 557). They are obviously destined to form the dorsal median and the other glial septa of the dorsal columns. Some of the cellular processes of these septa are pale, possess large numbers of glial filaments and clearly belong to mature astrocytes. Others have darker cytoplasm, with many organelles and no filaments (Figs 6B. C;

7A). Some may be immature astrocytcs or radial gliai ceils but others, associated with a basal lamina. are probably parts of invading capillaries. No oligodendrocytes can be distinguished in the dorsal columns until the onset of myelination in the cuneatc fasciculus.

There is no clear demarcation at any of the ages studied between the immature dorsal columns and the adjacent dorsal horns or dorsal gray commissure. Processes resembling those of mature astrocytcs are present but they do not form a conttnuous limiting membrane. Appeurancrs ut thr time oj orrirni of corticnspinul fihrrs. Arrival of the corticospinal fibers in a segment of the spinal cord is not accompanied by any overt structural reorganization. At a time when the corticospinal tract can be first detected in a thick section, electron-microscopy shows that it consists of a small number of fascicles of unmyelinated axons (Figs 7B. C). Most of the axons are of a fairly constant diameter (0.5 micron) and in a transverse section each fascicle contains 20-70 axons, though adjacent fascicles tend to fuse together. The fascicles are incompletely surrounded by the watery glial processes previously described (Fig. 7C). though the amount. number and orientation of the processes in relation to a single fascicle varies considerably. A few individual axons are occasionally observed within invaginations of single glial processes but this is rather rare. The majority of the unmyelinated fibers in a fascicle are not separated from their neighbors by glia but are in direct and intimate contact with one another. The small corticospinal axons usually contain no more than two or three microtubules but mitochondria are occcasionally seen and dense bodies are rclatively common (Figs 7C, F). By the time the corticospinal axons have grown into the cord. the axons of the gracile (Fig. 7D) and cuneatc fasciculi have become much larger (2~~3 microns). commonly contain large numbers of neurofitaments (Fig. 7D) and. in the case of the cuneate fasciculus. have commenced myetination. Within each fascicle of advancing corttcospinal axons a few larger. irregularly shaped prohtcs arc always visible (Figs 7F and 8). These typically contain a few large smooth-walled vesicle\ ctsternac oi smooth endoplasmic reticulum and .I trw filaments (Fig. 7F). They seem more difficult to lix than the small axons and often show ruptured membranes and washed-out cytoplasm when around them fixation appears good. We interpret these structures as growth cones on the ends of growing corticospinal axons. They have diameters similar to the labelled enlargcments on the ends of the axons demonstrated in the HRP material (2-3 microns). S&sequent appearances. In a segment at times mllowing passage of the advancing wave of corticospinal axons. further development is characterized by a great increase in numbers of the corticospinal axons and by marked reduction in the associated glint processes.

Growth and target findings in prenatal and postnatal rats Oligodendrocytes become obvious in the tract at PI0 and myelination commences on P12 (Figs 5D and SD). Up until the onset of myehnation, the diameters of the unmyelinated corticospinal axons remain remarkably uniform at approximately 0.5 microns (Fig, 8). The increase in numbers was assessed by counting the number of cross-sectioned axonal profiles in electron-micrographs from the mid thoracic region in animals at P5 and PlO. At P5. the tract on one side was estimated from plastic sections to measure 2.5 mm2 and from counts made on electronmicrographs an equivalent region was estimated to contain 1000 0.5 micron-diameter profiles. At PlO, the tract was estimated to measure 4.25 mm2 and from electron-micrographs was estimated to contain 3389 0.5 micron-diameter profiles. The internal structure of the unmyelinated axons is identical to that described at earlier times. At all ages, however. even at the time myelination commences. a considerable number of growth cones remain visible (Figs 7. 8). No obvious synapses or synapse-like contacts were detected in the corticospinal tract at any of the ages studied. Synapses commonly seen at the edges of the dorsal columns. even as early as Pl, were mostly made by dorso\entrally-oriented unmyelinated axons (Fig. 7E). They were interpreted as branches of primary atrerent fibers.

DISCUSSION One of the principal findings of the present study is that the corticospinal tract of the rat grows past a particular spinal segment for 2-3 days before sending fibers Into the gray matter of that segment. Using autoradiographic tracing, DonatelIe observed substantial ingrowth at cervical levels by P5 and reported growth into the gray matter at midthoracic levels at P7. with substantial spread of label across the dorsal horn at midthoracic and lower lumbar levels by P9. We have observed significantly earlier ingrowth at cervical and midthoracic levels possibly because of the greater sensitivity of the technique. Early growth toward a target. followed by a waiting period, with later growth into the target is clearly a feature of many de\,cloping pathways. It has now been demonstrated in corticofugal projections of the rat (present study: DonatelIe.‘) cat.32 hamster,*’ opossum14 and monkey,25 as well as in the afferent innervation of the cortex of the rat.‘3.34 cat3’ and monkey.“~‘* We need to confirm. in the spinal cord, however, that the late growth spurt into the gray matter is made by previously waiting fibers and not simply by continuously-growing but later-arriving fibers. In addition to the major bundle of corticospinal axons forming the principal corticospinal tract in the ventral angle of the dorsal columns. we have observed two other groups: an intermediate bundle of fasciculated axons running through the base of the dorsal

1851

horn in the cervical segments. and a lateral group of fibers extending as far as the lumbar enlargement in the lateral white columns. No ipsilateral corticospinal fibers were labelled except in cases of extensive contralateral spread of HRP from the injection site. A lateral corticospinal tract was reported in the rat by Goodman, Jarrard & Nelson’ though this report also described a ventral corticospinal tract and an extensive bilateral projection that was not evident in our reported labelled corticospinal study. Donatelle’ axons in the lateral white column by P7 at cervical levels, but did not identify them as a distinct projection path. Other studies did not mention corticospinal pathways outside the dorsal columns.‘,x,‘2 It is possible that the methods used in previous studies were not sensitive enough to identify these much smaller pathways. No other reports seem to mention the intermediate tract that we have described. It is also probable that with the methods of degeneration and autoradiography, the intermediate tract would be in-

distinguishable from PCST-derived terminal ramifications in the cervical gray matter. In the HRP material the distinction between thick parent axons and finer terminal ramifications is much clearer and also shows that the intermediate tract is not associated with the spinal trigeminal complex. The cerebral cortex is still very immature when corticofugal labelling is seen in the internal capsule following a cortical injection of HRP. Corticothalamic axons arising from cells in layer VI’ are probably included in our labelled population. Layer VI cells reach the cortical plate by El617, but cells that will populate layer Vb and give rise to corticospinal axons have only withdrawn from the mitotic cycle in the neuroepithelium at El7.’ If labelling of the internal capsule at E17.5 includes corticospinal axons. their parent cells are thus still migratory or just postmigratory at the time they send axons outside the cortex. This is also true for some cortical efferent cells in the cat32 and shows that axonal outgrowth by corticofugal cells is independent of the arrival of afferent connections and of the formation of a substantial dendritic tree.’ ’ Though corticospinal fibers grow out vcrq quickly and reach the spinal cord at about the time of birth. invasion of the spinal gray matter does not begin until P3. At this time, the cortex has just attained its final laminar organization of cells and has been diffusely invaded by thalamic afferents.“” However. columnar segregation of thalamo-cortical atTerents3’ and initial ingrowth of callosal afferents”” does not occur until P5. the time at which corticospinal axons have extended across the dorsal horn at cervical IeLels. The waiting period before gray matter incasion IS shorter and the extension across the dorsal horn quicker in the lumbosacral spinal cord. Innervation of these caudal levels occurs after thalamic and callosal afferents to sensori-motor cortex have taken on their adult laminar and columnar patterns of distribution, Therefore. an adult pattern of affercnt innervation in the

1852

D. J.

Schreyer and E. G.

cortex is not a prerequisite for invasion of the spinal gray matter by corticospinal axons. However, because thalamocortical fibers appear to form synapses as soon as they enter the cortex.‘3.3’ the possibility remains that synapse formation on layer V pyramids is the trigger for some signal that causes their axons to make a second growth spurt into the spinal grdy matter. Our use of the anterogrdde transport of HRP allowed good visualization of axons, with the labelling approaching solid filling in the best cases, We were thus able to identify expansions at the tips of growing axons which, from a correlation with the electron-microscopy, we regard as growth cones. These growth cones not only served as a marker for the earliest growth of the tract. but their appearance at all levels along the tract at later stages implies continuing fiber outgrowth at later times. WC deduced from the light- and electron-microscopy that additional axons continue to grow down the PCST m considerable numbers even after the colonization of the lumbosacral gray matter. There was at least a threefold increase in numbers at mid-thoracic levels between PS and PlO. Hicks & D’Amato’ found that the cross-sectional area of the pyramidal tract increased dramatically during the first two postnatal weeks also providing evidence for the late addition of axons. These late growing axons may be partly responsible for the greater morphological and functional recovery of the corticospinal system fotlowmg spinal injury in young animals as compared to adults. We do not yet know, however. whether the) represent axons growing out from additional layer V cells \>I whether they arise from collateral branching of existing axons. Nor do we know if any of thesr fail to establish synaptic connections. Wise & Jones33 and Wise et trl.“’ first showed that the distribution of cortical efferent cells is homogeneous across the neonatal rat sensorimotor cortex. But in the adult, their distribution is patchl.“‘.33 ” Recently it has been suggested that the non-connected patches of callosally-projecting cells develop hecausr: of loss of collaterals by cells in those patches.“.‘” Hence. some of the late growing corticospinal axon\ may not establish connections and presumably the axons would die so that their original parent cells. though remaining into adulthood. would not be rctrogradely-labelled by HRP injections in the spinal cord. We found little evidence for axon death in our electronmicroscopic preparations. There is no evidence that the rather prominent dense bodies that we observed in many axons necessarily betoken degeneration. A surprising finding was that the growth of cortlcospinal axons does not proceed at a constant rate. In the rat, extension of corticospinal axons iS first Sh in the brainstem, then more rapid in the spinal cord.

Jones

as is the case in the hamster.20 Our methods have also revealed growth spurts in the spinaf cord itself, at least as judged by the position of the end of the advancing tract in relation to the end of the cord. One might postulate fast-growing groups of corticospinal axons each overtaking in turn the front of corticospinal axons. We consider this explanation highly unlikely. We observed no morphological features that might distinguish between different classes of corticospinal axons with different growth rates It is not known what regional variations in spinai cord growth might influence the rate of axon growth but speeding up of the axons may indicate a response to lengthening of the cord during lengthening of the body as a whole. Our HRP cvperlments indicatt: th,lt gro\vth 01 rhe main wace of corticospinal axons Iq :&aq’s preceded by it small number of ‘pathfinder’ ax~~ns growing in ad\ancc of the rest. But the elccci.r)rl-mjcroszop\r strongly suggest\ that the main bodi advances 2s .L scrlt’s of fascicles and that later arriving :ixons prow down the cord along other axons within these fascicles. We cannot rule out that the ‘pathfinder’ fibers grow along some preferred glial channels ils has hecn suggested for the growth of optic writ fibers in the mouse” and for regenerating spinal fibers in ihc newt.” though the latter has been contested.” Channels or some other ordcrtl configuralIon of glia could not bc dctectetl by us a1 times prior iir lhc :trriv31 of Ihe corticospinal Ilact. In ;I camparabic 4tud> on that corpus cntlosum.‘” we have similari> r!ol been able tcj identify an) prefercntiallq oriented ,c!1:11guide5 t<’ axon growth. In preliminary cxprrimrnts involving partiat spinal transections it1 lumbar Ic\cls in ncwborn rats.” HC have shown tha1 rhc idler arrikinp corticospinat ;Lxons wilt prow past I/IL. lesion Oven though the dorsal columns habe hur,rt destroyed al thar point. This also tends to c)b\iatc? +ainsl the giin of the cart? dorsal columns playing ~II> bubstantiai role in directing the growth of thc corricospinal ;lkOIlX.

We were also not able to detect an:, morphological contacts of the synaptic or adhcstvc !>IX within the growing corticospinal tract. Though transient synapse-like contacts have been described in tht: dorsal columns of the early monkey fcluz.’ ’ in m1- malerinl such contacts were only detected 111situations where thcq could be attributed to primary :rH‘crcnts ending at the margins of the dorsal horn. M’c are Icci to the conclusion. therefore. that there arc no ~~\~c.‘(-t morphological features that could imply guidance by the iubstrate over which the corticospinal axon?, grow ol- an) that could imply interactions between ulbstrate :tnd growing axons. If such interactiollh OCCLII the> ilri’ presumably of a type that cannot bc visuatircd wtth

these techniques.

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