Citation
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
Creator:
Grafe, Marjorie Ruth
Publication Date:
Language:
English
Physical Description:
vi, 66 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Axons ( jstor )
Brain ( jstor )
Golden hamsters ( jstor )
Hamsters ( jstor )
Lesions ( jstor )
Olfactory bulb ( jstor )
Olfactory pathways ( jstor )
Pups ( jstor )
Rats ( jstor )
Regrowth ( jstor )
Central Nervous System -- physiology ( mesh )
Dissertations, Academic -- Neuroscience -- UF ( mesh )
Nerve Regeneration ( mesh )
Neuroscience thesis Ph.D ( mesh )
Regeneration ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis(Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 60-65.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Marjorie Ruth Grafe.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
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08260580 ( OCLC )
ABX8733 ( NOTIS )
AA00006114_00001 ( sobekcm )

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Full Text













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




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


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


ACKNOWLEDGMENTS
I thank the members of my supervisory committeeDrs. William
Brownell, Marieta Heaton, Kenneth Heilman, and Christiana Leonard,
and Dr. William Luttge, acting chairman of the Department of
Neurosciencefor 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 Reskofor giving me a positive introduction to science; and
Dr. Robert Schimpfffor 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 parentsfor their constant love and support in
everything I did; and to Markfor 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.
iii


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 transection of the lateral
olfactory tract 17
Data collection and analysis ......18
III RESULTS 27
IV
Experiment I 27
Experiment II 30
Experiment III 37
DISCUSSION
47
Time of mitral and tufted cell formation 47
Correlation of cell birthdate and axonal outgrowth 49
Reinnervation after transection 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
iv


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.
v


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.
3
The second experiment involved the combination of H-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
3
reached the level of the cut. Animals were given H-thymidine on Ell
or E13, and a transection 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.
3
Evidence was found for a mildly toxic effect of H-thymidine
injections. The positions of cells destined for the EPL were
3
specifically affected by the incorporation of H-thymidine on E13.
vi


CHAPTER I
INTRODUCTION
In the past twenty years, the use of H-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, tuxwever, 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.
3
This study combines H-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.
3
The combination of H-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.
1


2
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).


3
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
pilthe 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; 0N=
olfactory nerve layer; S= subventricular layer. A,C: bar= 250y.
B,D: bar= 50y.


5


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 ir>
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 in olfactory
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).


7
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
3
its projection areas. This required the combination of H-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 cellsPickel et al., 1974; olfactory system
Devor, 1976b; pyramidal tractKalil 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


9
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
3
H-thymidine labeling of the times of formation of the mitral and
tufted cells, transection 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
I
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
3
intraperitoneal injections of H-thymidine (5-10yCi/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= P0). Several pups were each
3
injected subcutaneously with H-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 (E0), 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
11


Table 1. Animals and treatments
Animal
number
3
Day of H-thy
Total amount
LOT section
HRP
Day of
injection
n-thy(uCi/gm)
sacrifice
1382L
(E)a
E10
10b
_ _
D10
1470L
(E)
E10
10b


D49
1471L
(E)
E10
iob


D49
1128L
(A)
Ell
5
D30
1324L
(B)
Ell
10


D26
1325L
(B)
Ell
10


D26
1511L
(F)
E12
10b
D10
1512L
(F)
E12
10b


D10
1560L
(F)
El 2
10


D29
1560R
(F)
E12
iob


D29
1381L
(D)
E13
8b
D10
1468L
(D)
E13
8b


D49
1469L
(D)
E13
8b


D49
1354L
(C)
E14
10
D36
1354R
(C)
E14
10


D36
1353L
(C)
E14
10


D36
1353R
(C)
E14
10


D36
1546L
(I)
Ell
10b
D3
D4
1547L
(I)
Ell
igb

D3
D4
1553L
(J)
Ell
9b

D3
D4
1554L
(J)
Ell
9b

D3
D4


Table 1continued.
1502L
(H)
E13
8b
D3
D4
1503L
(H)
E13
8b

D3
D4
1505L
(H)
E13
8b

D3
D4
1556L
(K)
E13
8b

D3
D4
1487L
(D)
E13
8b
_
D53
D54
1601L
(H)
E13
8b

D30
D31
1687L
(K)
E13
8b

D36
D37
1749L
(L)
E13
8b

D26
D27
17501
(L)
E13
8b

D26
D27
1670L
(J)
Ell
9b
D3
D34
D35
1674L
(J)
Ell
9
D3
D37
D38
1675L
(J)
Ell
9b
D3
D37
D38
1684L
(J)
Ell
9b
D3
D39
D40
1579L
(G)
E13
8b
D3
D30
D31
1580L
(G)
E13
8b
D3
D30
D31
1743L
(L)
E13
8b
D3
D25
D26
1747L
(L)
E13
8b
D3
D26
D27
aLitter identification.
bIn two doses, one hour apart.


14
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 8y 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
4C, 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
3
For this experiment, we chose two days for H-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


15
tubercle, LOT, and/or piriform cortex of animals in both groups on
3
day 3 postnatally. Several animals with H-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 50y 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 4C, 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
50y 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% C0CI2 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 4C (60min/3hr).
7) 0.01-0.02% ^2^2 (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
3
Animals which had received H-thymidine on Ell or E13 were given
a transection 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
transection. 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
3
H-thymidine labeling, histologically verified complete LOT sections,
and satisfactory HRP injections in the olfactory projection areas.


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
3
at 600X for the presence of cells labeled with H-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 H-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
3
on the basis of their locations in the cell ( H-thymidine in the
3
nucleus, HRP in cytoplasm), color and size ( H-thymidine is small,
distinct black grains, HRP is larger, dark brown particles), and
3
plane of focus (silver grains for H-thymidine are in the emulsion
above the section, HRP reaction product is within the cell). The
3
positions of H-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 tissu^, 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 (arrox^head, upper right), and one double-
labeled cell (arrow and arrowhead). DAB reaction, counterstained
with neutral red. Bar= lOp.


2r\
U


21
mid-OB were drawn for each brain, since Ho 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
3
mitral to tufted cells determined. The proportion of H-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).
3
Cell counts for H-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 ofHRP 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), at the level of the caudal olfactory tubercle. B. Animal
1556; H-thymidine E13, HRP D3. HRP placement in and surrounding
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; 0T= olfactory tubercle; V=
lateral ventricle. All brains reacted with DAB; D is lightly
counterstained with neutral red, others have no counterstain.
Bars= 500y.




24
of LOT transection 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
3
presence of H-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
3
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
3
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) transection.
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 transection. 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 transection
is somewhat reduced, and the glial nuclei are more disordered.
C-F. Sections through the 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= 500y.
E,F: bars= lOOp.


26
l'. Jm
ML A
\ -m
I l w-.j
hmfc
fi -yWw


CHAPTER III
RESULTS
Experiment I
Results
3
Heavily-labeled mitral and tufted cells were found with H-
thymidine injections on E10, 11, 12, 13, and 14, but not on PI or
3
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
27


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= 25y.
Right. Representation of MCL and EPL, ^ndicating average positions
(r) of heavily-labeled cells following JH-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.


Glom
f- ,
3u-
H"thymidine injection


30
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
3
Animals with H-thymidine injections on Ell or E13 were each
given injections of HRP into the olfactory bulb projection areas on
3
day 3 postnatally. The numbers of mitral and tufted cells with H-
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
3
projection areas, the proportion of cells with both H-thymidine and
HRP label would be equal to the proportion of HRP-labeled cells
3
multiplied by the proportion of H-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
3
HRP and H-thymidine-labeled cells, and the predicted and actual
proportions of cells containing both labels. For each animal in the


Table 2. Numbers of
Treatment3 Brain //
cells labeled with HRP
HRP-labeled cells
///section (%)
3
and H-thymidine after
2H-thy-labeled cells
///section (%)
HRP placement
Double-labeled
Predicted
on day 3.
cells: %xl02
Actual
Actual vs.
Predicted
1546L
21.2 (5.9)
7.3 (2.0)
10.0
20.0
>
Ell/HRP D3 1547L
9.2 (2.8)
6.0 (1.8)
5.0
9.0
>
1553L
31.1 (7.3)
22.0 (5.0)
35.0
67.0
>
1554L
30.7 (7.3)
18.0 (4.0)
28.0
74.0
>
1502L
3.7 (0.8)
18.0 (3.7)
3.0
0.3
<
E13/HRP D3 1503L
4.0 (0.8)
22.0 (4.9)
3.9
1.2
<
1505L
2.8 (0.6)
30.5 (6.8)
4.2
0.9
<
1556L
23.1 (5.0)
28.0 (5.6)
26.0
0
<
aTreatment= age at thymidine injection/ age at HRP placement.


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.
3
A comparison of the depth distribution of H-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
3
ratio of mitral to tufted cells for H-thymidine Ell animals is 4.14
3
at day 4, 0.74 at one month (t= 13.1, p<.001). The ratio for H-
thymidine E13 animals is 6.23 at day 4, 0.72 at one month (t= 16.4,
pC.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 H-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.


1.0
Average
depth of
3H-thymidine
labeled Q.5
cells (?)
0
i
OOP
Sacrifice
3H-thymidine
D4 I month
D4 I month
E13


Figure 7. Change in the ratio of mean total number, + SEM (labeled
and unlabeled) of mitral to tufted cells between 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.


36
10.
Mitral cells
per unit
perimeter
0
Tufted cells
per unit
perimeter
5
PI sacrificed D4
Ml sacrificed I month


37
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
3
the MCL if the animal received a H-thymidine injection on E13 rather
3
than Ell. The injection of H-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 lasta 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


38
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
3
labeled with both H-thymidine and HRP should be much greater in the
3
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-
3
ceived H-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.
3
Tract section on day 3 in animals with H-thymidine injections on
3
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
3
animals with tract sections injected with H-thymidine on Ell.


39
Table 3.
Proportions
of double-labeled cells after
LOT
transection.
Double-labeled
cells: %xl02
Actual vs.
Treatment3
Brain #
Predicted
Actual
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/
1579L
2.2
0
<
LOT D3/
1580L
4.8
0
<
HRP
1743L
0.04
0
<
1747L
0
0
£
Treatment
3
= age of H-thymidine injection/ age of LOT transection/
HRP placement at one month of age.


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.


41
E 13
3H-fhymidine
Ell


42
3
Both the Ell and E13 H-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.
3
The number of tufted cells is increased in the E13 H-thymidine
animals, but not in the Ell animals. Recall that animals in Experi-
3
ment II injected with H-thymidine on E13 and sacrificed on day 4
3
had more cells in the MCL than Ell H-thymidine animals. The in-
3
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
3
their final division. Examination of the numbers of H-thymidine-
labeled cells in brains with and without tract sections reveals that
3
there is no loss of either Ell H-thymidine-labeled cells or E13
3
H-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 El2, 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 ^r without day 3 LOT section (LOT D3). Animals were injected
with ii-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.


44
Mitral cells
per unit
perimeter
Tufted cells
per unit
perimeter
Ell
EI3
LOT D3


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


46
Sacrifice D4 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.
47


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


49
of about 6 hours (Shimada and Langman, 1970). It is not known if CNS
3
cells cycle in synchrony. Injection of H-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.
3
The combination of the H-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 transection 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
transection, 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 transection (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


51
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-
3
jected with H-thymidine on E13 than on Ell. Transection of the LOT
at day 3 results in a change of position within the EPL of cells
3
labeled with H-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
3
after LOT section in animals given H-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
3
In preliminary studies, animals were given 5yCi/gm of H-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


53
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 5yCi/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
3
and thus be heavily labeled, so the dose of H-thymidine was increased
to up to 10yCi/gm, given either in a single injection or in two
3
injections, one hour apart. Since intraperitoneally injected H-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
3
incorporation of thymidine. Lower doses of H-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,


54
3
however, suggest that the injection of H-thymidine on E13 has an
effect on the position of cells destined for the EPL that incorpor-
3
ate the H-thymidine at this time. Four pieces of evidence led to this
3
conclusion. 1) At 4 days of age, cells labeled with H-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 cellslabeled and
3
unlabeledin the MCL at day 4 is greater after H-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
3
groups. Injection of H-thymidine on E13 appears to delay or prolong
the migration of cells in which it is incorporated. 3) Tract section
3
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
3
of H-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
3
reported after doses of H-thymidine as low as 1-lOyCi/gm (Baserga et
al., 1962; Samuels and Kisieleski, 1963; Kisieleski et al., 1964).
3
The biological effect of H-thymidine is related to the dose of
3
H-thymidine incorporated into the nucleus, which depends upon the
dose administered, the specific activity, and the age of the animal


55
(Baserga et al., 1962; Samuels and Kisieleski, 1963; Bond and
Feinendegen, 1966; Blenkinsopp, 1967). The doses of 5-10yCi/gm
commonly used in studies of CNS histogenesis are well within the
range known to cause damage, but effects less severe than cell death
3
may not be readily detected. Olsson (1976) has shown that H-thymidine
(lyCi/gm) produces a temporary inhibition of DNA synthesis and a delay
in mitotic activity of labeled cells in mouse epidermis. Mitotic
3
activity of unlabeled cells was increased by H-thymidine injection.
Hicks and d'Amato (1968) found a decreased number of neurons in
layers II and III of rat cortex after injection of 16.4yCi/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 detectable by 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
3
animals. It is likely that the effective incorporated dose of H-
thymidine was less in the Ell animals due to a relative immaturity
3
of the circulation. Even with higher doses of H-thymidine, cells
were more lightly labeled after Ell injections, which also suggests
3
that less H-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


56
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
"^H-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
3
there is an additional effect of H-thymidine injection on E13.
These results suggest that studies of time of cell formation and
migration should be thoughtfully interpreted, since the measuring
3
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 transection 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;


58
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 Puchal 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, 1954,10 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.
66


I certify that I have read this study and that in ray 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, is a dissertation for the degree
of Doctor of Philosophy. / J
III l
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.
S'-ft
^Kenneth 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
Dean for Graduate Studies and Research


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3 -J262 08554 3725


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

Bound by DOBBS BROS. LIBRARY BINDING CO., INC., St. Augustine,
You can live to be 100 and still learn something about melons.
-Anonymous

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.
iii

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 transection of the lateral
olfactory tract 17
Data collection and analysis ......18
III RESULTS 27
IV
Experiment 1 27
Experiment II 30
Experiment III 37
DISCUSSION 47
Time of mitral and tufted cell formation 47
Correlation of cell birthdate and axonal outgrowth 49
Reinnervation after transection 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
iv

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.
v

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.
3
The second experiment involved the combination of H-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
3
reached the level of the cut. Animals were given H-thymidine on Ell
or E13, and a transection 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.
3
Evidence was found for a mildly toxic effect of H-thymidine
injections. The positions of cells destined for the EPL were
3
specifically affected by the incorporation of H-thymidine on E13.
vi

CHAPTER I
INTRODUCTION
In the past twenty years, the use of H-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, tuxwever, 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.
3
This study combines H-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.
3
The combination of H-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.
1

2
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).

3
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; 0N=
olfactory nerve layer; S= subventricular layer. A,C: bar= 250y.
B,D: bar= 50y.

5

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 ir>
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 in olfactory
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).

7
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
3
its projection areas. This required the combination of H-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

9
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
3
H-thymidine labeling of the times of formation of the mitral and
tufted cells, transection 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
I
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
3
intraperitoneal injections of H-thymidine (5-10yCi/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= P0). Several pups were each
3
injected subcutaneously with H-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 (E0), 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
11

Table 1. Animals and treatments
Animal
number
3
Day of H-thy
Total amount
LOT section
HRP
Day of
injection
JH-thy(uCi/gm)
sacrifice
1382L
(E)a
E10
10b
_ _
D10
1470L
(E)
E10
10b
—
—
D49
1471L
(E)
E10
iob
—
—
D49
1128L
(A)
Ell
5
D30
1324L
(B)
Ell
10
—
—
D26
1325L
(B)
Ell
10
—
—
D26
1511L
(F)
E12
10b
D10
1512L
(F)
E12
10b
—
—
D10
1560L
(F)
El 2
10Í
—
—
D29
1560R
(F)
E12
iob
—
—
D29
1381L
(D)
E13
8b
D10
1468L
(D)
E13
8b
—
—
D49
1469L
(D)
E13
8b
—
—
D49
1354L
(C)
E14
10
D36
1354R
(C)
E14
10
—
—
D36
1353L
(C)
E14
10
—
—
D36
1353R
(C)
E14
10
—
—
D36
1546L
(I)
Ell
10b
D3
D4
1547L
(I)
Ell
igb
—
D3
D4
1553L
(J)
Ell
9b
—
D3
D4
1554L
(J)
Ell
9b
—
D3
D4

Table 1—continued.
1502L
(H)
E13
8b
D3
D4
1503L
(H)
E13
8b
—
D3
D4
1505L
(H)
E13
8b
—
D3
D4
1556L
(K)
E13
8b
—
D3
D4
1487L
(D)
E13
8b
_
D53
D54
1601L
(H)
E13
8b
—
D30
D31
1687L
(K)
E13
8b
—
D36
D37
1749L
(L)
E13
8b
—
D26
D27
17501
(L)
E13
8b
—
D26
D27
1670L
(J)
Ell
9b
D3
D34
D35
1674L
(J)
Ell
9Í
D3
D37
D38
1675L
(J)
Ell
9b
D3
D37
D38
1684L
(J)
Ell
9b
D3
D39
D40
1579L
(G)
E13
8b
D3
D30
D31
1580L
(G)
E13
8b
D3
D30
D31
1743L
(L)
E13
8b
D3
D25
D26
1747L
(L)
E13
8b
D3
D26
D27
aLitter identification.
bIn two doses, one hour apart.

14
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 8y 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
3
For this experiment, we chose two days for H-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

15
tubercle, LOT, and/or piriform cortex of animals in both groups on
3
day 3 postnatally. Several animals with H-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 50y 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 4°C, 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
50y 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% C0CI2 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 4°C (60min/3hr).
7) 0.01-0.02% ^2^2 (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
3
Animals which had received H-thymidine on Ell or E13 were given
a transection 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
transection. 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
3
H-thymidine labeling, histologically verified complete LOT sections,
and satisfactory HRP injections in the olfactory projection areas.

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
3
at 600X for the presence of cells labeled with H-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 H-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
3
on the basis of their locations in the cell ( H-thymidine in the
3
nucleus, HRP in cytoplasm), color and size ( H-thymidine is small,
distinct black grains, HRP is larger, dark brown particles), and
3
plane of focus (silver grains for H-thymidine are in the emulsion
above the section, HRP reaction product is within the cell). The
3
positions of H-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 tissu^, 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 (arrox^head, upper right), and one double-
labeled cell (arrow and arrowhead). DAB reaction, counterstained
with neutral red. Bar= 10p.

2r\
u

21
mid-OB were drawn for each brain, since Ho 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
3
mitral to tufted cells determined. The proportion of H-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).
3
Cell counts for H-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„HRP 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), at the level of the caudal olfactory tubercle. B. Animal
1556; H-thymidine E13, HRP D3. HRP placement in and surrounding
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;
â– ^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; 0T= olfactory tubercle; V=
lateral ventricle. All brains reacted with DAB; D is lightly
counterstained with neutral red, others have no counterstain.
Bars= 500y.


24
of LOT transection 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
3
presence of H-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
3
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
3
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) transection.
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 transection. 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 transection
is somewhat reduced, and the glial nuclei are more disordered.
C-F. Sections through the 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= lOOp.

26

CHAPTER III
RESULTS
Experiment I
Results
3
Heavily-labeled mitral and tufted cells were found with H-
thymidine injections on E10, 11, 12, 13, and 14, but not on PI or
3
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
27

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= 25y.
Right. Representation of MCL and EPL, ^ndicating average positions
(r) of heavily-labeled cells following JH-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.

EIO Ell E12 EI3 EI4
^H*thymidine injection
ho

30
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
3
Animals with H-thymidine injections on Ell or E13 were each
given injections of HRP into the olfactory bulb projection areas on
3
day 3 postnatally. The numbers of mitral and tufted cells with H-
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
3
projection areas, the proportion of cells with both H-thymidine and
HRP label would be equal to the proportion of HRP-labeled cells
3
multiplied by the proportion of H-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
3
HRP and H-thymidine-labeled cells, and the predicted and actual
proportions of cells containing both labels. For each animal in the

Table 2. Numbers of
Treatment3 Brain //
cells labeled with HRP
HRP-labeled cells
///section (%)
3
and H-thymidine after
2H-thy-labeled cells
///section (%)
HRP placement
Double-labeled
Predicted
on day 3.
cells: %xl02
Actual
Actual vs.
Predicted
1546L
21.2 (5.9)
7.3 (2.0)
10.0
20.0
>
Ell/HRP D3 1547L
9.2 (2.8)
6.0 (1.8)
5.0
9.0
>
1553L
31.1 (7.3)
22.0 (5.0)
35.0
67.0
>
1554L
30.7 (7.3)
18.0 (4.0)
28.0
74.0
>
1502L
3.7 (0.8)
18.0 (3.7)
3.0
0.3
<
E13/HRP D3 1503L
4.0 (0.8)
22.0 (4.9)
3.9
1.2
<
1505L
2.8 (0.6)
30.5 (6.8)
4.2
0.9
<
1556L
23.1 (5.0)
28.0 (5.6)
26.0
0
<
aTreatment= age at thymidine injection/ age at HRP placement.

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.
3
A comparison of the depth distribution of H-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
3
ratio of mitral to tufted cells for H-thymidine Ell animals is 4.14
3
at day 4, 0.74 at one month (t= 13.1, p<.001). The ratio for H-
thymidine E13 animals is 6.23 at day 4, 0.72 at one month (t= 16.4,
pC.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 H-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.

1.0
Average
depth of
3H-thymidine
labeled Q.5
cells (?)
0
i
OOP •••
Sacrifice
3H-thymidine
D4 I month
D4 I month
E13

Figure 7. Change in the ratio of mean total number, + SEM (labeled
and unlabeled) of mitral to tufted cells between 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.

36
10.
Mitral cells
per unit
perimeter
0
Tufted cells
per unit
perimeter
5
|"~1 sacrificed D4
HI sacrificed I month

37
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
3
the MCL if the animal received a H-thymidine injection on E13 rather
3
than Ell. The injection of H-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

38
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
3
labeled with both H-thymidine and HRP should be much greater in the
3
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-
3
ceived H-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.
3
Tract section on day 3 in animals with H-thymidine injections on
3
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
3
animals with tract sections injected with H-thymidine on Ell.

39
Table 3.
Proportions
of double-labeled cells after
LOT
transection.
Double-labeled
cells: %xl02
Actual vs.
Treatment3
Brain #
Predicted
Actual
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/
1579L
2.2
0
<
LOT D3/
1580L
4.8
0
<
HRP
1743L
0.04
0
<
1747L
0
0
£
Treatment
3
= age of H-thymidine injection/ age of LOT transection/
HRP placement at one month of age.

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.

41
E 13
5H"thymidine
Ell

42
3
Both the Ell and E13 H-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.
3
The number of tufted cells is increased in the E13 H-thymidine
animals, but not in the Ell animals. Recall that animals in Experi-
3
ment II injected with H-thymidine on E13 and sacrificed on day 4
3
had more cells in the MCL than Ell H-thymidine animals. The in-
3
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
3
their final division. Examination of the numbers of H-thymidine-
labeled cells in brains with and without tract sections reveals that
3
there is no loss of either Ell H-thymidine-labeled cells or E13
3
H-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 El2, 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 ^r without day 3 LOT section (LOT D3). Animals were injected
with JH-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.

44
5
Mitral cells
per unit
perimeter
Tufted cells
per unit
perimeter
Ell
EI3
â–¡ LOT D3

Figure 10. Mean width, + SEM, of the EPL in animals injected
with JH-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.

46
Sacrifice D4 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.
47

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

49
of about 6 hours (Shimada and Langman, 1970). It is not known if CNS
3
cells cycle in synchrony. Injection of H-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.
3
The combination of the H-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 transection 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
transection, 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 transection (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

51
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-
3
jected with H-thymidine on E13 than on Ell. Transection of the LOT
at day 3 results in a change of position within the EPL of cells
3
labeled with H-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
3
after LOT section in animals given H-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
3
In preliminary studies, animals were given 5yCi/gm of H-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

53
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 5yCi/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
3
and thus be heavily labeled, so the dose of H-thymidine was increased
to up to 10yCi/gm, given either in a single injection or in two
3
injections, one hour apart. Since intraperitoneally injected H-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
3
incorporation of thymidine. Lower doses of H-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,

54
3
however, suggest that the injection of H-thymidine on E13 has an
effect on the position of cells destined for the EPL that incorpor-
3
ate the H-thymidine at this time. Four pieces of evidence led to this
3
conclusion. 1) At 4 days of age, cells labeled with H-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
3
unlabeled—in the MCL at day 4 is greater after H-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
3
groups. Injection of H-thymidine on E13 appears to delay or prolong
the migration of cells in which it is incorporated. 3) Tract section
3
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
3
of H-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
3
reported after doses of H-thymidine as low as 1-lOyCi/gm (Baserga et
al., 1962; Samuels and Kisieleski, 1963; Kisieleski et al., 1964).
3
The biological effect of H-thymidine is related to the dose of
3
H-thymidine incorporated into the nucleus, which depends upon the
dose administered, the specific activity, and the age of the animal

55
(Baserga et al., 1962; Samuels and Kisieleski, 1963; Bond and
Feinendegen, 1966; Blenkinsopp, 1967). The doses of 5-10yCi/gm
commonly used in studies of CNS histogenesis are well within the
range known to cause damage, but effects less severe than cell death
3
may not be readily detected. Olsson (1976) has shown that H-thymidine
(lyCi/gm) produces a temporary inhibition of DNA synthesis and a delay
in mitotic activity of labeled cells in mouse epidermis. Mitotic
3
activity of unlabeled cells was increased by H-thymidine injection.
Hicks and d'Amato (1968) found a decreased number of neurons in
layers II and III of rat cortex after injection of 16.4yCi/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 detectable by 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
3
animals. It is likely that the effective incorporated dose of H-
thymidine was less in the Ell animals due to a relative immaturity
3
of the circulation. Even with higher doses of H-thymidine, cells
were more lightly labeled after Ell injections, which also suggests
3
that less H-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

56
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
"^H-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
3
there is an additional effect of H-thymidine injection on E13.
These results suggest that studies of time of cell formation and
migration should be thoughtfully interpreted, since the measuring
3
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 transection 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;

58
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 Puchalá 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.
66

I certify that I have read this study and that in ray 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, is a dissertation for the degree
of Doctor of Philosophy. / J
(/¡A í R
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.
//jf
'Kenneth 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
Dean for Graduate Studies and Research

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