Developmental factors affecting regeneration in the central nervous system

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Title:
Developmental factors affecting regeneration in the central nervous system early but not late formed mitral cells reinnervate olfactory cortex after neonatal tract section
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vi, 66 leaves : ill. ; 29 cm.
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Grafe, Marjorie Ruth
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Central Nervous System -- physiology   ( mesh )
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Thesis:
Thesis(Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 60-65.
Statement of Responsibility:
by Marjorie Ruth Grafe.
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Photocopy of typescript.
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Vita.

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DEVELOPMENTAL FACTORS AFFECTING REGENERATION
IN THE CENTRAL NERVOUS SYSTEM:
EARLY BUT NOT LATE FORMED MITRAL CELLS REINNERVATE
OLFACTORY CORTEX AFTER NEONATAL TRACT SECTION








BY

MARJORIE RUTH GRAFE


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1981



















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P
SYou can live to be 100 and still learn something about melons.
-Anonymous










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ACKNOWLEDGMENTS


I thank the members of my supervisory committee--Drs. William

Brownell, Marieta Heaton, Kenneth Heilman, and Christiana Leonard,

and Dr. William Luttge, acting chairman of the Department of

Neuroscience--for the various forms of assistance and interest they

provided. I especially thank Tiana for her endless enthusiasm, for

caring and understanding, and for letting me do what I wanted to do.

I want to acknowledge the influence of two other persons: Dr.

John Resko--for giving me a positive introduction to science; and

Dr. Robert Schimpff--for providing a challenge. I thank Dr. John

Scott for prompting the use of the depth analysis in these experiments.

I appreciate the friendship and tolerance of everyone in the lab.

Special thanks to my parents--for their constant love and support in

everything I did; and to Mark--for the happiness he gave me during

these years.

I have been supported by a National Science Foundation pre-

doctoral fellowship and a traineeship from the Center for Neuro-

biological Sciences. This research was also supported by NIH grant

NS 13516 to Dr. Leonard.















TABLE OF CONTENTS


CHAPTER PAGE


ACKNOWLEDGMENTS.................................................. iii

ABSTRACT.............. ......................... .............. ..v

I INTRODUCTION............................................... 1

II MATERIALS AND METHODS...................................... 11

Experiment I. Time of cell formation...................11
Experiment II. Correlation of cell birthdate and
axonal outgrowth...................................14
Experiment III. Early transaction of the lateral
olfactory tract......................................17
Data collection and analysis...........................18

III RESULTS ....................... .............. ..... ......... 27

Experiment I................................ ...........27
Experiment II.........................................30
Experiment III........................................37

IV DISCUSSION...... ................................................47

Time of mitral and tufted cell formation...............47
Correlation of cell birthdate and axonal outgrowth......49
Reinnervation after transaction of the LOT.............50
Other effects of lateral olfactory tract transection....52
Aberrations in developmental interactions as possible
consequences of mild H-thymidine toxicity...........52
Possible mechanisms of reinnervation after early
lesions ..............................................57

REFERENCES................................... .............. 60

BIOGRAPHICAL SKETCH...............................................66















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



DEVELOPMENTAL FACTORS AFFECTING REGENERATION
IN THE CENTRAL NERVOUS SYSTEM:
EARLY BUT NOT LATE FORMED MITRAL CELLS REINNERVATE
OLFACTORY CORTEX AFTER NEONATAL TRACT SECTION



By

Marjorie Ruth Grafe

December, 1981



Chairman: Christiana M. Leonard
Major Department: Department of Neuroscience



The olfactory system of the neonatal golden hamster has a known

capacity for behavioral and neuroanatomical plasticity. If the lateral

olfactory tract (LOT) is transected in the first postnatal week, axons

grow through the cut and reinnervate the terminal regions. Functional

recovery occurs only when the terminal regions are reinnervated.

These experiments examined the possibility of a specific anatomical

principle regulating axonal regrowth and reinnervation after early

lesions. The original hypothesis was that the reinnervation arises

from continued growth of newly-formed axons which were not severed

by the lesion.








The first experiment determined the time of cell formation of

neurons in the hamster olfactory bulb. Mitral cells are formed on

gestational days 11 and 12 (Ell and E12), and tufted cells on Ell to

E14. There is an outward progression of the positions within the

external plexiform layer (EPL) of cells formed from E10 to E14.

The second experiment involved the combination of 3H-thymidine

labeling for time of cell formation with the retrograde transport of

horseradish peroxidase (HRP). Axons of early-formed cells reach the

olfactory cortex before those of later-formed cells. This is believed

to be the first demonstration of the correlation of times of cell

formation and axonal projection in individual cells.

The third experiment examined the possibility that axons which

grow through an early LOT transection are new axons which had not yet

reached the level of the cut. Animals were given 3H-thymidine on Ell

or E13, and a transaction of the LOT on day 3. After a recovery

period sufficient to allow axonal regrowth, HRP was placed in the

olfactory projection regions caudal to the prior LOT section. The

original hypothesis was not supported. Cells which are formed early,

and send out their axons early, reinnervate the olfactory cortex, while

late-formed cells do not. Early LOT section decreases the total

number of mitral cells, and affects the positions of tufted cells.

Evidence was found for a mildly toxic effect of 3H-thymidine

injections. The positions of cells destined for the EPL were

specifically affected by the incorporation of H-thymidine on E13.














CHAPTER I
INTRODUCTION



In the past twenty years, the use of 3H-thymidine autoradio-

graphic techniques has provided much information about the develop-

ment of the central nervous system (CNS). This method has revealed

the time and place of origin of CNS cells, the cell lines from which

neurons and glia are derived, and the paths which these cells follow

in their migrations from site of origin to final position. The

technique is limited, however, in that, by itself, it cannot give

information about the connections of cells or the development of these

connections, or how these processes are related to the functional

development of the brain.

This study combines 3H-thymidine autoradiography with the retro-

grade transport of horseradish peroxidase (HRP) and neonatal lesions

in an attempt to directly investigate possible correlations in the

time of cell formation and the development of their axonal projections.

The combination of 3H-thymidine and HRP has been used previously to

examine whether cells within a given region with different birth-

dates ultimately project to different, or the same regions (Nowakowski

et al,, 1975). They found that mouse hippocampal neurons formed on

gestational days 13 and 14 have similar projections to the contra-

lateral hippocampus. The study we are now reporting looks at whether

individual cells within a region, which are known to ultimately pro-

ject to similar areas, do so in a sequence which correlates with their

time of origin.







The olfactory bulb, and its projection to the olfactory cortical

regions of the basal forebrain, is a convenient system in which to

examine this question. The olfactory bulb sits in the front of the

brain, is well laminated, and has a discrete, easily identifiable

population of cells which project centrally. The axons of the mitral

and tufted cells leave the bulb in a distinct bundle, the lateral

olfactory tract (LOT), which travels on the surface of the brain to

its projection areas in the basal forebrain. Along the course of

the LOT, many collaterals branch off and project into the anterior

olfactory nucleus, olfactory tubercle, piriform cortex, and other

basal forebrain regions (Devor, 1976a). The bulb, LOT, and olfactory

cortex are all easily accessible with little disruption of the rest

of the brain.

The rodent olfactory system is behaviorally functional in the

neonate (Devor and Schneider, 1974; Cornwell, 1975; Blass et al.,

1977; Rudy and Cheatle, 1977; Crandall and Leonard, 1979), but under-

goes both functional and anatomical changes during the first few

weeks of postnatal life (see, for example: Devor and Schneider, 1974;

Leonard, 1975; 1978; Singh and Nathaniel, 1977; Schwob and Price,

1978; Crandall and Leonard, 1979). The time of origin of olfactory

bulb cells, their pattern of migration, and their differentiation

have been extensively studied in the mouse by Hinds (1968a,b; 1972;

Hinds and Ruffet, 1973). The postnatal establishment and expansion of

the LOT projections in rat and hamster have been described using

degeneration (Leonard, 1975; Singh, 1977), autoradiographic (Schwob

and Price, 1978), and electron microscopic methods (Westrum, 1975).

The LOT projection also has a capacity for functional and anatomical

plasticity during the neonatal period (Devor, 1975; 1976b; Small, 1977).








Devor (1975) showed that recovery of male hamster mating behavior,

which is olfactory-dependent, occurs only when the terminal fields

caudal to the cut are reinnervated after an early LOT section.

Following early LOT sections, pups which showed functional recovery

on a thermal behavior test were found to have reinnervation of the

piriform cortex-and the lateral part of the olfactory tubercle

(Small, 1977).

Much of the behavioral work on olfactory development has been

done with the golden hamster, including Devor's work showing behavior-

al and neuroanatomical plasticity. For this reason, we chose to

use the hamster in our studies of the LOT projections. The hamster

offered the additional advantage of a very short gestational period,

so that it was possible to make postnatal, rather than fetal manip-

ulation of the system.

Before detailing the experimental rationales, a description of

the anatomy of the olfactory bulb and its projection neurons is

necessary. The rodent olfactory bulb consists of the following

layers (Figure 1): 1) the superficial olfactory nerve layer,

2) the glomerular layer, where the olfactory nerve axons synapse

with olfactory bulb dendrites, 3) the external plexiform layer (EPL),

4) the mitral cell body layer (MCL), 5) a cell-poor region of neuro-

pil--the internal plexiform layer, 6) the granule cell layer, with

densely packed granule cell perikarya, and 7) the ventricular/sub-

ventricular layer (Ramon y Cajal, 1911). The subventricular zone

persists in the adult olfactory bulb (Hinds, 1968b; Altman, 1969),

and neurogenesis continues to at least 90 days of age in the mouse

(Kaplan and Hinds, 1977). The accessory olfactory bulb, which

receives sensory input from the vomeronasal nerve, sits in the caudal






























Figure 1. Coronal sections through the olfactory bulb of hamster
at one month of age (A and B) and four days of age (C and D). Note
the change in size and shape of the bulb between these ages, and the
changes in the width and cellular density of the mitral cell layer
(MCL) and external plexiform layer (EPL). B and D are higher power
views of the MCL and EPL at each age. Abbreviations: EPL= external
plexiform layer; Glom= glomerular layer; GrCL= granule cell layer;
IPL= internal plexiform layer; MCL= mitral cell body layer; ON=
olfactory nerve layer; S= subventricular layer. A,C: bar= 250p.
B,D: bar= 50p.















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6

dorsal part of the bulb. The primary projection neurons from the bulb

to olfactory cortex are the mitral cells, and the tufted cells, which

are located primarily in the EPL. Classically, the tufted cells have

been divided into three categories: inner tufted cells, located in

the inner part of the EPL, middle tufted cells, located in the mid-

to outer parts of the EPL, and outer tufted cells, which occupy the

outer edge of the EPL and the regions bordering the glomeruli (Ramon

y Cajal, 1911). The size and shape of the tufted cells are highly

variable, but inner tufted cells tend to be about the same size as

mitral cells, while outer tufted cells are generally smaller

(Haberly and Price, 1977). The tufted cells were originally believed

to project to the contralateral olfactory bulb via the anterior limb

of the anterior commissure (Ramon y Cajal, 1911). Lohman and Mentink

(1969), however, made lesions of the bulb which included either both

mitral and tufted cells or tufted cells only, and were able to demon-

strate that both cell types project in the LOT to the olfactory cor-

tex. The commissural projections connect the anterior olfactory

nuclei of the two sides (Lohman and Mentink, 1969). Retrograde

labeling of olfactory bulb cells after HRP injections inolfactory

cortex has shown that all three types of tufted cells project to the

olfactory cortex, although the proportion of labeled outer tufted

cells was usually less than that of inner and middle tufted cells

(Haberly and Price, 1977). Tufted cells tend to be labeled more

frequently from injections in the olfactory tubercle than from the

piriform cortex (Haberly and Price, 1977; Scott et al., 1980). The

results of these studies are still consistent with the possibility that

some tufted cells, especially outer tufted cells, do not project out of

the bulb (Lohman and Mentink, 1969; Haberly and Price, 1977).








Because the gestational period of the hamster (16 days) is

considerably shorter than that of the mouse (19 days), we felt it

necessary to investigate the time of cell origin in this species.

It was conceivable that in the hamster there could be either increased

postnatal histogenesis or a more compressed prenatal development.

The first experiment reported here defined the times of cell forma-

tion of the mitral and tufted cells in the hamster olfactory bulb.

The second experiment was designed to determine if the date of cell

formation correlates with the time the axon enters and innervates

its projection areas. This required the combination of 3H-thymidine

labeling for time of cell formation with the retrograde transport of

HRP at a time when the olfactory bulb projections are not yet complete.

Specifically, the hypothesis tested was that the axons of early-

formed cells reach the olfactory cortex before the axons of later-

formed cells.

The last experiment proceeds from the results of the first two

experiments, to explore the possibility of a developmental principle

related to functional recovery and neuroanatomical plasticity in

young animals. Devor (1976b) found that when the LOT is cut prior

to day 7, axons grow back through the cut and reinnervate the terminal

regions. Functional recovery occurs only when the terminal regions

are reinnervated (Devor, 1975; Small, 1977). When the LOT is cut

after day 7, this reinnervation does not occur, and there is no

functional recovery. The extensive literature on regneration (for

reviews see: Ramon y Cajal, 1928; Windle, 1956; Bernstein and

Goodman, 1973; Guth and Clemente, 1975; Puchala and Windle, 1977)

suggests that there are three possible sources of the reinnervating






8

axons: 1) True regeneration: The regenerative capacities of the CNS

following axotomy have been described by Ramon y Cajal (1928). Some

fibers proximal to the cut show some regenerative ability, with the

formation of a growth cone and subsequent arborization of the

growing terminal. The axons grow toward the necrotic zone, but rarely

enter either the necrotic zone or the connective tissue scar before

the growth is halted and the growing ends are resorbed. The regen-

erative attempts are much stronger in newborn animals than in the adult.

There is evidence for axonal regeneration of the central adrenergic

neurons both in the adult (Bjorklund et al., 1971; Stenevi et al.,

1973) and younger animals (Nygren et al., 1971), but occurs faster

in the younger animals. Regeneration of pryamidal tract axons has

been reported after lesions made in the first week of life (Kalil

and Reh, 1979). 2) Collateral sprouting: reinnervation could occur

by the fibers sprouting from the proximal portion of the transected

axons (regenerative sprouting) or by collateral sprouting from

nearby uninjured axons. Collateral sprouting from uninjured axons

has been reported in many parts of the nervous system (for example:

Hicks and d'Amato, 1970; Lynch et al., 1973; Price et al., 1976).

Regenerative sprouting from the proximal axons occurs after lesions

of the central adrenergic axons (Bjorklund et al., 1971; Nygren

et al., 1971). In several systems, sprouting has been found from

both the proximal part of the cut axons and from other nearby

fibers (adrenergic cells--Pickel et al., 1974; olfactory system--

Devor, 1976b; pyramidal tract--Kalil and Reh, 1979). Most of these

studies find sprouting to occur either exclusively, or to a much

greater extent, in the young animal. 3) Neogenesis: the axons








passing through the cut may represent continued growth of normal,

newly-formed axons which had not yet reached the level of the cut at

the time of the tract section. A well-documented example of this

process is the olfactory nerve, where there is a "reconstitution"

of the nerve and its projections by axons of newly-formed receptor

cells following olfactory nerve section (Graziadei et al., 1979).

Axonal neogenesis as an explanation for CNS axonal regeneration in

young animals was first suggested by Ranson (1903). In the visual

system, aberrant projections resulting from early lesions appear to

be due to continually growing new axons which are no longer receiving

the appropriate positional signals (Guillery, 1972; Lund et al., 1973;

So, 1979). Since axon section must occur early in the period when

the LOT projection is establishing its distribution if functional

recovery is to occur, it seemed likely that those fibers which grow

through the LOT section prior to day 7 are new axons which had not yet

reached the level of the section, rather than regenerating axons or

collateral sprouts. The design for the third experiment thus includes

3H-thymidine labeling of the times of formation of the mitral and

tufted cells, transaction of the LOT at day 3, a recovery period of

approximately one month to allow any regrowth and reinnervation of

the olfactory cortex projection, and retrograde transport of HRP

placed in the projection region caudal to the prior LOT section.

If the hypothesis of continued growth of new axons as the

explanation for LOT reinnervation is confirmed, severe limitations

are placed on the possibilities for CNS regeneration in the adult.

Regardless of the source of the axons, regrowth and reinnervation

occur much more easily, and to a greater extent, in young animals





10

than in the adult. There are critical changes in development that

either allow or prevent axonal regrowth, but it is not yet known if

these changes are in the cells of origin, or in the tissue into which

the axons are growing. It is likely that there are multiple inter-

actions in the development of the system which influence the capacity

for regrowth.














CHAPTER II
MATERIALS AND METHODS


Experiment I
Time of Cell Formation


A series of pregnant hamsters (Mesocricetus auratus) was given

intraperitoneal injections of H-thymidine (5-lOuCi/gm) on gestational

days 10 (E10), 11, 12, 13, or 14. We did not wish to compromise the

pregnancies, so did not inject on E15 or 16. The pups are born after

a gestational period of 16 days (E16= PO). Several pups were each

injected subcutaneously with 3H-thymidine on postnatal days 1 or 2,

but no heavily labeled mitral or tufted cells were seen with these

injections, and their data are not included in this report. A

summary of the animals from which data were obtained and their

specific treatments is given in Table 1.

Animals were mated in the mid-afternoon (EO), thymidine injections

were also given mid-afternoon on the days indicated, and birth

usually occurred on the morning of E16. The pups were housed with

their mothers in solid-bottom cages containing hardwood shavings until

the time of sacrifice. At approximately one month of age (see Table

1) the pups were sacrificed by transcardial perfusion of fixative

(1% paraformaldehyde, 1.25% glutaraldehyde) for one-half hour, followed

by one-half hour of 10% sucrose in 0.1M phosphate buffer. The brains

were removed, and the olfactory bulbs were separated from the rest of

the brain. The bulbs were rinsed in phosphate buffer for 1-2 hours,

then dehydrated in a graded series of methanol and methanol: ethylene

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glycol monomethyl ether (1:1), cleared in methyl benzoate until sink-

ing and in toluene (5-10 minutes only), then embedded in Paraplast

Plus. The tissue was processed in this manner rather than the conven-

tional formalin fixation and ethanol dehydration to correspond with

the brains in Experiments II and III. Ethanol dehydration of brains

fixed with the paraformaldehyde-glutaraldehyde mixture resulted in

very brittle, over-hardened tissue.

Coronal sections were cut at 8p through the entire olfactory

bulb. Serial sections were mounted onto acid-cleaned chrom-alum

slides, deparaffinized, and prepared for autoradiography with Kodak

NTB-3 emulsion. The slides were allowed to expose for 8 weeks at

4 C, then developed in Kodak D-19 developer and lightly counter-

stained with cresyl violet or neutral red. Exposure times of 3-6

weeks were considered initially, but the labeling was generally

inferior compared to the 8 week exposure time.



Experiment II
Correlation of Cell Birthdate and Axonal Outgrowth


For this experiment, we chose two days for 3H-thymidine injec-

tions: one early in the period of mitral and tufted cell formation

(Ell), and one late in this period (E13), as determined by the results

of Experiment I. On E10 so few mitral or tufted cells were labeled

that the probability of finding double-labeled cells would be extremely

low. On E14 mostly outer tufted cells were labeled, and it is not

certain if all of these cells send efferent projections to the olfac-

tory cortex (Lohman and Mentink, 1969; Haberly and Price, 1977).

Horseradish peroxidase (Sigma, Type VI) was placed in the olfactory








tubercle, LOT, and/or piriform cortex of animals in both groups on

day 3 postnatally. Several animals with 3H-thymidine injections on

E13 received HRP injections at about one month of age (see Table 1).

For the HRP placement, a small pellet of 80% HRP in saline

was dried onto the tip of a 50p diameter wire. One eye was removed

and a small hole was made in the medial wall of the orbit, and the tip

of the wire containing the HRP was placed in the desired region

under direct visualization. In the older animals it was most

efficient to make a small slit in the pia and underlying LOT before

inserting the HRP. The insertion wire was left in place for 2-3

minutes to allow the HRP to dissolve away from it, then gently re-

moved. The animals 3 days of age were anesthetized by immersion in

crushed ice; the older animals were anesthetized by intraperitoneal

injection of Chloropent, .0038ml/gm. Bleeding was controlled as

necessary with saline- or thrombin-soaked Gelfoam. The wounds were

closed with Steristrips or sutures, and the skin was carefully

cleaned to reduce post-surgical cannibalism by the mother. When all

pups in a litter had recovered from the anesthesia, the younger pups

were returned to the mother. The older animals were housed separ-

ately after surgery. All animals were allowed to survive for 24

hours after the HRP placement.

The animals were sacrificed as in Experiment I. After removal

from the skull, the olfactory bulbs were separated from the rest of

the brain and processed separately. The brains were immersed in

sucrose-buffer overnight at 40C, then embedded in gelatin-albumen

with glutaraldehyde (0.5ml glutaraldehyde per 10ml gelatin-albumen)

for 2-4 hours. Frozen sections were cut on a sliding microtome at





16

50p in the coronal plane. To determine the location of the HRP in-

jection site, equidistant sections were reacted with either tetra-

methylbenzidine (TMB; Mesulam, 1978) or diaminobenzidine (DAB) with

cobalt chloride enhancement (Adams, 1977) and counterstained with

neutral red.

To identify retrogradely-labeled cells, the olfactory bulbs

were reacted with DAB, en bloc, as described below (modified from

Moody and Heaton, 1981). The times indicated for each step are for

4 and 30 day old olfactory bulbs, respectively. The en bloc reaction

works well in embryonic and neonatal tissue, but the DAB does not

penetrate older tissue well, requiring extremely long reaction times.

The size of the tissue block did not seem to be a major factor,

within the range of this experiment. The tissue should be agitated

during all steps, when possible.

1) Rinse twice in phosphate buffer, pH 7.3 (15/30 min each).

2) Rinse in 0.1M Tris-HCl buffer, pH 7.6 (15/30 min).

3) 0.5% CoCl2 in Tris buffer (60min/2hr).

4) Rinse twice in 0.1M Tris buffer (15/30 min each).

5) Rinse in 0.1M phosphate buffer, pH 7.3 (15/30 min).

6) 0.05-0.1% DAB, in phosphate buffer, at 40C (60min/3hr).

7) 0.01-0.02% H202 in DAB (60min/4hr or more).

8) Rinse twice in phosphate buffer (15/30 min each).

The tissue was then dehydrated and embedded, and prepared for auto-

radiography as in Experiment I.








Experiment III
Early Transection of the Lateral Olfactory Tract


Animals which had received 3H-thymidine on Ell or E13 were given

a transaction of the lateral olfactory tract on day 3 postnatally.

The approach for the surgery was the same as that described for

HRP placement into this region in the previous experiment. The

region around the LOT was exposed, and a small cut through the LOT

was made using a microknife, taking care not to cause unnecessary

additional damage. After surgery the pups were returned to the mother

for 3-4 weeks, an interval more than sufficient to allow reinner-

vation of the olfactory cortex by the olfactory bulb efferents

(Devor, 1976b; Small, 1977). When the pups were about a month old, they

received injections of HRP into the olfactory projection areas caudal

to the prior LOT section. The location of the LOT section was iden-

tified first by scar formation on the skull, and then by direct

visualization of the residual portion of the LOT rostral to the cut.

The LOT was not visible as a distinct bundle caudal to a complete

transaction. The animals were sacrificed and the tissue reacted

with DAB and processed for autoradiography as described in the two

previous experiments. Because of the multiple procedures combined

in these experiments, large numbers of animals were used to produce

the groups from which data were obtained, as listed in Table 1. The

animals from which data are reported are only those with adequate

3H-thymidine labeling, histologically verified complete LOT sections,

and satisfactory HRP injections in the olfactory projection areas.






18

Data Collection and Analysis


Cmaera lucida drawings were made of olfactory bulb sections at a

magnification of 125X. Mitral and tufted cells were identified on the

basis of their size, large nucleus and abundant cytoplasm, and position

in the MCL or EPL. No distinction was made between the different types

of tufted cells. Only cells whose nucleus was clearly in the plane of

section were counted. The sections were examined under the microscope

at 600X for the presence of cells labeled with 3H-thymidine and/or HRP

(Figure 2). Cells which had many silver grains over the nucleus

(about 5 times background or more) were considered "heavily-labeled"

with thymidine and were presumed to have undergone their last division

on the day of 3H-thymidine injection. A thorough discussion of the

problems in defining "Heavily-labeled" cells can be found in Sidman

(1970). The HRP reaction product is seen as a dark brown, particu-

late substance in the cytoplasm of the cell body and proximal den-

drites. The two labels are easily distinguished under the microscope

on the basis of their locations in the cell (3H-thymidine in the

nucleus, HRP in cytoplasm), color and size ( H-thymidine is small,

distinct black grains, HRP is larger, dark brown particles), and

plane of focus (silver grains for H-thymidine are in the emulsion

above the section, HRP reaction product is within the cell). The

positions of 3H-thymidine and HRP-labeled cells were indicated on

the camera lucida drawings. Labeled granule cells and glia were not

included on the drawings. For each olfactory bulb analyzed in

Experiment I, five sections were drawn: rostral OB, two sections in

mid-OB, caudal OB, and a section which contained the accessory

olfactory bulb. In Experiments II and III, two sections in the






























Figure 2. High power photomicrograph of cells in the mitral cell
layer of an animal injected with H-thymidine on Ell, HRP on day 3.
The plane of focus in the upper half of the figure is through the
emulsion above the tissue, while that in the lower half is through
the tissue itself. The H-thymidine label is seen as silver grains
in the emulsion (upper). Heavily-labeled cells have many silver
grains above their nuclei. The HRP reaction product is a particulate
substance in the cytoplasm (lower). There are many unlabeled cells,
a cell labeled with H-thymidine only (arrow, lower left), a cell
with HRP label only (arrowhead, upper right), and one double-
labeled cell (arrow and arrowhead). DAB reaction, counterstained
with neutral red. Bar= 10p.















U



II.
r .I *










a 4. *


U


4


I V*A






21


mid-OB were drawn for each brain, since no rostral-caudal differences

were found in Experiment I.

The position of each labeled cell within the EPL was determined

in the following manner. The distance from the outer edge of the MCL

to the labeled cell and the distance from the MCL to the inner edge

of the glomerular layer were measured. The ratio (r) of these dis-

tances was calculated. An r of 0 indicates a cell in the MCL, an r

of 1.0 a cell in the glomerular layer (see figure 5). For each

section drawn, the mean r (r) and mean EPL depth were calculated. An

overall r was then determined for each olfactory bulb.

Additional measurements were made on the sections from Experi-

ments II and III, and on the mid-OB sections from the Ell and E13

brains in Experiment I. The total number of all mitral and tufted

cells per section was counted (both labeled and unlabeled cells),

and the perimeter of the outer edge of the MCL was measured. The

numbers of mitral cells per unit perimeter, tufted cells per perimeter,

and total cells per perimeter were calculated, and the ratio of

mitral to tufted cells determined. The proportion of 3H-thymidine-

labeled cells to total mitral and tufted cells was also calculated

for these sections (p(thy)).

The location and extent of the site of HRP placement was

determined for each animal in Experiments II and III (Figure 3).

Cell counts for 3H-thymidine-labeled and total cells, as well as

HRP-labeled cells,were made only in brains which had HRP injections

in the olfactory projection regions which were of a size sufficient

to produce retrograde labeling of a reasonable number of olfactory

bulb cells (Grafe and Leonard, 1981). In Experiment III, the site






























Figure 3. Examples of 3HRP placement sites for Experiments II and
III. A. Animal 1553; H-thymidine Ell, HRP D3. HRP placement in
ventral piriform cortex and just deep to the lateral olfactory tract
(LOT),3at the level of the caudal olfactory tubercle. B. Animal
1556; H-thymidine E13, HRP D3. HRP placement in and sur wounding
LOT, at a more rostral level than in A. C. Animal 1674; H-thymidine
Ell, LOT D3, HRP at one month. HRP placement in and surrounding
LOT, at the level of mid-olfactory tubercle. D. Animal 1743;
H-thymidine E13, LOT D3, HRP at one month. HRP placement in caudal
piriform cortex. Abbreviations: BST= bed nucleus of the stria
terminalis; CPU= caudate-putamen; OT= olfactory tubercle; V=
lateral ventricle. All brains reacted with DAB; D is lightly
counterstained with neutral red, others have no counterstain.
Bars= 500p.

























S .
CPU





C,: "-:31


CPU


- .4
a


a

di


C





24

of LOT transaction was verified in histological sections, and only

those animals with complete tract sections were included in the analysis.

Caudal to the level of the cut, the LOT was completely absent as a

bundle on the surface of the brain, and there was usually some dis-

tortion of the underlying cortex at the cut (Figure 4).

For Experiments II and III, approximately 200 (Experiment II) to

400 (Experiment III) sections from each olfactory bulb were examined

under the microscope at 600X for the presence of HRP-labeled and

double-labeled cells. Using the total number of cells per section

counted as above, the proportion of HRP-labeled cells to total cells

was calculated (p(HRP)). If there was no interaction between the

presence of 3H-thymidine and that of HRP in the cells, the incidence

of both labels in the same cell can be predicted, based on the pro-

portions of each label individually. The predicted proportion of

double-labeled cells for each animal is calculated by multiplying the

proportion of HRP-labeled cells by the proportion of H-thymidine-

labeled cells: p(double-labeled, predicted) = p(HRP) X p(thy). This

was compared to the actual number of double-labeled cells identified.

Deviation from the predicted value indicates some interaction of

the presence of H-thymidine label (time of cell formation) and HRP

label (time the axon reaches the projection areas). If the number of

double-labeled cells is greater than predicted, there is a positive

correlation between the particular time of cell formation and the time

of axonal outgrowth. Fewer than predicted double-labeled cells

indicates a negative correlation between these events.


























Figure 4. Site of lateral olfactory tract (LOT) transaction.
Animal 1743, LOT D3, sacrificed at one month of age. A, C, and
E are from the right (normal) side of the brain. B, D, and F are
from the left (operated) side. A and B. Sections through the
olfactory peduncle, rostral to the site of LOT transaction. The
LOT can be seen between the two arrowheads. In A, the normal LOT
is a pale region on the surface of the brain, easily identified by
the rows of glial nuclei. In B, the LOT rostral to the transaction
is somewhat reduced, and the glial nuclei are more disordered.
C-F. Sections through ithe rostral olfactory tubercle, at the level
of the cut. In C, the LOT is a discrete bundle of fibers (between
arrowheads). E. Higher power view of region between arrowheads
in C. D. The LOT is completely absent on the operated side. There
is some glial scarring near the pial surface, and some aberrant cell
groups are seen deep to where the LOT would be found (asterisks).
F. Higher power view of region between arrowheads in D. All
sections counterstained with neutral red. A-D: bars= 500p.
E,F: bars= 100p.



















































''
''
r; ..
z.. c~
~~rCT.'C

;s,: Y..


C T


L1


Ir



V


V


i1~ I





I


L' ~I

r














CHAPTER III
RESULTS


Experiment I

Results

Heavily-labeled mitral and tufted cells were found with 3H-

thymidine injections on E10, 11, 12, 13, and 14, but not on P1 or

P2. Following H-thymidine injection on E10, only a very few mitral

cells were heavily labeled (5 cells in 14 sections, 3 animals).

Mitral cells apparently undergo their last division primarily on

Ell and E12. No labeled mitral cells were seen following injections

on E13 or E14. Tufted cells were formed on Ell to E14. Inner

tufted cells were formed predominantly on Ell and E12, middle tufted

cells on E12 and E13, and outer tufted cells on E13 and E14. We

did not inject animals on E15 or 16 (day of birth), so the length

of the period of outer tufted cell formation is uncertain.

The depth analysis shows that there is an outward progression

of the position of cells formed from E10 to E14 (Figure 5). The

overall r values for each date of injection are E10= 0; Ell= .03

(n= 25-37, S.E.M.= .003-.020); E12= .39 (n= 164-208, S.E.M.= .024-

.028); E13= .72 (n= 93-219, S.E.M.= .012-.023); E14= .86 (n= 90-

152, S.E.M.= .011-.014). At each age there was a range of cell

positions, but the overall variability was quite low. In Figure 5,

the standard errors of the means are within the boundaries of the

circles for all animals. We found no consistent regional differences






























Figure 5. Left. Photomicrograph of MCL. and EPL of hamster olfactory
bulb, showing locations of mitral and tufted cells. Abbreviations:
EPL= external plexiform layer; Glom= glomerular layer; MC= mitral
cell; MCL= mitral cell body layer; TC= tufted cell. Bar= 25p.
Right. Representation of MCL and EPL, indicating average positions
(r) of heavily-labeled cells following H-thymidine injections on
E10 to E14. All animals were sacrificed at about one month of age.
Each circle represents r for one animal (SEM is within the boundaries
of each circle for this experiment). See text (methods) for
explanation.













0 i)
d OIL


0 0




0
e W

*- 4

N
0 c

L .
*0




O I
-r"
4'


-. t._ "-. -



1' PI 4 ;
--- -



.* A
A -. > &I a .rt. .
0 t.' 8 'I







within the bulb in the times of mitral and tufted cell formation, but

our sample size may have been too small to demonstrate subtle differ-

ences.



Experiment II

Rationale

Animals with 3H-thymidine injections on Ell or E13 were each

given injections of HRP into the olfactory bulb projection areas on

day 3 postnatally. The numbers of mitral and tufted cells with 3H-

thymidine label, HRP label, and both thymidine and HRP labels, and

the total numbers of unlabeled mitral and tufted cells were counted.

At day 3, the efferent projections from the bulb are still incomplete

(Leonard, 1975; Schwob and Price, 1978). The HRP injection on day 3

will label the olfactory bulb cells whose axons enter the lateral

olfactory tract and innervate the terminal regions early in the course

of development of this projection. If there is no interaction be-

tween the time of cell formation and the time the axon reaches the

projection areas, the proportion of cells with both 3H-thymidine and

HRP label would be equal to the proportion of HRP-labeled cells

multiplied by the proportion of 3H-thymidine-labeled cells. Our

hypothesis was that the earlier-formed cells send out their axons

earlier, thus we predicted that there would be more double-labeled

cells in the Ell group than in the E13 group.



Results

Table 2 indicates for each animal the number and percent of

HRP and 3H-thymidine-labeled cells, and the predicted and actual

proportions of cells containing both labels. For each animal in the



















A A A A









0000

NC \Or-








0000

r-4 en C14















0 00 C 0
H clO 0















00o00



~0000
'- \D CM CO
rtDc r'c















I..1 0-
C rl


1-1 1- 1-1
r,. en -It
-IT r) Lr
Lr n inin
r-i r-1 r-4


VVVV









en CM1O










0 > CM0

00~0





















SCoc'10Coz




0 O in O










r- 00 -

cM4


ooin
L,-L- -I-
HHHr


H
'CO
to





0
no
r-I1






1 C
'U


4





U)
u c










ca



! v
cn






32

Ell, HRP D3 group, the actual number of double-labeled cells is about

twice the predicted value. In the E13, HRP D3 group, three animals

had fewer than predicted double-labeled cells (about one-fifth the

predicted value), and one animal had no cells containing both labels.

The presence of double-labeled cells in brains of the E13 group which

had HRP injections at one month of age confirmed that cells formed

on E13 send their axons into the projection areas and maintain these

connections. These results support the hypothesis that the time of

axonal innervation of terminal regions correlates with the time of

cell birth.

A comparison of the depth distribution of 3H-thymidine-labeled

cells between the animals in this experiment sacrificed on day 4 and

those in this and the previous experiment sacrificed at one month shows

that r is significantly lower in the E13, D4 group than E13, 1 month

(Figure 6; .59 versus .72, t= 3.26, p<.005). There was no difference

in r between Ell, D4 and Ell, 1 month (r= .04, r= .03, respectively).

The cells formed on E13 have apparently not yet reached their final

positions in the EPL by day 4, while those formed on Ell have already

completed their migration.

When all the olfactory bulb projection neurons (labeled and

unlabeled) are considered, the ratio of all mitral cells to tufted

cells also changes significantly between 4 days and one month. The

ratio of mitral to tufted cells for 3H-thymidine Ell animals is 4.14

at day 4, 0.74 at one month (t= 13.1, p<.001). The ratio for 3H-

thymidine E13 animals is 6.23 at day 4, 0.72 at one month (t= 16.4,

p<.001). Figure 7 shows that at day 4 there are many more mitral than

tufted cells, but by one month this ratio is reversed so that there

are more tufted than mitral cells. This reflects the fact that the































Figure 6. Positions of heavily-labeled cells following 3H-thymidine
injections on Ell or E13. Animals were sacrificed at either 4 days
of age, or about one month. Each circle represents r, + SEM for
one animal. The value of r for E13, D4 is reduced compared to
that for E13, 1 month. Details are in text.
























0
0

^0-I
cOs
SO~


I.-


> o -
0 o


E

ro






























Figure 7. Change in the ratio of mean total number, + SEM (labeled
and unlabeled) of mitral to tufted cells be ween 4 days of age and
one month. Animals received injections of H-thymidine on Ell or E13.
At day 4, there are many more mitral than tufted cells, but the ratio
is reversed at one month. At 4 days of age, the number of cells in
the mitral cell layer is greater in the E13 animals than the Ell
animals.













Mitral cells

per unit

perimeter
5-





0-
Tufted cells

per unit
perimeter
5. Ell E 13
O sacrificed D4
M sacrificed I month








MCL is several cells thick at 4 days of age, but thins to a single

cell layer by one month (see Figure 1). The changes in the direction

of the ratio indicate that some cells in the MCL at day 4 are no

longer in that position at one month, and the thinning of the MCL is

not simply due to an expansion of the perimeter of the bulb. Sur-

prisingly, at 4 days of age, there are more total cells present in

the MCL if the animal received a 3H-thymidine injection on E13 rather

than Ell. The injection of 3H-thymidine on E13 affects the positions

of cells in (or passing through) the MCL on day 4. This effect will

be discussed later in relation to the consequences of LOT section in

Experiment III.



Experiment III

Rationale

Transection of the LOT at 3 days of age allows some bulb effer-

ents to reinnervate the olfactory cortex caudal to the cut, and the

density of innervation rostral to the cut is increased (Devor, 1976b).

Our hypothesis was that the fibers which grow through the cut are

new fibers which had not yet reached the level at which the tract

was sectioned. Experiment II demonstrated that the earlier-formed

cells send their axons into the projection areas first. For this

experiment, the assumption is that the later-formed cells send their

axons out last--a hypothesis which could not be directly tested in

Experiment II, since it is not possible to selectively label the later-

arriving axons with HRP in an intact animal. The axons of cells

formed on Ell are in (and may be expanding within) their projection

regions by day 3. Very few axons of cells formed on E13 have

reached this level by day 3. By adding a tract section on day 3, the








early-arriving axons would be cut. If the late-arriving axons grow

through the cut, these fibers and their cells of origin will be prefer-

entially labeled by HRP injection in the olfactory cortex one month

later. If our hypothesis is to be supported, the number of cells

labeled with both 3H-thymidine and HRP should be much greater in the

group with H-thymidine injection on E13 than on Ell.



Results

Our results do not support the hypothesis. They demonstrate,

instead, that cells formed on Ell, but not on E13, are able to re-

innervate the olfactory cortex (Table 3). Two of the Ell animals

had more double-labeled cells than would be predicted based on a

random association of the birthdate and axonal uptake of HRP. One

animal had fewer than predicted double-labeled cells, while the fourth

animal had no double-labeled cells. None of the animals that re-

ceived 3H-thymidine injections on E13 had any double-labeled cells.

Few cells formed on E13 were projecting through the level of the

tract section at day 3 (the time of the cut), and none passed

through the cut and subsequent scar.

Several other effects of LOT section on day 3 were observed.

Tract section on day 3 in animals with 3H-thymidine injections on

E13 produced a decrease in the average depth of H-thymidine-labeled

cells in the EPL, as compared to animals with no tract section

(Figure 8). The value of r in LOT-section animals is 0.59, as compared

to 0.72 for intact animals (t= 4.67, p<.001). The cells formed on

E13 do not move as far into the EPL after tract section as they do in

the normal animal. There was no change in the r of labeled cells in the

animals with tract sections injected with 3H-thymidine on Ell.














Table 3. Proportions
transaction.


of double-labeled cells after


Treatment


Double-labeled cells: %xlO2
Brain # Predicted Actual


Actual vs.
Predicted


Ell/ 1670L 1.6 0.3 <
LOT D3/ 1674L 0.4 0.9 >
HRP 1675L 0.1 0 <
1684L 0.8 1.4 >


E13/
LOT D3/
HRP


1579L
1580L
1743L
1747L


2.2
4.8
0.04
0


aTreatment= age of 3H-thymidine injection/ age of LOT transaction/
HRP placement at one month of age.


LOT





























Figure 8. Positions of heavily-labeled cells after transection of
the LOT at day 3 (LOT D3). Animals received injections of H-thymidine
on Ell or E13. All animals were sacrificed at about one month of
age. Each circle represents r, SEM for one animal. The value of
r is reduced following LOT section in the E13 animals.












1.0

Average
depth of
3H-thymidine
labeled 0.5.
cells (F)





0.

3H-thymidine


0= LOT D3












69000


E 13


^ 0


E ll


o00







Both the Ell and E13 3H-thymidine groups have a decreased number

of total (unlabeled) mitral cells per unit perimeter following LOT

section (Figure 9). The mitral cells are formed early (Ell and E12),

and are likely to have had their axons severed by the LOT section.

The number of tufted cells is increased in the E13 3H-thymidine

animals, but not in the Ell animals. Recall that animals in Experi-

ment II injected with 3H-thymidine on E13 and sacrificed on day 4

had more cells in the MCL than Ell H-thymidine animals. The in-

jection of H-thymidine on E13 appears to have an effect on the

positions of cells destined for the EPL that have yet to undergo

their final division. Examination of the numbers of 3H-thymidine-

labeled cells in brains with and without tract sections reveals that

there is no loss of either Ell 3H-thymidine-labeled cells or E13

3H-thymidine-labeled cells. The loss of mitral cells following LOT

section is apparently due to the loss of cells which were born between

these two injection times, or on E12, broadly speaking.

Tract section also had no effect on the mean width of the EPL

(Figure 10). A normal, age-related change in width occurs between

day 4 and one month, but this increase was not affected by tract

section on day 3.






























Figure 9. The ratio of total mitral to tufted cells in animals
with Sr without day 3 LOT section (LOT D3). Animals were injected
with H-thymidine on Ell or E13, and all were sacrificed at about
one month of age. Both Ell and E13 groups have a decreased
number of mitral cells, and the E13 animals have an increased
number of tufted cells after LOT section.















Mitral cells
per unit

perimeter

Tufted cells
per unit
perimeter


Ell E13

0 LOT D3





























Figur 10. Mean width, + SEM, of the EPL in animals injected
with H-thymidine on Ell or E13 and sacrificed at day 4 or one
month. Tract section at day 3 (LOT D3) had no effect on the width
of the EPL.












20-



15


Width

of

EPL


10.


5





3H-thymidine

Sacrifice


I) LOT D3


- V B


Ell E13

D4


E13 EII E13

I month














CHAPTER IV
DISCUSSION


Time of Mitral and Tufted Cell Formation


The mitral and tufted cells of the hamster olfactory bulb

undergo their last divisions between E10 and E14. Mitral cells are

formed primarily on Ell and E12, and tufted cells are formed on Ell

to E14. In the mouse olfactory bulb (Hinds, 1968a) mitral cells are

formed on E10-E15, peaking at E13, and tufted cells are formed on

E10-E18, peaking at E16. The period of histogenesis in the hamster

is both earlier and more compact than that of the mouse. The

golden hamster is born after an extremely short gestational period

of 16 days. This is one of the shortest gestational periods of all

rodents (Graves, 1945). The mouse is born after 19 days of gestation.

It has been reported that the hamster is born in a more immature

state than other rodents (Schneider and Jhaveri, 1974), but studies

of the general prenatal development of the hamster have found that the

fetal hamster is one of the most rapidly developing mammals known

(Graves, 1945; Ferm, 1967). In view of the hamster's immaturity at

birth, it was expected that mitral and/or tufted cells would continue

to divide in the early postnatal period. The results support, instead,

the concept of a more compressed, rapid period of prenatal develop-

ment. The olfactory bulbs of both hamster and mouse appear to be

at a similar stage of histogenetic development at the time of birth.





48

In a preliminary analysis of the data, an attempt was made to

classify the large cells in the external plexiform layer as either

inner, middle, or outer tufted cells, as has been done previously

(Ramon y Cajal, 1911; Hinds, 1968a). The initial analysis showed

that the inner tufted cells were more closely related to the mitral

cells in their time of origin than to the middle and outer tufted

cells. The histological appearance in Nissl and Golgi stains of the

inner tufted cells is very similar to the mitral cells. The depth

analysis in this experiment demonstrates that there is also no clear

histogenetic distinction between mitral and tufted cells, but rather

a continuum of cells from the MCL through the EPL. The distinction

between mitral and tufted cells was originally based on their positions

in different layers of the bulb (Ramon y Cajal, 1911). Although it

is now known that tufted cells project to olfactory cortical regions

along with the mitral cells, rather than to the contralateral

olfactory bulb, there are differences in the distribution of their

projections. Evidence from HRP and electrophysiological studies

indicates that tufted cells, especially middle and outer tufted

cells, project more heavily to the olfactory tubercle than to piri-

form cortex (Haberly and Price, 1977; Scott et al., 1980, Scott,

1981). The relationship of these differential projections to the

functional organization of the bulb or projection areas is yet to be

determined.

One limitation to the present analysis is that H-thymidine has

only been injected at 24-hour intervals. The cell cycle of hamster

cortical neurons has been estimated to be about 12 hours, with an S

phase (the time of DNA synthesis when 3H-thymidine is incorporated)







of about 6 hours (Shimada and Langman, 1970). It is not known if CNS

cells cycle in synchrony. Injection of 3H-thymidine at 24-hour inter-

vals illuminates fairly narrow "windows" of cell formation, and

labels only a fraction of the cells which actually go through their

last S phase on that day.



Correlation of Birthdate and Axonal Projection


The second experiment demonstrated that in the hamster olfactory

system, the axons of early-formed olfactory bulb cells reach the

olfactory cortex before the axons of late-formed cells. The corre-

lation of time of cell formation with axonal projections has been

suggested in the rat retina, based on indirect evidence from di-

verse techniques. Cell formation in the retina generally proceeds

from central to peripheral regions (Sidman, 1961), and occurs both

pre- and postnatally. There is evidence from Golgi studies that

axons from the central retina project into the optic tract when the

peripheral retinal axons are still in the retinal optic nerve layer

(Morest, 1970). Removal of one eye at birth produces an aberrant

increased projection to the ipsilateral superior colliculus. Enuclea-

tion at 5 days of age results in an ipsilateral projection only to

those areas innervated by the peripheral retina, again suggesting

that the peripheral axonal projection develops later than the central

projection (Lund et al., 1973). The present report is believed to

be the first demonstration of the correlation of time of cell form-

ation and axonal projection in individual cells.

The combination of the 3H-thymidine and HRP labeling, each of

which labels only a fraction of the possible cell population, results





50
in a fairly low expected proportion of double-labeled cells. In

Experiment II the average predicted proportion of double-labeled

cells was about 0.15%. There were about 350-400 mitral and tufted

cells per section, which gives an expected frequency of approximately

one double-labeled cell every two sections. Since about 200 sections

were examined for each olfactory bulb in this experiment, the very

low expected percentages translate into ample numbers of cells. The

early-formed cells (Ell) were double-labeled with a higher probability

than predicted, while the late-formed cells were labeled consid-

erably less than predicted.



Reinnervation after Transection of the LOT


After transaction of the LOT at day 3, axons of cells formed

on Ell are able to grow through the cut and reinnervate the olfactory

cortex. Axons of cells formed on E13, on the other hand, rarely

reinnervate the region caudal to the cut. Many of the Ell cells have

axons which are already in the projection areas at the time of the

transaction, and would thus be cut by this procedure. Some axons

of Ell cells may be well arborized along the course of the LOT, with

established connections, while others may be just entering the

region on their way to their final targets. Few axons of E13 cells

have reached the level of the transaction (which was generally at the

level of the rostral olfactory tubercle) by day 3. Using autoradio-

graphic methods to determine the LOT projection, Schwob and Price

(1978) found that the LOT in the rat first innervates the region deep

to the LOT, and then expands laterally, caudally, and medially. The

data reported here suggest that the axons with the most extensive






projections (from Ell cells), rather than those that have not yet

reached the region (from E13 cells), are the ones able to grow through

the site of an early tract section. The projections rostral to

the level of the cut (to the anterior olfactory nucleus and rostral

olfactory tubercle) are probably not extensively arborized at 3

days of age, but may be sufficient to provide "sustaining collater-

als" to support the cell. There may be other more mature features of

the early-formed cells and their axons which help to maintain the

cell and allow axonal regrowth. It is likely that the terminal fields

are reinnervated by collateral sprouts from proximal branches of

the transected fibers. The formation of collateral sprouts from

both the proximal branches of the severed axons and nearby undamaged

axons after lesions in young animals is now a well-established

phenomenon (see, for example: Bjorklund et al., 1971; Lynch et al.,

1973; Schneider, 1973; Pickel et al., 1974; Devor, 1976b; Kalil and

Reh, 1979). Following an early LOT section, Devor (1976b) found

both proximal collateral sprouting (rostral to the cut), and sprouting

from the association system fibers caudal to the cut. It is possible

that some of the proximal sprouts are able to continue through the

cut into the more caudal projection regions. Axons of the later-

formed cells (E13), which apparently cannot grow through the cut or

subsequent scar may contribute to the increased density of innerva-

tion rostral to the cut, since these cells do not die. There was a

loss of mitral cells following LOT section, apparently due to the loss

of mitral cells formed on E12. These cells may have had their axons

severed by the cut, but did not have sufficient proximal arborization

(or maturity) to maintain the cell's viability.





52

Other Effects of Lateral Olfactory Tract Transection


When a tract section is done on day 3, some of the cells of origin

in the bulb, especially tufted cells, have not yet completed their

migration (as shown in Figures 6 and 7). At day 4, there are more

cells in the mitral cell layer than in the EPL. As the perimeter of

the MCL increases, the thickness of the MCL decreases. The reversal

in the ratio of mitral to tufted cells, such that at one month of

age there are more tufted than mitral cells, indicates that many cells

in the MCL at day 4 are actually passing through the MCL and will

eventually be located in the EPL. Figure 7 shows additionally that

the number of cells in the MCL at day 4 is greater in animals in-

jected with 3H-thymidine on E13 than on Ell. Transection of the LOT

at day 3 results in a change of position within the EPL of cells

labeled with 3H-thymidine on E13. These cells have not moved as far

into the EPL as they normally would have (Figure 8). In addition,

the number of total (labeled and unlabeled) tufted cells is increased

after LOT section in animals given 3H-thymidine on E13, but not those

injected on Ell. This suggests that the altered position at the time

of tract section of a cell destined for the EPL affects the final

position the cell achieves. A possible mechanism for this effect is

proposed in the following section.



Aberrations in Developmental Interactions as Possible
Consequences of Mild H-thymidine Toxicity


In preliminary studies, animals were given 5pCi/gm of 3H-thymidine

on various days of gestation and in the early postnatal period. This

dose is higher than that required to label cells in other organ







systems, but it is a common dose for studies of the CNS (Sidman, 1970).

Although the hamster has not been found to have any deficiencies in

thymidine incorporation (Adelstein et al., 1964; Adelstein and Lyman,

1968), the dose of 5pCi/gm resulted in extremely light labeling of

cells, even after long exposure times (up to 13 weeks). The labeling

was especially light in animals injected early in the gestational

period (E10-11). The embryonic circulation is still developing at

this time, and it is likely that less thymidine is available to the

embryo for incorporation than later in development, given equivalent

intraperitoneal injections to the mother (Atlas et al., 1960; Taber

Pierce, 1967; Boyer, 1968). We were fairly confident that some cells

in the population examined ought to be undergoing their last divisions

and thus be heavily labeled, so the dose of 3H-thymidine was increased

to up to 10pCi/gm, given either in a single injection or in two

injections, one hour apart. Since intraperitoneally injected 3H-thy-

midine is available for nuclear incorporation for about one-half to

one hour (Sidman, 1970), giving two injections (in the context of an

S phase of about 6 hours) effectively increases the exposure time for

incorporation of thymidine. Lower doses of 3H-thymidine result in more

effective uptake and retention of the thymidine, so that incorporation

after two smaller doses is greater than if the same total amount was

given in one dose (Samuels and Kisieleski, 1963). The higher dose

produced identifiable labeling of all cell types.

As in many other studies of CNS histogenesis (for example: Hinds,

1968a; Altman, 1969; Fujita, 1967), there was no increase in the inci-

dence of abortion or maternal or pup mortality, and no gross devel-

opmental defects were observed. The results of this experiment,







however, suggest that the injection of 3H-thymidine on E13 has an

effect on the position of cells destined for the EPL that incorpor-

ate the 3H-thymidine at this time. Four pieces of evidence led to this

conclusion. 1) At 4 days of age, cells labeled with 3H-thymidine on

E13 have not yet reached their final positions in the EPL (as shown

in Figure 6). This is likely to partially reflect their normal

course of migration, but 2) the number of total cells--labeled and

unlabeled--in the MCL at day 4 is greater after 3H-thymidine injection

on E13 than on Ell (Figure 7). The normal course of migration of

unlabeled cells would not be expected to be different in these two

groups. Injection of 3H-thymidine on E13 appears to delay or prolong

the migration of cells in which it is incorporated. 3) Tract section

on day 3 also affects the positions of E13 H-thymidine-labeled cells

(Figure 8). 4) Tract section on day 3 causes an increase in the number

of tufted cells, many of which are formed on or after E13 (Figure 9).

These results have led to the speculation that perhaps the injection

of 3H-thymidine on E13 has a mildly toxic effect on cells that undergo

their last division on or after that time. This effect is expressed

as delayed migration or a decreased rate of migration, although the

initial effect could be to lengthen the cell cycle or interfere with

mitosis.

Cytological damage, cell death, and tumor induction have been

reported after doses of 3H-thymidine as low as 1-10OCi/gm (Baserga et

al., 1962; Samuels and Kisieleski, 1963; Kisieleski et al., 1964).

The biological effect of 3H-thymidine is related to the dose of

H-thymidine incorporated into the nucleus, which depends upon the

dose administered, the specific activity, and the age of the animal








(Baserga et al., 1962; Samuels and Kisieleski, 1963; Bond and

Feinendegen, 1966; Blenkinsopp, 1967). The doses of 5-10pCi/gm

commonly used in studies of CNS histogenesis are well within the

range known to cause damage, but effects less severe than cell death

may not be readily detected. Olsson (1976) has shown that 3H-thymidine

(lCi/gm) produces a temporary inhibition of DNA synthesis and a delay

in mitotic activity of labeled cells in mouse epidermis. Mitotic

activity of unlabeled cells was increased by 3H-thymidine injection.

Hicks and d'Amato (1968) found a decreased number of neurons in

layers II and III of rat cortex after injection of 16.4pCi/gm on

E17 (22 day gestation). They were not able to determine if this was

due to a delayed migration or cell death. Because of its lamination

and distinct developmental pattern, the olfactory bulb provided an

ideal system where it was possible to count both labeled and unlabeled

cells, and to follow the relative positions of cells with known times

of origin without the presence of labeled thymidine in the cells.

The toxic effect was only detectableby the comparison of injections

on Ell and E13. Although there may have been an effect of Ell in-

jection, the relatively greater effect was seen in the E13-injected

animals. It is likely that the effective incorporated dose of 3H-

thymidine was less in the Ell animals due to a relative immaturity

of the circulation. Even with higher doses of 3H-thymidine, cells

were more lightly labeled after Ell injections, which also suggests

that less 3H-thymidine was incorporated.

A delayed migration or decreased rate of migration of E13 cells

could explain both the increase in total tufted cells and the decreased

r of thymidine-labeled cells after LOT section in E13 animals. At 3








days of age, cells formed on E13, in their course of migration, are

likely to be passing by earlier-formed cells in the MCL and lower EPL.

Under normal circumstances, they would pass by E12 cells and proceed

to the appropriate level in the EPL. Cells formed on E14 would also

proceed in this manner, some of which may continue up into the

glomerular layer, where they are not easily identifiable as tufted

cells. In Ell or uninjected animals, following LOT section on day 3,

some E12 cells die, accounting for the decreased number of mitral

cells, but the E13 and E14 cells are already passing the E12 cells

and continue to their normal positions. When the animal has received

3H-thymidine on E13, the E13 and E14 cells arrive at the MCL later,

and may not have reached the E12 cells by the time the E12 cells die.

If one of the cues for termination of migration comes from the positions

of other cells which are being passed along the course of migration,

the E13 and E14 cells might assume lower positions in the EPL when the

population of E12 cells is reduced. The presence of some E14 cells in

the EPL which otherwise would have passed up into the glomerular layer

could result in the increased number of tufted cells in this case.

Both an increase in the duration of the cell cycle (Kauffmann,

1968; Hoshino et al., 1973) and a decrease in the rate of migration

(Hicks and d'Amato, 1968; Hinds, 1968b) are found to occur during

normal embryonic development. The differences in the positions of

the total cells between animals injected on Ell and E13 indicate that

there is an additional effect of 3H-thymidine injection on E13.

These results suggest that studies of time of cell formation and

migration should be thoughtfully interpreted, since the measuring

instrument ( H-thymidine) may influence the phenomenon being

measured (Olsson, 1976).






57

Possible Mechanisms of Reinnervation after Early Lesions


We have found that the axons of early-formed cells, which have

reached the olfactory projection regions early, are able to reinnervate

these regions after tract section on day 3. One question which has

not been answered by these experiments is why the LOT axons are not

able to regrow and reinnervate the projection regions when the tract

is cut after day 7. The results of these experiments show that it is

not due to the normal termination of new axons growing into the area

at this time. Other experiments have shown that while the capacity

for regrowth progressively decreases to day 7 (Devor, 1976b), there

is a large increase in the innervation of the olfactory cortex after

day 7 (Grafe and Leonard, 1981). From the experiments reported here,

it appears that the proximal parts of the more mature axons are

better able to survive the transaction and then move through the

cut or scar. Mature axons do retain some capacity for collateral

sprouting (Lynch et al., 1973) and Barker and Ip (1966, p. 550) found

that "motor axons of mammals undergo collateral and ultraterminal

sprouting under normal conditions" as well as when deafferented. The

critical changes in development which prevent axonal regrowth after

day 7 appear to be not in the cells of origin and their axons, but

rather in the tissue into which the axons are growing.

The end of the first postnatal week is approximately the time

when long-lasting degeneration argyrophilia appears in the olfactory

projections (Leonard, 1975). The persistence of degenerating fibers

could influence the ability of axons to reinnervate the area (Westrum,

1980). During this time there is also an increase in the growth of

the olfactory assocaition projections (Price et al., 1976; Singh, 1977;







Schoenfeld et al., 1981). Regrowing axons may not be able to compete

as well for synaptic space with association axons which had never been

interrupted. The glial and connective tissue scar which forms after

CNS injury has been recognized for many years to interact with axonal

regenerative attempts (Windle, 1956). The scar may serve not only as

a barrier (Windle, 1956; Puchala and Windle, 1977), but may also

actively deviate growing axons. Regenerating axons have been found

to associate with loose connective tissue and be deflected by dense

connective tissue (Fertig et al., 1971). In the developing and regen-

erating newt spinal cord, axons are guided by channels formed by the

ependymal cells and their processes (Singer et al., 1979). The scar

is not an absolute barrier, however, since regenerating adrenergic

axons can grow through the scar (Bjorklund et al., 1971; Nygren et

al., 1971). The early descriptions of regenerative attempts by

Ramon y Cajal (1928) report that axons can grow into the scar, but

then stop their growth. Windle (1956) and Puchala and Windle (1977)

report. pharmacological manipulations which allow axons to grow

into the region of the scar, but these also do not survive. There

may be developmental changes in the density or composition of the scar,

or in the presence and form of phagocytic cells in the region of the

scar, but the effects these factors may have on axonal regeneration

are not clear.



If the potential for reinnervation had been shown to be limited

by the period of axonal growth, the possibility for functional

recovery would be quite limited. The results of this study thus add

a positive note to the current views of the restorative capacities





59

of the CNS. The difficult questions of what the critical developmental

factors influencing axonal regrowth are, and whether they can be

modified in a functionally beneficial manner remain to be inves-

tigated.













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BIOGRAPHICAL SKETCH


Marjorie Grafe was born April 28, 1954,in Portland, Oregon,

to the Reverend Robert F. and Helen R. Grafe. She was raised in

Portland, and graduated from Woodrow Wilson High School in 1972.

She entered Stanford University, and in 1976 received the Bachelor

of Arts degree in psychology, with honors and distinction. She

entered the Department of Neuroscience at the University of Florida

as a graduate student in the fall of 1976, and received the Ph.D.

in 1981. In the fall of 1981 she is returning to Stanford University

to enter medical school on her way to becoming a neuropathologist.












I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.



Christiana M. Leonard, Chairman
Associate Professor of Neuroscience


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, s a dissertation for the degree
of Doctor of Philosophy. /



William E. Brownell
Assistant Professor of Neuroscience


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.



Marieta B. Heaton
Associate Professor of Neuroscience


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.



7Kinneth M. Heilman
Professor of Neurology












This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Phiolosophy.
December, 1981


't14 Z


ean, College of Medicine '



Dean for Graduate Studies and Research


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