THE AUTORADIOGRAPHIC DEMONSTRATION OF AXONAL CONNECTIONS, Cowan, 1972

BRAIN RESEARCH 21
Research Reports
T H E A U T O R A D I O G R A P H I C D E M O N S T R A T I O N OF A X O N A L CONNEC-
TIONS IN T H E C E N T R A L NERVOUS SYSTEM
W. M. COWAN, D. I. GOTTLIEB, ANITA E. HENDRICKSON, J. L. PRICE AND

T. A. WOOLSEY
Department of Anatomy, Washington University School of Medicine, St. Louis, Mo. 63110 and
Department of Ophthahnology, University of Washington, Seattle, Wash. 98105 (U.S.A.)
(Accepted August 9th, 1971)
INTRODUCTION
Most of the published work on the phenomenon of axoplasmic transport has

been directed towards determining the site of synthesis and the chemical nature of
materials involved, the mechanisms responsible for their movement, and the rates
at which they are transported (see refs. 1, 23, 44, 49, 53 for reviews). However, follow-
ing the suggestion of Taylor and Weiss 68, several workers, notably Lasek et al. 45,
have shown that the transport of radioactively labeled materials can be used to trace
axonal connections autoradiographically. This approach has thus far been applied
to the study o f the central connections of certain peripherally located neurons (spe-
cifically the dorsal root ganglion cells45, the ganglion cells of the retina 19,a°,aa,5°,65
and the olfactory receptorsT1). Although this method does not appear to have been
used for tracing pathways arising and terminating within the central nervous system,
it should be possible to do this by locally injecting radioactively labeled precursors
of proteins or other macromolecules into the brain or spinal cord. Indeed, there
are several reasons for thinking that this method may offer a number of advantages
over other currently available techniques4L
Nearly all experimental neuroanatomical techniques (including the various cell
degeneration methods, the reduced silver techniques for impregnating degenerating
axons - - such as the Nauta-Gygax method and its recent variants - - and the electron
microscopic examination of degenerating axons and their terminals) are dependent
upon a sequence of pathological changes following injury to the nervous system.
This essential feature underlies a number of the problems encountered in the use and
interpretation of these methods. Among the most significant difficulties is the fact
that experimental lesions are usually non-specific, and destroy not only the neurons
within the intended focus but also the fibers passing through that region and the
axons terminating within it. Since degeneration inevitably alters the normal morphol-
Brain Research, 37 (1972) 21-51

22 w.M. COWAN et al.
ogy of the injured neurons and their processes, at the site of the lesion or at the origin
of the pathway being studied, the normal cytoarchitecture is generally disrupted, and
at the termination of the fiber system it may be impossible to relate the morphology
of the degenerating axons and terminals to that seen in normal material. In addition,
most neuropathological changes are known to be extremely variable, both in their
severity and in their time course. The variability of the cellular reaction to axon sec-
tion, for example, has resulted in so many interpretative difficulties that cell degene-
ration methods are now quite limited in their usefulness. Similarly, because the rate of
axonal degeneration may vary markedly in different fiber systems, it is often difficult,
without carrying out a large number of experiments, to establish the optimum post-
operative survival period for the pathway under study. In many systems a further
problem is posed by the occurrence of retrograde fiber degeneration, and where recip-
rocal connections exist it may be extremely difficult to distinguish between the antero-
grade and retrograde changes.
Theoretically many of these problems could be obviated by the use of axo-
plasmic transport together with autoradiography to trace central connections. Be-
cause axoplasmic transport is a physiological phenomenon, the morphology of the
neuronal system being studied will not be altered by the experimental procedures
needed to label the axons. And, as the neuronal soma has been found to be the prin-
cipal site of synthesis of the proteins and other materials which are transported down
the axonS,10,11,44, the site of injection of the radioactive precursors should clearly mark
the origin of the labeled pathway: axons passing through, or terminating within, the in-
jected area should not transport significant amounts of the labeled precursor. It is
also known that materials synthesized within the neuronal soma are transported
along the axon in at least two phases which have different rates and appear to be
distributed within different components of the axon. Much of the rapidly transported
material reaches, and may accumulate within, the axon terminals, while the slower
phase (which constitutes the bulk of the transported material) is distributed more or
less uniformly over the length of the axon22,3°,ao,65,66; it should thus be possible to
selectively label either the terminal projection field of a pathway or its entire extent.
The purpose of the present paper is to validate these aspects of axoplasmic transport
in so far as they are applicable to morphological studies and to present observations
from a number of experiments which illustrate the use of autoradiography as a method
for tracing neuronal connections.
METHODOLOGY
In this section the general aspects of the methods we have used will be consider-
ed*. It should be emphasized that this is simply a description of a proven and prac-
tical method which can be readily adopted in most neuroanatomical laboratories;
similar methods have been used by other workers and further modifications of these
approaches can be anticipated as the autoradiographic method is applied to different
situations.
* A detailed protocol for the method we have used is available upon request to the authors.

AUTORADIOGRAPHIC TRACING OF C N S PATHWAYS 23
(1) Preparation and delivery of the radioactively labeled precursor
We have used tritiated leucine (L-[4,5-aH]leucine) in all of our experiments;

this amino acid is readily incorporated into proteins which are transported along the
anon, and the energy spectrum of tritium is well suited to autoradiography. However,
the method is equally applicable to other isotopes and to other precursor substances.
Most commercially available solutions of tritium-labeled amino acids are in concen-
trations of up to 1 mCi/ml. While this concentration is adequate for neural systems
where a relatively large volume of fluid can be accommodated at the site of injection
(e.g., for intraocular injection), for intracerebral injections it is generally desirable to
concentrate the isotope to permit the injection of a relatively large quantity of labeled
amino acid in a small volume. The desired concentration can be readily obtained by
evaporating the solvent in an evacuated desiccator and then redissolving the labeled
amino acid in sterile distilled water or saline. Since concentrated aqueous solutions of
tritiated compounds are unstable, they should be used as soon after concentration as
possible.
The actual injection technique and the amount of isotope administered will
obviously vary according to the system under study. In some of our experiments we
have injected the isotope through fine bore hypodermic needles (27-33 gauge) mounted
on a 50/B Hamilton syringe, or when smaller volumes were required, through the
needles supplied with the 10/zl and 1 #1 Hamilton syringes. Still finer delivery systems,
such as micropipettes, have been used by other workers45; these produce less damage
at the site of injection and allow for more restricted injections. The ultimate refine-
ment of this approach is the intracellular delivery of isotope to single neurons as
recently demonstrated by Globus and his colleagues 18 in the spinal cord.
It is advisable to administer the isotope over a period of some minutes to
permit the maximum incorporation of precursor into transported material and to
minimize tissue damage at the site of injection. To facilitate this, the anesthetized
animal should be immobilized and the injection system mounted on a carrier, such
as a conventional stereotaxic electrode holder.
(2) Postinjection survival times
As with degeneration methods, it is important to determine the optimum sur-

vival time for each system studied. The reported rates of transport for the rapid and
slow phases vary over a wide range 44 but, as a rule of thumb, material transported in
the rapid phase can be assumed to travel at a speed in excess of 100 ram/day, and that
in the slow phase at a rate of less than 5 ram/day in most warm-blooded animals.
After estimating the length of the fiber system under study, an approximate survival
time can be calculated using these rates. For more detailed studies it is advisable to use
a timed survival series; for this purpose the levels of radioactivity in the system may
be determined either by liquid scintillation counting 3-~ or by autoradiography.
As we shall show, the rapidly transported materials can be used to provide an
indication of the sites of terminal projection fields, and for this purpose relatively

short survival periods (generally between 2 and 72 h) are required. Most of the mate-
rial which is transported moves in the slow phase and is distributed throughout the
axons, making longer survival periods (up to 3 or 4 weeks) more useful for tracing
fiber pathways, especially where the number, or concentration, of fibers is low.
(3) Fixation and histological processing
We have used a variety of routine histological fixatives for light microscopic

autoradiography, including 1 0 ~ formalin, Bouin's and Carnoy's solutions. For
electron microscopic autoradiography the fixative of choice is 4 ~ buffered para-
formaldehyde. Glutaraldehyde-containing solutions should be avoided since these
are known to bind free amino acids 56.
For light microscopic autoradiography the tissues can be processed using
standard methods for embedding in paraffin, carbowax or plastic. Frozen sections
can also be used; this is particularly useful when autoradiography is to be com-
bined with the study of axonal degeneration. The choice of section thickness will
depend upon a number of considerations: when the identification of cytoarchitec-
tonic features is of prime importance it is generally advisable to use relatively thick
sections (10-20 #m); where fine cytological detail is needed, and for optimal auto-
radiographic resolution, 1-2 #m plastic embedded sections have been found to give
the best results. If thick sections are used it should be remembered that since the /3
emissions from tritiated compounds are of low energy, only label in the upper portion
of the section (at the most, the upper 5 #m) will contribute to the autoradiographic
image.
In most respects the processing of tissue for autoradiography requires no special
precautions, but if paraffin embedded material is used it is advisable to deparaffinize
the sections for at least 12 h in fresh xylene, before hydration and coating with emul-
sion.
(4) A utoradiography
As the techniques of autoradiography are well established and have been thor-
oughly reviewed elsewhere39, 62, we will deal only with certain aspects of the proce-
dures which we have used. For light microscopic autoradiography we have used
Kodak NTB2 and NTB3 emulsions because of their ready availability and generally
consistent quality. NTB2 is preferred since it is more stable, has a larger grain size
and is of adequate sensitivity for use with tritium. Autoradiographic emulsions have
a relatively short shelf life but this can be prolonged by storing them in the cold (but
not at freezing temperatures).
For coating, the emulsion should be melted in a water bath at 40°-45°C and
diluted in the ratio of one or two parts of emulsion to one part of distilled water.
Due to the low energy of the/3 particles emitted by tritium, an emulsion thickness of
slightly more than a monolayer will capture virtually all the emissions which enter it,
and for quantitative comparisons such emulsion coatings can be treated as if they

AUTORADIOGRAPHIC TRACING OF CNS PATHWAYS 25
were of unitbrm thickness. The emulsion should be tested before use by dipping blank
slides and processing them by the same procedures as are to be used for the experi-
mental material. If the number of grains in the test slides exceeds a reasonable level
(e.g., ~ 10 grains/100× oil immersion field with NTB2), the emulsion should be
discarded. The slides with the experimental sections can be dipped either individually
or back to back; the latter procedure conserves time and emulsion, and in our ex-
perience does not cause slippage of the emulsion coat. After allowing the emulsion
to set for 1-3 h - - in a humid atmosphere to prevent cracking - - the slides are packed
in light-tight boxes containing desiccant and stored in a refrigerator or freezer during
exposure. A number of additional slides with sections containing the area under study
should be coated and packed in a separate box; these will serve as test slides which
can be developed at various intervals until the optimum exposure time has been deter-
mined. Exposure times of 1-3 weeks have been found to be adequate for most of the
light microscopic material discussed in this paper.
For light microscopic autoradiography we have developed the coated slides for
2-3 min in fresh, undiluted K o d a k D-19 developer. The temperature of all the
photographic solutions must be strictly maintained between 14°C and 19°C39; above
these temperatures background levels increase very sharply. After rinsing in running
water, the slides are fixed for 5-10 min in a rapid fixer, such as K o d a k Ektaflo, in
the dilution recommended for films, and then washed in gently running tap water
for at least 10 rain.
(5) Staining
A wide variety of standard histological stains are applicable to autoradio-

graphy 6z,67,69. The material which will be presented below has been routinely stained
through the emulsion with either thionine or cresyl violet because our primary con-
cern has been to relate the pathways under study to cytoarchitectonic structure.
The sections may be stained either directly after washing, or they may be dried and
stained at a later time. Differentiation can be carried out in the usual way; some stain-
ing of the emulsion is inevitable but this should present no problem when the sections
are examined.
(6) Electron microscopic applications
One of the principal advantages of the autoradiographic method for tracing

connections is that it can be readily extended to the fine structural level. This permits
the precise identification of the terminal projections of the labeled neurons and has
one important advantage over the degeneration methods in that the normal morphol-
ogy of the axon terminals and their synaptic relations are unaltered. The procedures
most commonly used are those of Salpeter and Bachmann 63 and of Granboulan z6,
to whose papers reference should be made for details of the technique. The papers
of Salpeter et al. 64, of Hendrickson30, 31, and of Haddad etal. ~8 should be consulted
for a discussion of the interpretation of electron microscopic autoradiography. An

abbreviated account of the technique we have used is included with the protocol
referred to in the footnote on page 22.
RESULTS
We have used the autoradiographic method for tracing neuronal connections

in recent studies of the visual pathways in the monkey and chick, the cortico-thalamic
connections in the mouse, and the olfactory and hippocampal systems in the rat.
A number of selected observations will be presented from this material to illustrate
certain features of the autoradiographic method: (1) the localization of isotope at the
site of the injection; (2) the transport and distribution of labeled materials in systems
in which the connections are well known from earlier studies; (3) the localization
of the labeled material in axons and their terminals; (4) the absence of transport of
labeled materials by fibers passing through the injection site; and (5) the absence of
retrograde transport of labeled materials.
(1) The injection site
Following the local injection of [3H]-labeled amino acids into the brain, there
is prompt uptake and incorporation of precursors by the neurons in the vicinity of
the injection. Autoradiographs of this area in animals sacrificed shortly after injection
show a heavy concentration of silver grains over the neuronal somata and relatively
few grains over the intervening neuropil (Fig. 1A). With longer post-injection times
the gcains come to be more evenly distributed over the somata and neuropil (Fig. 1B,
~k:' 3"
~;~
Fig. 1. This figure indicates the changes in the distribution of silver grains at the site of injection of
the labeled precursor with time. In each case, 5 #Ci of [aH]leucine were injected into the somatosenso-
ry cortex of a mouse; the animals were allowed to survive for the following times: A, 1.5 h; B, 4 days;
C, 18 days. At the shortest survival time the grain density is greatest over the neuronal perikarya;
with progressively longer survival periods the grains come to be more evenly distributed over the
neuropil, and at 18 days there appear to be relatively few grains over the neuronal somata.

C) and there is a progressive decrease in the total number of grains. This sequence of
changes in the distribution of labeled material at the site of local injection is essentially
the same as that seen by Droz and Leblond ~1 following the systemic administration
of labeled amino acids. These workers have shown that a considerable proportion of
B ~lmm
Fig. 2. The site of injection of radioactive precursors into the brain can be readily identified even at
low power, as shown by these autoradiographs of frontal sections through the somatosensory cortex
immediately adjacent to the needle track in two mice. In each cas~, 5 #Ci of [ZHlleucine in 0.5/~1 were
injected through a 1//1 Hamilton syringe, and the animals were allowed to survive for 24 h. The emul-
sion was exposed for two weeks. In A the injection is in the medial $1 motor cortex; in B the injection
is in the SI face area. The thalamic projections in these two animals are illustrated in Fig. 6.
Fig. 3. Since the concentration of label at the site of injection is particularly heavy, it is possible to
identify the labeled neurons in this region after very short exposure times. A is a low-power photo-
micrograph of an unstained autoradiograph from the site of injection of [ZHlleucine into the cerebral
cortex of a rabbit (10/~Ci; 3 days survival); this, and the autoradiograph shown in B, were developed
24 h after coating the sections with Kodak NTB2 emulsion. Although the silver grains are not so
prominent in stained preparations after such short exposure times, the injection site can still be
readily identified at higher magnifications, as shown in B.

the newly-synthesized proteins are not transported but remain within the cell soma
(the so-called 'sedentary proteins') and that these have a relatively slow turnover
rate which may be on the order of several weeks.
The extent of spread of the injected amino acids can be determined by noting
the distribution of neuronal somata which have been labeled (Fig. 2); the precise
location, and even the approximate number of labeled cells, can thus be accurately
defined. Neuronal injury at the site of injection is not generally a complicating factor
since only living neurons are capable of synthesizing materials for transport down
their axons. Furthermore, as the injection itself causes relatively little damage, the
architectonic structure at the site of the injection is not disrupted and so it is possible
to identify with certainty the cells of origin of the pathway being studied; this is not
generally possible with lesion methods. A final point of some practical value is that
because of the high concentration of radioactivity at the injection site, the accuracy
of placement of the injection can be determined, in selected sections, as early as one
or two days after coating (Fig. 3). In this way the usefulness of a given experiment
can be assessed before processing the entire series of sections.
(2) A utoradiographic study of known projection systems
A critical test of any new method is that when it is applied to systems whose
organization is well established, it can faithfully reproduce the pattern of connections
demonstrated by other established methods. Lasek et al. 45 have shown that after
injection of [3H]leucine into the dorsal root ganglia of toads and cats, the transported
proteins can be followed through the dorsal roots into the dorsal columns and hence
to the termination of these fibers in the dorsal column nuclei. Similarly, the pattern
of radioactivity which was found in the gray matter of the spinal cord corresponded
very closely to the distribution of degenerating fibers stained by the Nauta method
after dorsal root section.
The central connections of the monkey retina were recently re-examined in a
comparative study using the Nauta-Gygax and the Fink-Heimer methods following
eye removal, and light microscopic autoradiography after intraocular injection with
[aH]leucine a3. There was an excellent correspondence in the findings with the two
methods; radioactivity was found in all sites in which terminal degeneration could be
seen in the silver-stained preparations, and conversely, labeled material was never
seen in sites which could not be shown to receive a retinal projection by the degenera-
tion methods. In addition, in the pretectal region and in the pregeniculate nucleus,
the autoradiographic method showed details of the retinal projection with greater
clarity than had been seen in earlier degeneration studies.
The projection of the retina has also recently been examined in the pigeon 65
and chick 37 by this method and the distribution of radioactivity after intraocular
injection of [3H]leucine compared with observations based on a variety of reduced
silver methods. The clarity with which the major fiber bundles can be traced in auto-
radiographs is particularly well shown in the region of the optic chiasm where the
labeled fiber bundles from the injected eye interdigitate with the unlabeled fibers

AUTORADIOGRAPHICTRACING OF CNS PATHWAYS 29
.f.'.., ~ .:,~..;,,,:., ,~:, .: :
q'L -I ., "~L" ¢" "~, "r~ ~.
rd:J¢_ "
Fig. 4. The contrast between labeled and unlabeled fibers is especially well demonstrated in the optic
chiasm of the chick where the ipsi- and contralateral fibers interdigitate(A); similarly, the localized
distribution of the retinal fibers within the outer layers of the stratum griseltm et fibrosum superficiale
of the tectum (through layer f) is quite distinct (B). Both of these autoradiographs were from the
brain of a 3-week-old chick which survived 8 days after the intraocular injection of 50 #Ci of [ZH]-
leucine.
from the control eye (Fig. 4A). Similarly, no difficulty has been found in determining
the terminal projection fields of the chick retina, and labeled material has been traced
to all the visual relay nuclei identified by degeneration methods 6,37. The pattern of
labeling in the optic tectum in these experiments will be referred to later, but it may
be noted here that it corresponds exactly with the distribution of degenerating axon
terminals seen with the electron microscope in the outer layers of the stratum griseum
et fibrosum superficiale (Fig. 4B).
The subsequent evidence shows that with appropriate injection methods labeled
amino acids can also be introduced intracerebrally to trace central connections. The
injection of [ZH]leucine into the olfactory bulb of rats leads to the selective labeling
of all those central olfactory structures which have been defined by degeneration
methods, including the anterior olfactory nucleus, the olfactory tubercle, the piriform
cortex and the cortical amygdaloid nucleus. The laminar pattern of termination of
the axons from the olfactory bulb in these areas as seen with the autoradiographic
method is exactly comparable to that demonstrated by the reduced silver methods 29,59
(Figs. 5 and 14). Using more restricted injections of isotope it has also been possible
to demonstrate the organized projection of the bulb upon the olfactory tubercle re-
cently shown by reduced silver methods ~9. Thus, when the injected isotope is con-
fined to the dorsal portion of the bulb, labeled material is only found in the antero-

30 w . M . COWAN et al.
'- . "-7 " " "Z, ~% "" ":''
50p
I I I
Fig. 5. In systems in which it has been possible to compare fiber projections studied with the auto-
radiographic and the Nauta or Fink-Heimer methods, there has always been a good correspondence
between the two approaches. The laminar distribution of silver grains in an autoradiograph of the
piriform cortex taken from a rat which had survived 5 days after injection of 50/~Ci of pH]leucine
into the olfactory bulb, at which time the slow component would have reached the cortex, is shown
in A. This is very comparable to the distribution of degenerating fibers and terminals seen with the
Fin~Heimer method after a lesion of the bulb (B). C is a photomicrograph of a Golgi-Cox prep-
aration to show the position of the neurons and their dendrites in this part of the cortex.
lateral p a r t o f the olfactory tubercle, b u t if mitral cells are labeled t h r o u g h o u t the

bulb, the t r a n s p o r t e d m a t e r i a l is f o u n d over the entire extent o f the tubercle.
The cornmissural connections o f the r a t h i p p o c a m p u s a n d dentate gyrus have
also been studied, using stereotaxic injections o f [3H]leucine20, zl. The details o f these
experiments will be given below, b u t at this p o i n t it m a y be n o t e d t h a t the projections
which they show (see Figs. 9 a n d 10) are in g o o d agreement with the p a t t e r n o f degen-
e r a t i o n seen with the electron m i c r o s c o p e a n d with r e d u c e d silver methods3, 61. In
a d d i t i o n , the a u t o r a d i o g r a p h i c m e t h o d has m o r e readily revealed the h o m o t o p i c
o r g a n i z a t i o n within the c o m m i s s u r a l connections o f the h i p p o c a m p u s a n d has p r o -
vided strong evidence t h a t the crossed p r o j e c t i o n to the dentate gyrus has its origin
within the h i p p o c a m p u s itself.
Finally, the o r g a n i z a t i o n o f c o r t i c o - t h a l a m i c fibers has been studied in the
m o u s e 6°,7z. W i t h d e g e n e r a t i o n m e t h o d s it has been difficult to conclusively establish

A U T O R A D I O G R A P H I C T R A C I N G OF C N S P A T H W A Y S 31
Gralns / 3600p2 ~ _ _ _ _ ~ ~ ~ Grains / 3600p2
• ....o - - . . ~ m ~ .-~. U~ . . . .
o ~ / ; o 7 . ~.;<U . . . . . •, ,..~ ....... I

o~o o ~/o ~,C~o ,~N . . . . • • • • .I . . . . . . . I o
onooo[oooo .... • .oo.olo. .... o •
o,o o, . . . . . . . o,,o . . . . . . ....... , ..... !:
. . . . . . . . . . . . ., . . . . . . . •
,o,o1~ ....... ~o ~ . • :1 . . . . . . . U ~ (
~ojo ........ -,, .... ~ ....... • I~o ~ ....... .o ......
~oo%o~ ...... ~o~oo' . . . . . . . • ~-o.~..I .... ooo~o.'~ ...... \
. . . . . . '. •. . . . . . o ~.a~-o~o '

~ o o o o o o o . . . kk:l:
. .
. . . .
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o o • o • • •
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155 18 36 598
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Fig. 6. These experiments on the cortico-thalamic system of the mouse indicate the precision with
which terminal fields may be localized in nonlaminar structures. The arrows on the brain outlines
in the upper part of the figure indicate the center of the sites of the injections into the somatosensory
cortex (SI) in the two experiments illustrated in Fig. 2 (the outline on the left corresponds to Fig. 2A;
that on the right to Fig. 2B). Both labeled areas have a diameter of approximately 1 m m (Fig. 2) and
extend rostrally into the related motor cortex. Superimposed on the outline of the ventral nuclei
of the thalamus in these two cases (in the central portion of the figure) are grids of open circles and
dots of various sizes to indicate the grain density in each 3,600 sq.#m area of the ventrobasal complex
(VB), the ventromedial nucleus (VM), the ventrolateral nucleus (VL), the reticular nucleus (R) and
the zona incerta (ZI). In the lower part of the figure are 4 representative autoradiographs of the 60
t t m × 60/~m areas marked on the nuclear outlines; the numbers above each autoradiograph indicate
the actual grain counts in each of these areas.
The injection into the medial part of SI has resulted in a very localized distribution of grains over
the external portion of VB; the larger labeled area over VL is due to the labeling of the adjoining
motor area by the injection. The injection into the face area of SI has given a larger, but equally well-
defined, area of labeling in the arcuate portion of the VB complex.
the existence of such fibers because of the inevitable retrograde changes in the thala-
mo-cortical neurons after neocortical ablations and the possibility of concomitant
d e g e n e r a t i o n i n r e c u r r e n t c o l l a t e r a l s o f t h e s e cells ~7. A s w e s h a l l s h o w l a t e r , t h e a u t o -
r a d i o g r a p h i c m e t h o d o b v i a t e s t h i s c o m p l i c a t i o n . F o r p r a c t i c a l p u r p o s e s , t h e r e is n o
retrograde transport of labeled material, hence any labeled material found in the

32 w.M. COWAN el al.
thalamus after local injection o f isotope into the cortex can only be due to transport in
cortico-thalamic fibers. By injecting [aH]leucine into the first somatic sensory (SI)
area o f the mouse (Fig. 2), a topographically organized projection u p o n the ventrc-
basal complex o f the thalamus has been found. With injections into the medial
portion o f SI, in which the tail, hind limb and trunk are represented 72, autoradio-
graphs show that the labeled material is limited to the external part o f the ventrobasal
nucleus (Fig. 6). On the other hand, after injections into the lateral portion o f the
somatosensory cortex (the barrel field,) which contains the p r i m a r y representation
of the face 74, the transported material is found only in the medial portion o f the nucleus.
Grain counts over the ventrobasal complex o f the thalamus, such as those shown in
Fig. 6, indicate that the n u m b e r o f grains over the center o f the projection field is
approximately an order o f magnitude above background. Using this approach, the
extent o f the thalamic projection from this cortical area has been defined with con-
siderable precision.
Fig. 7. Much of the rapidly transported material accumulates within axon terminals where it can be
demonstrated by electron microscopic autoradiography. This micrograph is from lamina 6 of the
lateral geniculate nucleus of a monkey 3 days after the injection of 100 ttCi of [aH]leucine into the
vitreous of the contralateral eye. Most of the silver grains (some of which are indicated by arrows)
are over axon terminals (t), although one is over a myelinated axon at the bottom of the figure (a).
Dendrites are labeled (d). Note that the morphology of the retino-geniculate axon terminals is un-
altered, which is one of the principal advantages of this method as compared to degeneration
techniques.

(3) The localization of the rapidly and slowly transported materials
Several electron microscopic autoradiographic studies have served to establish

that the transported materials are contained within axons 10,32, but in the present
context it is important to emphasize that they are transported down the entire length
of the axon and into the axon terminals. Direct evidence for this has been adduced
from electron microscopic autoradiographic studies of the monkey 31 and pigeon 66
visual systems.
Three days after the intraocular injection of [3H]leucine in the monkey, auto-
radiographs of the 3 laminae of the lateral geniculate nucleus which receive fibers from
the injected eye show that more than 50 ~ of the silver grains are over optic nerve
terminals (Fig. 7). Only about one quarter of the grains attributable to this rapidly
transported material are found over myelinated axons in the same layers. At survival
times long enough for the slow component to reach the lateral geniculate (27 and 30
days), the pattern of grain distribution is almost exactly reversed; between one-half
and two-thirds of the grains are found over myelinated axons, while the optic nerve
terminals account for only one-fourth to one-third of the grains. The remaining grains,
after both long and short survival times, are distributed over neuronal somata,
dendrites, glial cells and blood vessels (see Discussion) 31.
Schonbach et al. 66 have examined the distribution of the rapidly transported
materials in the pigeon optic tectum by electron microscopic autoradiography 12 h
after injecting [3H]leucine into the contralateral eye. In the outer layers of the tectum
in which the retinal fibers are known to terminate 6, 67 ~ of the silver grains were
localized over axon terminals and only about 1 8 ~ over axons. Their evidence,
together with that cited above for the monkey al, suggests that labeled material trans-
ported in the rapid phase can provide a clear indication of the sites of termination of
projection pathways.
A number of light microscopic autoradiographic studies have also provided
indirect evidence bearing on the distribution of the rapid and slow components.
The earliest of these is McEwen and Grafstein's 50 study of the distribution of trans-
ported materials in the goldfish optic tectum. Here, the material transported in the
rapid phase is preferentially located in those layers in which the retinofugal fibers
terminate; the material transported in the slow phase, on the other hand, has been
found both in the layers which contain the incoming optic nerve fibers and the layers
in which they terminate. Essentially the same observations have been made in studies
on the visual system of the pigeon 65, and of the chick 37. Twenty-four hours after the
injection of [aH]leucine into the eye, the highest concentration of silver grains in the
contralateral optic tectum is found over the layers of the stratum griseum etfibrosum
superfieiale in which most of the retinal fibers terminate. One week after eye injection
(at which time much of the slowly transported material has reached the tectum) the
highest grain densities are found over the retinal fibers in the stratum opticum, and
relatively fewer grains are seen in the layers containing the terminal segments of the
retinal fibers (Figs. 4 and 8; ef Figs. 4 and 5 of Ref. 65).
A third system in which the distribution of materials transported in the rapid

34 w . M . COWAN et a[.
"010
o SO a b c d e f g h
9
c /\~8 days post injection
: 8 k,~..~ t slow )
o
7
6 \
¢:
L.
(3 4 i nje,© Itl,o n 1 \I I 1~
-- 3
2
0
I- 1
Ioop ~ ~--e
~pia
Fig. 8. To illustrate differences in the distribution of silver grains in the contralateral optic tectum
1 and 8 days after the injection of 50 ffCi of [3Hlleucine into the contralateral eyes of two 3-week-old
chicks. For convenience of comparison the grain densities in the various laminae are presented as
percentages of the total number of grains counted in perpendicular traverses across the stratum
opticum (SO) and the outer layers of the stratum griseum etfibrosum superficiale and the cytoarchitec-
tonic layers of one brain have been matched to the other (the stratum opticum and the plexiform layers
b, d, f and h are stippled; the predominantly cellular layers a, c, e and g are shown as clear bands).
The vertical arrows indicate the extent of the terminal projection field of the retinal fibers as deter-
mined by the Nauta method. It is clear that the rapidly transported material is relatively more con-
centrated in this region while the slow component has labeled the retinal fibers in the SO most
heavily.
phase has been studied with the light m i c r o s c o p e is the c o m m i s s u r a l p r o j e c t i o n o f the

h i p p o c a m p u s 20,21. T w e n t y - f o u r to 48 h after injecting a small q u a n t i t y o f [3H]leucine
into the h i p p o c a m p u s o f one side, l a b e l e d m a t e r i a l is f o u n d only in those regions o f the
c o n t r a l a t e r a l h i p p o c a m p u s a n d dentate gyrus which are k n o w n f r o m d e g e n e r a t i o n
studies to receive c o m m i s s u r a l fibers3, 61. Systematic counts o f the n u m b e r o f grains
in selected traverses across the d e p t h o f fields CA1 a n d CAz o f the h i p p o c a m p u s show
a very striking degree o f localization. F o r example, in field CA1 the n u m b e r o f grains
f o u n d over the alveus ( t h r o u g h which certain o f the c o m m i s s u r a l fibers reach the h i p p o -
c a m p u s ) is close to b a c k g r o u n d levels (Fig. 9). Just b e y o n d the b o u n d a r y between the
alveus and the stratum oriens there is a b o u t a 20-fold increase in the n u m b e r o f silver
grains; this agrees well with electron m i c r o s c o p i c findings t h a t between 20 ~ a n d 40
o f the synapses in this region degenerate after lesions w h i c h i n t e r r u p t the c o m m i s s u r a l
fibers. The n u m b e r o f grains falls to a b o u t o n e - t h i r d o f this level over the stratum
pyramidale where electron m i c r o s c o p i c o b s e r v a t i o n shows only a small n u m b e r o f
c o m m i s s u r a l terminals. There is a n o t h e r increase in the n u m b e r o f grains over the
inner four-fifths o f the apical dendritic field (the stratum radiatum) in which the re-
m a i n d e r o f the c o m m i s s u r a l fibers t e r m i n a t e .Over the o u t e r one-fifth o f this field
(the stratum moleculare) the grain c o u n t is again close to b a c k g r o u n d levels. In field

hi ppocampal fissure -~
-atD molecu la re &

0
I lacunosum
, 4
~ . .I~. ~
i
radiatum
~ - 2 0
pyramidale
oriens
l o 0 j~ I~ • al veus ,~_ q ~ ; z . ' r ~ L ~ ,j

o 20 40 60 80 l o o 12o14o
ar ai n s / 476 jj2
Fig. 9. This figure illustrates the use of the autoradiographic method for localizing terminal pro-
jection fields. Ten ~tCi of [3H]leucine were stereotaxically injected into the hippocampus of a rat which
was allowed to survive for 48 h. The distribution of silver grains was determined by a series of grain
counts across the depth of field CA1 of the contralateral hippocampus. A segment of an autoradio-
graph of field CA~ is shown on the right of this figure; the arrowheads mark the width of the area over
which grains were counted. The distribution of grains over the different strata is shown in the graph
in the middle part of the figure: ovei the alveus (through which cert,.in of the cemmissural fibers reach
the hippocampus), and in the distal part of the apical dendritic field (the stratum lacunosum moleculare)
the number of grains is at background levels and is approximately 1/20th of that in the stratum orielts
and about 1/10th that in the stratum radiatum where the commissural fibers have been shown to
terminate by light and electron microscopic degeneration studies. By contrast, electron microscopic
studies have shown that few commissural fibers terminate in the stratum pyramidale, and the counts
show that few silver grains are found there. The tracing of a Golgi impregnated neuron on the left
indicates the arrangement of the basal and apical dendrites upon which most of the commissural
fibers terminate.

hippocampal fissureJ.
20f
5011 ?:r
v ,
I
J
w v
¢n
i
>
15 molecu lare kr •- ti
1Q
¢D
v-
granu|osum
0 5 10 15 20 25
Grains/ 4 7 6 jp2
Fig. 10. To show the distribution of silver grains in the dentate gyrus contralateral to the injection
in the same experiment used for Fig. 9. Here the great majority of the silver grains are found over a
narrow (40 #m) segment of the dendritic field of the granule cells• The arrowheads in the autoradio-
graph mark the site of the grain counts which are plotted in intervals of 12.6/~m on the left. The
boundaries of the stratum granulosum and the stratum moleculare are also indicated.
CAs a similar pattern of grain distribution is found although, interestingly, in this field
there are very few grains over the stratum lucidum, which is m a i n l y occupied by the
mossy fibers from the dentate gyrus, and their large axon terminals• The distribution
o f the labeled material in the dentate gyrus in these experiments is particularly striking;
here the grains are confined to a narrow band approximately 40 # m wide in the inner
one-third of the molecular layer, as are degenerating terminals after lesions of the
contralateral fimbria 21 (Fig. 10).
In all of these experiments the localization of the transported material to the
terminal fields after short postinjection survival times suggests that the rapidly
t r a n s p o r t e d material can be used to determine the site of t e r m i n a t i o n o f fiber path-
ways with some confidence. With longer survival periods, material transported in the
slow c o m p o n e n t can be utilized to d e m o n s t r a t e the fiber pathways themselves, as

~ ~r~
qpOe
O
I
,Ollb
o a
q
t
Fig. 11. A light microscopic autoradiograph of a frontal section through the corpus callosum of a
mouse to demonstrate the usefulness of the slow component of axoplasmic transport for tracing
fiber pathways. In this experiment 5 #Ci of [aH]leucine were injected into the face area of the primary
somatosensory cortex and the animal was allowed to survive for 9 days. A well-defined fascicle of
fibers in the ventral portion of the corpus callosum is heavily labeled. The arrowhead marks the
midline.
well as their terminal projections. We have already indicated in the descriptions of the
electron microscopic study of the retino-geniculate projection in the monkey 31 and
of the light microscopic studies of the retinotectal projection in the pigeon 65 and in
the chick 37, that the amount of radioactive labeling in the axon increases with time
after the injection of [aH]leucine (Fig. 8; cf. Figs. 3 and 5 of ref. 65). This point has
also been systematically studied in the mouse cortico-thalamic system, where after
short postinjection survival periods (2-48 h), some labeled material is found in the
internal capsule but this is considerably less than the amount seen in the terminal
projection sites such as the ventrobasal nucleus. On the other hand, in animals which
survived from 4 to 16 days after injection, the entire pathway from the cortex, through
the internal capsule, to the ventrobasal complex, is clearly labeled, as are other cor-
tical projections, including that via the corpus callosum to the contralateral soma-
tosensory cortex (Fig. 11).
Thus, by carefully selecting the post-injection survival times, advantage can be
taken of the rapid and slow components of axonal transport to demonstrate either
terminal fields or the whole extent of the fiber pathway under study. However, it
should be emphasized that the rapidly transported material is not exclusively distrib-
uted within axon terminals. At all times after the passage of the rapid phase some la-
beled material is found within the axons of every fiber system we have examined.
The ratio of the label in the axons to that in the terminals at short survival periods
appears to change with different experimental conditions, and in our experiments has
varied from approximately one-twentieth in the rat hippocampus (Fig. 9) to about

two-thirds in the chick optic tectum (Fig. 8). In all cases the a m o u n t o f radioactive
material in the axon markedly increases with longer survival times, making the demon-
stration o f the fiber p a t h w a y practicable.
(4) Evidence for the absence of transport by fibers of passage
A n i m p o r t a n t question which must be considered is whether or not fibers passing

t h r o u g h the area labeled by the injected isotope are able to incorporate the labeled
precursor and to transport the newly synthesized materials. Three experiments bearing
on this problem may be cited. In the first, [ZH]leucine was injected into the corpus
callosum of a rabbit and the animal was sacrificed 3 days later. A u t o r a d i o g r a p h s o f
this brain show that at the site o f the injection there is label in the corpus callosum
(most o f which appears to be due to uptake by glial cells), but beyond the site
o f the injection the level o f radioactivity drops off rapidly. There is no label in
the contralateral limb o f the corpus callosum, and there is a complete absence o f
label in the related areas o f the cerebral cortex on either side (Fig. 12). Similarly,
injection o f [ZH]leucine into the depths o f the olfactory bulb (without substantial
labeling o f the mitral cell somata) or into the lateral olfactory tract at the level o f the
anterior olfactory nucleus does not result in the transport o f labeled material to the
~dh,.,~"~'z" O ..~ .2 ~ "wwret
e" "-"
It "~,
Fig. 12.A, A low-power photomicrograph of a frontal section through the brain of a rabbit into which
10/~Ci of [aH]leucine in 0.5 #1 of saline were injected into the corpus callosum at the site marked by
the large arrowhead; the animal survived for 3 days following the injection. B, C and D, Autoradio-
graphs of the 3 areas marked in A, to show the local uptake of labeled material near the site of in-
jection (D), and the absence of labeled material in the corpus callosum on the ipsilateral (B) and
contralateral sides (C). This suggests that there is no significant transport of labeled material by
axons passing through the site of injection.
Brain Research, 37 (1972) 21-5l

PC ~ ~=' ~b ~, '~
e
ti 8 •
|,,
" 6
Fig. 13. To demonstrate that fibers of passage do not incorporate significant amounts of precursor
for transport, 6 #Ci of [aH]leucine were injected into the lateral olfactory tract (LOT) and the
anterior olfactory nucleus (AON) of a rat which survived for 3 days. In A, a parasagittal section
through the olfactory peduncle shows the appearance of the LOT and the AON close to the injection
site. Anterior (a), posterior (p), dorsal (d) and ventral (v) directions are given at the right. B is a higher
power view of the lateral olfactory tract and shows that at this level there is a considerable amount of
label in the tract. C is an autoradiograph of the piriform cortex caudal to the site of injection. There
is no label in the terminal projection field of the olfactory tract fibers in the molecular layer of the
piriform cortex (PC) (cf. Fig. 5).
t e r m i n a l p r o j e c t i o n sites o f the bulb. The absence o f labeling over the p i r i f o r m cortex

in these experiments is in striking c o n t r a s t to the heavy labelling in the p i r i f o r m area
in experiments in which the mitral cell s o m a t a are labeled (Fig. 13).
(5) The absence of retrograde transport of labeled materials
It r e m a i n s to be shown t h a t w h a t e v e r i n c o r p o r a t i o n o f a m i n o acids m a y occur

in axon t e r m i n a l s at the site o f injection, there is no a u t o r a d i o g r a p h i c evidence for
r e t r o g r a d e t r a n s p o r t o f labeled m a t e r i a l t o w a r d s the cell soma.
I n experiments in which the o l f a c t o r y b u l b was injected with [3H]leucine, with-
o u t i n v o l v e m e n t o f the a n t e r i o r o l f a c t o r y nucleus, we have f o u n d no evidence o f
r e t r o g r a d e t r a n s p o r t in the cells o f origin o f either the ipsilateral o r the c o n t r a l a t e r a l
centrifugal fibers to the bulb. The absence o f label over the ipsilateral nucleus o f the
h o r i z o n t a l l i m b o f the d i a g o n a l b a n d (which has been identified as the nucleus o f origin
o f the ipsilateral centrifugal fibers aS) is p a r t i c u l a r l y obvious by c o m p a r i s o n with the

40 w . M . COWAN et al.
Fig. 14. Light microscopic autoradiographs to show (1) the use of this method for tracing connections
in a well-established fiber system, and (2) the absence of retrograde transport of labeled materials in
the same system. A, Following the injection of 50/~Ci of [3H]leucine into the olfactory bulb of the
right side, the lateral olfactory tract (LOT) and the superficial one-half of the plexiform layer of the
anterior olfactory nucleus (AON) were heavily labeled on the injected side 5 days after the injection,
but there was no label in the contralateral A O N in which the crossed centrifugal fibers to the olfactory
bulb have their origin, nor in the anterior commissure (AC) in which the centrifugal fibers run. B,
Similarly, in this brain there was no labeled material in the nucleus of the horizontal limb of the
diagonal band (HDB) from which the ipsilateralcentrifugal fibers to the olfactory bulb arise, although
the outer portion of the plexiform layer of the olfactory tubercle (OT) was heavily labeled.

AUTORADIOGRAPHICTRACING OF C N S PATHWAYS 41
500p
Fig. 15. A low-power autoradiograph of a frontal section through the caudal part of the diencephalon
of a mouse into which 5 pCi of [aH]leucine were injected into the anterior thalamic and habenular
nuclei of the left side. Although the animal survived for only 24 h the efferent fibers of the habenular
nuclei in the habenulo-peduncular tract (HP) are heavily labeled. The afferent fibers to the anterior
nuclei in the mammillo-thalamic tract (MT) are not labeled. The absence of label in the mammillo-
thalamic tract shows quite clearly that there is no significant retrograde axoplasmic transport (see
also Fig. 14).
heavy label in the olfactory tubercle immediately ventral to this nucleus (Fig. 14B).
The centrifugal fibers in the anterior commissure are clearly unlabeled and there is no
indication o f label over the contralateral anterior olfactory nucleus from which these
arise 48 (Fig. 14A).
A n o t h e r example which m a y be cited is f r o m an experiment in which [3H]leucine
was injected directly into the dorsal thalamus o f a mouse, labeling the cells in the
anterodorsal and anteroventral nuclei and in the medial and lateral habenular nuclei.
The habenulo-peduncular tract which arises from the latter two nuclei is heavily
labeled and can be readily identified even at low magnifications (Fig. 15), as can the
interpeduncular nucleus in which it terminates. The mammillo-thalamic tract, which
terminates in the anterior thalamic nuclei, and the medial and lateral mammillary
nuclei, from which the tract arises, show only background levels o f radioactivity.
In another experiment [aH]leucine was injected into the lateral geniculate nucleus and
the superior colliculus: again no evidence o f retrograde transport was f o u n d in the
optic nerve, although the striate cortex was heavily labeled.
A similar finding has been made after intraocular injections o f [3H]leucine in
the pigeon 65 and in the chick 37. Neither the nucleus o f origin o f the centrifugal fibers

to the retina (the isthmo-oWic nucleus) nor its projection pathway (the isthmo-optic
tract) 7 was ever labeled, even though in some of the experiments sufficient time had
elapsed for the slowly transported materials in the optic nerve to reach the caudal
end of the optic tectum.
DISCUSSION
Our observations, together with those of previous workers, demonstrate that

the autoradiographic tracing of axonally transported radioactive materials can be
used as a neuroanatomical method for determining the origin, course and termination
of fiber pathways within the central nervous system. The basis of the method is that
radioactively labeled amino acids and certain other precursor substances injected
into specific regions of the brain are taken up and incorporated into protein and other
macromolecules by neuronal somata in these regions and transported along the
axons where the label can be demonstrated by autoradiography. The most rapidly
transported materials are largely distributed to the axon terminals while the more
slowly transported materials are distributed along the whole length of the axons.
(1) The physiological basis of the method
(a) Site of precursor uptake and protein synthesis. The synthesis of injected pre-
cursors into proteins and other macromolecules by neurons depends first upon the
uptake of the precursors, and secondly upon their incorporation into the appropriate
macromolecules. Since the evidence relating to the sites of amino acid incorporation
is clearest, we shall only consider this subject here.
Ultrastructural and biochemical evidence indicates that neuronal ribosomes
are concentrated within the cell perikarya and large dendrites where they are found
as elements of the rough endoplasmic reticulum (or Nissl bodies) or as free ribosomes,
and that it is in these structures that most protein synthesis occurs. Droz 8 has shown
by electron microscopic autoradiography that 5 min after the systemic administration
of [3H]leucine, radioactivity in the cells of the ciliary and superior cervical ganglia
was associated with the rough endoplasmic reticulum. At later times, radioactive
material appeared in the Golgi apparatus, throughout the perikaryal neuroplasm,
and in the axon hillock. Similarly, in an earlier light microscopic study, Droz and
Leblond la had shown that 30 min after the systemic administration of this radioactive
amino acid in the mouse, labeled material in the cerebellum and hippocampus was
found primarily in the Purkinje and pyramidal cell layers. The molecular layers which
contain the dendrites of these cells, and the white matter through which their axons
pass, were relatively free of label after 30 rain but became labeled after longer survival
times. Our observations on the distribution of silver grains in light microscopic auto-
radiographs at the site of an injection into the cerebral cortex after various survival
periods similarly indicate that initially most of the labeled material is concentrated in
the neuronal somata and that there is a progressive decrease in the amount of this ma-
terial as it is metabolized and/or transported into the processes of the cells.

A U T O R A D I O G R A P H I C T R A C I N G OF CNS PATHWAYS 43
Although dendrites contain free ribosomes and rough endoplasmic reticulum,

there is little direct evidence relating to protein synthesis in dendrites. The evidence
of Droz and Leblond 11 cited above indicates that dendritic synthesis is normally
at a low level compared with that in the perikaryon (see also ref. 10). However, since
the dendritic volume may greatly exceed that of the perikaryon, even a relatively low
level of protein synthesis in the dendrites may contribute substantially to the total
production of protein by the neuron.
It is now clear that most axons are capable of very little protein synthesis 44.
Ribosomes are only rarely seen in electron micrographs of axons, and all direct che-
mical measurements of R N A in axons reveal a very low concentration relative to that in
the perikaryon (ranging from 1/40th in the Mauthner cell of fish 12, to 1/500-1/1,000th
in the accessory nerve of the catZS). Recently, Heuser and Miledi a4 have shown that
isotopically labeled leucine and glutamate, whether applied externally or injected
intra-axonally, are not incorporated into protein by portions of the squid giant axon
which are distant from the cell body. These results are particularly interesting because
they show that even though these axons may take up labeled amino acids, they do not
incorporate them into protein. Our own experiments suggest further that if amino
acids are taken up and incorporated into protein by axons in the mammalian nervous
system, this protein is not transported in sufficient amounts to be detectable auto-
radiographically (Figs. 12 and 13). Thus, any protein which may be synthesized by
axons is not likely to complicate the interpretation of experiments using the autoradio-
graphic method.
Axon terminals have been shown to be capable of local uptake of several sub-
stances such as norepinephrine, dopaminO 7 and choline 2, but whether axonal
endings are capable of significant amounts of protein synthesis is still an open question.
Droz and Barondes 9 have shown that labeled material may be found in cortical synap-
tosomes isolated shortly after intracortical injection of labeled amino acids. Although
this finding could be accounted for by the rapid transport of labeled proteins from the
somata of short axon cells, more recent work in which synaptosomes were first iso-
lated and then incubated with labeled amino acids has shown that axon terminals are
capable of some degree of protein synthesis in vitro 5.
The question as to which regions of the neuron are capable of amino acid up-
take for subsequent incorporation into transportable material by the soma or nearby
portions of the neuron is also of considerable interest to the morphologist since this
will determine the effective size of an extracellular injection site. For any given in-
jection the pool of labeled neurons will be larger if, for example, the processes are
capable of uptake than if uptake is limited to the perikaryon. Unfortunately there
appears to be no evidence bearing upon this important point at this time.
(b) Axoplasmie transport. Two major phases of axoplasmic transport have now
been identified in a wide variety of fiber systems and animal species. Although there are
considerable variations in the absolute values of both the rapid and slow components
in different species (see Lasek 44, pp. 300-304), the relative rates are quite similar,
the rate of the rapid component being approximately two orders of magnitude
greater than that of the slow phase. It should be pointed out, however, that in addi-

tion to these two well-defined phases, there is evidence for various intermediate phases
of transport 3~,42,4a, and for changes in the rates of transport during postnatal devel-
opment 32 and during nerve regeneration ')5.
Three closely related questions are posed by these findings: (1) What substances
are transported in each component? (2) What roles do the transported materials play
in the economy of the cell? (3) What are the mechanisms involved in their transport?
A full discussion of these problems is clearly outside the scope of this paper, but
some comments are pertinent in so far as they bear on the use of axoplasmic transport
for tracing connections in the nervous system.
The composition of the materials transported in the two phases of axoplasmic
transport appears to be heterogenous, and as yet has been only partially characterized 54.
The rapid component is generally considered to consist, in part at least, of particulate
matter, which may include mitochondria and synaptic vesicles, and is thought to be
implicated in some aspect of synaptic function. The slow component, on the other
hand, consists largely of soluble material and possibly some particulate matter and
may be primarily involved in the growth and maintenance of the axon 23,44. To date,
the most conclusive identification of a portion of the transported material comes from
work on sympathetic postganglionic fibers, where [all]labeled norepinephrine has
been shown to be transported in the rapid component, probably in association with
at least two of the protein constituents of the dense-core synaptic vesicles, dopamine-
fl-hydroxylase, and chromagranin A 17,46,47. The recent finding of Forman et al. 15
that glucosamine appears to be transported exclusively in the rapid component is of
particular interest in this context since it may well be used to selectively label axon
terminals. One further observation which merits comment is that of Miledi and Sla-
ter 52, who have shown that following interruption of the phrenic nerve at different
levels, the time of onset of failure of neuromuscular transmission is compatible with
the loss of materials from the axonal stump at rates comparable to the movement
of the rapid phase of axoplasmic transport.
The 'trophic' influence of neurons on postsynaptic neurons or other cells is a
well-known, but poorly understood, phenomenon. Recent studies have indicated that
materials transported in the rapid phase may enter postsynaptic cells. For example
Grafstein 24 has shown that after the intraocular injection of labeled amino acids in
mice, some radioactivity is found in the contralateral striate cortex (amounting to
about 3 ~ of the activity seen in the lateral geniculate nucleus). Similarly, by electron
microscopic autoradiography, after intraocular injection of [3H]leucine, Hendrickson 31
has found that a small, but significant, number of silver grains is localized over den-
drites in the monkey lateral geniculate nucleus after short postinjection periods. The
nature of the material found in the postsynaptic cells in this case is unknown. Sim-
ilarly, the mode of transfer of label from presynaptic fibers, if this occurs, remains
to be clarified, but these observations represent an important initial step in under-
standing the function of some component of the materials which are transported
down the axon in the rapid phase.
Several investigations dealing with the mechanism of axoplasmic transport
support the general conclusion that it is dependent upon oxidative metabolism 5a,

A U T O R A D I O G R A P H I C TRACING OF CNS PATHWAYS 45
and may involve the axonal microtubulesl, 35. However, apart from these features the
actual mechanism of transport is, as yet, unknown.
The question of transport in a retrograde or somatopetal direction along the
axon is of paramount importance if axoplasmic transport is to be used as a neuro-
anatomical tool. That some retrograde transport may occur is suggested by direct
observations on the movements of particulate material along axons51, 5v, and from
experiments in which peripheral nerves were either ligated or sectioned and materials
were found to accumulate in small quantities distal to the cut or ligation41, 44. More
recently it has also been reported that horseradish peroxidase may be transported
from an injection site in the gastrocnemius muscle into motoneuron cell bodies 40.
On the other hand, there is also a good deal of evidence to the contrary. For example,
while norepinephrine has been thought to accumulate in sympathetic fibers distal to
nerve ligation because of retrograde flow, Geffen et al. ~6 could find no radioactivity
in the cells of origin of the sympathetic fibers to the eye following intraocular injec-
tion of labeled norepinephrine. Our own evidence from at least 3 different neural
systems similarly indicates that uptake and retrograde transport of labeled leucine,
if it occurs at all in the central nervous system, does not significantly complicate the
use of axoplasmic transport as a neuroanatomical method (Figs. 14 and 15).
From this brief review of some of the functional parameters of axoplasmic
transport and from the evidence we have presented in the Results section, it is clear
that as a neuronatomical method the labeling of axoplasmically transported material
has a number of theoretical and practical advantages. Foremost among these is that,
in contrast to the degeneration techniques, the autoradiographic method is based
upon a physiological function of normal neurons. Only living neurons are capable of
incorporating precursor substances into proteins, and other macromolecules, and of
transporting them down their axons. This means that the cytoarchitectonic features
of the projection source are preserved, and at the electron microscopic level important
details of neuronal morphology, such as the form and distribution of synaptic vesicles,
are not lost. Since the neuronal soma appears to be the major site of incorporation
of labeled amino acids into transportable protein, and as there is no morphologically
significant retrograde transport of labeled material, the polarity of the neuronal
system under study is clear. Taken together with the observation that axons do not
manufacture significant amounts of transportable protein, this means that two of the
major difficulties inherent in the degeneration techniques - - the interruption of
fibers of passage and the interpretation of retrograde effects - - are obviated by the
autoradiographic method. Finally, by utilizing the two phases of axoplasmic transport
(by carefully selecting survival times) terminal fields can be distinguished from fiber
pathways.
A word of caution is necessary in view of the findings of Grafstein 24 and Hen-
drickson 31 that small but detectable amounts of label reach postsynaptic cells. If this
does not involve more than about 5 ~ of the transported label, transneuronal labeling
should not complicate the interpretation of light microscopic autoradiographs. How-
ever, as we have pointed out, if transneuronal movement of label occurs, this cannot
be so easily dismissed at the electron microscopic level since each grain can be

identified in relation to a specific structure. Of course, the origin of this label (from
transneuronal movement or due to systemic uptake) always presents an interpretative
problem.
(2) Practical considerations
Pragmatic advantages and disadvantages of an experimental method often

dictate its use. Autoradiography is a straightforward and reliable histological proce-
dure which lacks many of the vagaries inherent in the reduced silver methods. With
exposure times of 1-3 weeks, the total preparation time is not significantly different
from that of the Nauta method*. Some minimal precautions must be taken to secure
consistent results, however. Both the emulsion and the isotope are relatively unstable,
and care must be taken in preserving these materials and in determining their quality
before use. Much of the procedure must be carried out under a dim safelight but this
inconvenience becomes minimal with experience. For electron microscopic autoradio-
graphy there is less technical leeway and the added inconvenience of relatively long
exposure times.
The autoradiographic method has a number of distinct experimental advan-
tages. As we have shown, the high concentration of isotope at the injection site allows
rapid identification of the injection site prior to processing the entire brain (Fig. 3).
Because the two rates of axoplasmic transport differ by one to two orders of magni-
tude, it is possible, particularly in longer fiber systems, to differentiate terminals using
the rapid component without determining precise survival times. The speed of the rapid
phase of transport (generally in excess of 100 ram/day) makes it possible to carry
out acute experiments, and to combine the radioactive labeling of a system with
physiological studies involving stimulation or recording without elaborate aseptic
precautions. Injection may be done extracellularly, as we have done, or intracellu-
larly, as elegantly demonstrated by Globus and his colleagues 18. (The tracing of long
processes of a single neuron with autoradiography is, of course, difficult and except
in special cases this approach is likely to be less useful than the extracellular labeling of
small populations of neurons.)
A wide variety of stains can be used with autoradiography so that the localiza-
tion of the labeled pathway and its termination can be extremely precise in terms of
nuclear masses or major fiber tracts. One disadvantage of the autoradiographic
method as compared with the degeneration techniques is that the course, and to
some extent the characteristics, of individual fibers cannot be easily determined. In
addition, the experimental evidence overlies the actual tissue, and where high energy
isotopes are used problems of resolution can be serious.
With all methods utilizing radioactive tracers, serious consideration must be
given to background activity. The physical environment contributes this from a
variety of sources such as cosmic and ? irradiation, and thermal and light energy.
A more important consideration is the 'physiological' background which results from
* It appears that these exposure times can be markedly shortened by soaking the coated slides in
scintillation fluid7o.

A U T O R A D I O G R A P H I C TRACING OF CNS PATHWAYS 47
the entry of the isotope into the general circulation. Most of this is due to vascular
and/or CSF uptake of the radioactive isotopes in the vicinity of the injection, but
labeled materials taken up by neurons and other tissues of the body are also metabo-
lized and may be reutilized. In practice, the amount of label available to the neurons by
either systemic uptake or reutilization is very low and there is evidence that the 'physio-
logical' background can be reduced further by using proline or other amino acids in-
stead of leucine 13. The possibility of the accumulation of isotope in local pools, such as
in the immediate vicinity of the synapses, however, must be kept in mind, particularly
for electron microscopy 24,al.
A final word of caution concerns systems in which the projections are sparse.
Since the bulk of the transported material which is labeled with [3H]leucine moves
in the slow phase of axoplasmic transport, longer survival times, together with long
exposure times, may be necessary to demonstrate these projections.
(3) Anticipated developments
Throughout this paper several references have been made to potential exten-
sions of the autoradiographic technique or tracing fiber connections. Foremost
among these is that autoradiography lends itself particularly well to quantification.
We have presented several examples to show how grain counts can be used to demon-
strate the discreteness of a projection, the precision of the laminar termination of a
pathway, or the relation of autoradiographic grains to specific structures. The dense
and sharply particulate quality of silver grains should make automated image pro-
cessing and counting methods possible, thus eliminating much of the tedium of data
collection. At present it is only possible to make quantitative comparisons of different
projections from the same source, since the exact amount of label which is incorporated
is not standardized from one experiment to the next. Should a practical method for the
stoichiometric labeling of neurons be developed - - and the technique used by Globus
and his co-workers is is a promising step in this direction - - it should be possible to
use the autoradiographic method to quantitate precisely the relative numbers of fibers
or synapses originating from different sources. The combination of autoradiographic
and degeneration methods, by injecting one site after producing a lesion at another,
and observing the pattern of silver grains in relation to degenerating structures, may
be particularly useful in systems in which afferents converge upon a center from
different sources. Similarly, where some fraction of the total neuron population in a
given area can be destroyed by the lesion or made to undergo retrograde cell degener-
ation, the efferent projection of the remaining ceils could be determined autoradio-
graphically.
It is now known that a wide variety of substances in addition to amino acids
are taken up by neurons, and many of these are incorporated into material which is
transported somatofugally along dendrites and axons 13-15. Although up to the present
the method has been used in the central nervous system only with a limited number
of protein precursors, it should be possible to selectively label other transported ma-
terials and in this way to trace specific neural pathways, or to label differentially the

48 w.M. COWAN e t al.
rapid and slow components. For example, adrenergic pathways can be selectively
labeled with appropriately labeled precursors ~ and, as Forman e t al. 15 have shown,
some substances transported in the rapid phase can be preferentially labeled with
glucosamine. It is reasonable to suppose that specific precursors may be found that
are associated with discrete subcellular sl ructures and may in the future offer a method
of studying the behavior of such organelles as microtubules, neurofilaments or syn-
aptic vesicles. The use of these variations singly and in combination should permit
the morphological analysis of the central nervous system along truly functional lines.
SUMMARY
(1) The usefulness of autoradiography for tracing pathways in the central ner-
vous system has been examined critically in a number of neuronal systems, including
the central connections of the rat olfactory bulb, the cortico-thalamic projections in
the mouse, the commissural connections of the rat hippocampus and the retinal pro-
jections in the monkey and chick.
(2) The technique is based upon the autoradiographic demonstration of axo-
plasmically transported materials synthesized from a radioactively-labeled precursor
in the neuronal somata. The methods we have used are described in detail and their
advantages and limitations discussed.
(3) Neurons at the site of injection promptly take up and incorporate the labeled
precursor; thus, the precise origin of a pathway can be determined by identifying
the number and distribution of the labeled cells. The normal cytoarchitecture at the
site of injection is preserved.
(4) Evidence is presented from light and electron microscopic autoradiography
that the transported materials travel down the axons at, at least, two well-defined rates.
Much of the rapidly transported material (which moves at a rate generally in excess
of 100 mm/day) reaches and accumulates within axon terminals; the slower compo-
nent (with rates between 1 and 5 mm/day) constitutes the bulk of the transported
material and is distributed throughout the length of the axon. By the selection of
appropriate postinjection survival times it is therefore often possible to selectively
demonstrate the terminal projection field(s) or to trace the pathway from its origin
to its termination.
(5) Where the autoradiographic method has been applied to neuronal pathways
which have been intensively studied by other experimental neuroanatomical tech-
niques (such as the Nauta-Gygax and the Fink-Heimer methods, and the electron
microscopic study of terminal degeneration) it has usually demonstrated the pathways
and their termination with equal clarity and in some cases has revealed additional
details of the projection.
(6) Theoretically, one of the major advantages of the method is that it should
distinguish between fibers arising from a region and axons passing through it. Mor-
phological evidence is presented in support of the view that fibers of passage do not
synthesize significant amounts of transported material.
(7) Similarly, there is no morphological evidence for the retrograde transport

of labeled material from the axon terminals towards the neuronal soma, so that the
functional polarity of a fiber pathway can be readily established.
(8) Certain theoretical and practical considerations in the use of the method
are discussed and the advantages of the technique (which is based upon an established
physiological property of neurons) over other currently used techniques (which are
dependent upon pathological changes in the neuronal somata, axons, or their ter-
minals) are reviewed.
ACKNOWLEDGEMENTS
The work reported in this study was supported in part by Grants EY 00599,
EY 00491 and NS 09518 of the United States Public Health Service. Dr. Woolsey
was in receipt of an N.I.H. postdoctoral fellowship (1 F10 NS 2394).
REFERENCES
1 BARONDES, S. M., Axoplasmic transport. A report of an N.R.P. work session, Neurosci. Res.
Progr. Bull., 5 (1967) 307-419.
2 BIRKS, R., AND MACINTOSh, F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad.
J. Biochem., 39 (1961) 787-827.
3 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special
reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-538.
4 BLOOM, F. E., HOFFER, B. J., AND SIGGINS, G. R., Studies on norepinephrine-containing afferents
to Purkinje cells of rat cerebellum. I. Localization of the fibers and their synapses, Brain Research,
25 (1971) 501-521.
5 COTMAN,C. W., AND TAYLOR,O. A., Autoradiographic analysis of protein synthesis in synapto-
somal fractions, Brain Research, 29 (1971) 366~372.
6 COWAN,W. M., ADAMSON,L., AND POWELL, T. P. S., An experimental study of the avian visual
system, J. Anat. (Lond.), 95 (1961) 545-563.
7 COWAN, W. M., AND POWELL, T. P. S., Centrifugal fibers in the avian visual system, Proc. roy.
Soc. B, 158 (1963) 232-252.
8 DROZ, B., Synth6se et transfert des prot6ines cellulaires dans les neurones ganglionnaires: t~tude
radioautographique quantitative en microscopie 61ectronique, J. Microscopie, 6 (1967) 201-228.
9 DROZ, B., AND BARONDES, S. M., Nerve endings; rapid appearance of labeled protein shown by
electron microscope radioautography, Science, 165 (1969) 1131-1133.
10 DROZ, B., AND KOENIG, H. L., Localization of protein metabolism in neurons. In A. LAJTHA
(Ed.), Protein Metabolism of the Nervous System, Plenum, New York, 1970, pp. 93-108.
11 DROZ, B., AND LEBLOND, C. P., Axonal migration of proteins in the central nervous system and
peripheral nerves as shown by radioautography, J. comp. Neurol., 121 (1963) 325-346.
12 EDSTROM, A., The ribonucleic acid in the Mauthner neuron of the goldfish, J. Neurochem., 11
(1964) 30%314.
13 ELAM, J. S., AND AGRANOFF, B. W., Rapid transport of protein in the optic system of the goldfish,
J. Neurochem., 18 (1971) 375-387.
14 ELAM, J. S., GOLDBERG,J. M., RADIN, N. S., AND AGRANOFF,B. W., Rapid axonal transport of
sulfated mucopolysaccharide protein, Science, 170 (1970) 458-459.
15 FORMAN, I). S., McEWEN, B. S., AND GRAFSTEIN, B., Rapid transport of radioactivity in goldfish
optic nerve following injections of labeled glucosamine, Brain Research, 28 (1971) 119 130.
16 GEFFEN, L. B., HUNTER, C., AND RUSH, R. A., Is there bidirectional transport of noradrenaline
in the sympathetic nerves? J. Neurochem., 16 (1969) 469-474.
17 GEEFEN, L. B., AND LIVETT, B. G., Synaptic vesicles in sympathetic neurons, Physiol. Rev., 51
(1971) 98-157.
18 GLOBUS, A., Lux, H. D., AND SCHUBERT,P., Somadendritic spread of intracellularly injected
tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1968) 440-445.

50 W . M . COWAN et al.
19 GOLDBERG,S., AND KOTANI, M., The projection of optic nerve fibers in the frog, Rana catesbeiana,
as studied by radioautography, Anat. Rec., 158 (1967) 325-332.
20 GOITLIEU, D. I., EM and autoradiographic studies of the commissural connections of the hippo-
campus and dentate gyrus, Anat. Rec., 169 (1971) 327-328.
21 GOTTLIE~, D. I., AND COWAN, W. M., Studies on the commissural connections of the hippocampal
formation, in preparation.
22 GRAESTEIN, B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198.
23 GRAFSlEXZ~,B., Axonal transport; communication between soma and synapse, Advanc. Biochem.
Psychopharmacol., 1 (1969) 11-25.
24 GRAFSTEIY, B., Transneuronal transfer of radioactivity in the central nervous system. Science,
172 (1971) 177-179.
25 GRAFSTEIN,B. AND MURRAY, M., Transport of protein in goldfish optic nerve during regeneration,
Exp. Neurol., 25 (1969) 494-508.
26 GRANBOULAN, P., Comparison of emulsions and techniques in electron microscope radio-
autography. In C. P. LEBLOND AND K. B. WARREN (Eds.), The Use of Radioautograp,~y in In-
vestigating Protebt Synthesis, Academic Press, New York, 1965, pp. 43 63.
27 GUILLERY, R. W., Discussion of paper. In W. J. H, NAUTA AND S. O. E. EBBESSON (Eds.), Con-
temporary Research Methods in Neuroanatomy, Springer, New York, 1970, p. 250.
28 HADDAD, A., SMtTH, M. D., HERSCOWCS, A., NADLER, N. J., AND LEBLOND, C. P., Radioauto-
graphic study of in vivo and in vitro incorporation of fucose-3H into thyroglobulin by rat thyroid
follicular ceils, J. Cell Biol., 49 (1971) 856 882.
29 HEtMER, L., Synaptic distribution of centripetal and centrifugal fibres in the olfactory system of
the rat, J. Anat. (Lond.), 103 (1968) 413-432.
30 HENDRICKSON, A., Electron microscopic radioautography: Identification of origin of synaptic
terminals in normal nervous tissue, Science, 165 (1969) 194 196.
3l HENDRICKSON, A. E., Electron microscopic distribution of axoplasmic transport, J. comp.
Neurol., in press.
32 HENDRICKSON, A. E., AND COWAN, W. M., Changes in the rate of axoplasmic transport during
postnatal development of the rabbit's optic nerve and tract, Exp. Nenrol., 30 (1971) 403 422.
33 HENDRICKSON, A., W~LSON, M. E., AND TOYNE, M. J., The distribution of optic nerve fibers in
Macaca mulatta, Brain Research, 23 (1970) 425~427.
34 HEUSER, J., AND MTLEDI, R., Autoradiography of labeled amino acids injected iontophoretically
into the giant squid synapse, J. Physiol. (Lond.), 208 (1970) 55-57P.
35 KARLSSON, J. O., AND SJOSTRA~D, J., The effect of colchicine on the axonal transport of protein
in the optic nerve and tract of the rabbit, Brain Research, 13 (1969) 617-619.
36 KARLSSON, J. O., AND SJOSrRAND, J., Synthesis, migration and turnover of protein in retinal
ganglion cells, or. Neurochem., 18 (1971) 749-767.
37 KELLY, J. P., AND COWAN, W. M., The central connections of the retina in the chick, in prep-
aration.
38 KOENIG, E., Synthetic mechanisms in the axon. II. R N A in myelin-free axons of the cat, J.
Neurochem., 12 (1965) 357-361.
39 KOPRLWA,B. M., AND LEBLOND, C. P., Improvements in the coating technique of radioautography,
J. Histoehem. Cytochem., 10 (1962) 269-284.
40 I'(RISTENSSON, K., AND OLSSON, Y., Retrograde axonal transport of protein, Brain Research, 29
(1971) 363-365.
41 LASEK, R. J., Bidirectional transport of radioactively labeled axoplasmic components, Nature
(Lond.), 216 (1967) 1212-1214.
42 LASErC, R., Axoplasmic transport of labeled proteins in rat ventral motoneurons, Exp. Neurol.,
21 (1968) 41-51.
43 LASEK, R., Axoplasmic transport in cat dorsal root ganglion cells; as studied with [3H]-L-leucine,
Brain Research, 7 (1968) 360-377.
44 LASErC, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324.
45 LASEK, R., JOSEPH, B. S., AND WHITLOCK, O. G., Evaluation of a radioautographic neuro-
anatomical tracing method, Brain Research, 8 (1968) 319-336.
46 LIVETT, B. G., GErFEN, L. B., AND AUSTIN, L., Proximo-distal transport of [~aC]noradrenaline
and protein in sympathetic nerves, J. Neurochem., 15 (1968) 931-939.
47 LIVETT, B. G., GEFFEN, L. B., AND RUSH, R. A., Immunohistochemical evidence for the transport
of dopamine-fl-hydroxylase and a catecholamine binding protein in sympathetic nerves, Biochem.
Pharmaeol., 18 (1969) 923-924.
48 LOHMAN, A., The anterior olfactory lobe of the guinea pig. A descriptive and experimental ana-
tomical study, Acta anat. (Basel), 53, Suppl. 49 (1963) 1-109.
49 LUBI~SKA, L., Axoplasmic streaming in regenerating and in normal nerve fibers. In M. SINGER
AND J. P. SCHADI~(Eds.), Meehan&ms of Neural Regeneration, Progress in Brain Research, Vol. 13,
Elsevier, Amsterdam, 1964, pp. 1-66.
50 McEWEN, B. S., AND GRAFSTEIN, B,, Fast and slow components in axonal transport of protein,
J. Cell Biol., 38 (1968) 494-508.
51 McMAHAN, U.J.,ANDKuFrLER, S.W.,Visualidentificationofsynaptic boutons on living ganglion
cells and of varicosities in postganglionic axons in the heart of the frog, Proc. roy. Soc. B, 177
(1971) 485 508.
52 MILEDI, R., AND SLATER, C. R., On the degeneration of rat neuromuscular junctions after nerve
section, J. Physiol. (Lond.), 207 (1970) 507-528.
53 OCHS, S., Axoplasmic flow in neurons. In J. GMTO (Ed.), Macromolecules and Behavior, Appleton-
Centur~Crofts, New York, 1966, pp. 20-39.
54 Ocns, S., Fast axoplasmic transport of proteins and polypeptides in mammalian nerve fibers.
In A. LAJTHA (Ed.), Protein Metabol&m in the Nervous System, Plenum, New York, 1970, pp.
291-304.
55 OCHS, S., AND HOLLINGSWORTH, D., Dependence of fast axoplasmic transport in nerve on
oxidative metabolism, J. Neurochem., 18 (1971) 107-114.
56 PETERS, T., AND ASHLEY, C. A., An artefact in radioautography due to binding of free amino
acids to tissues by fixatives, J. Cell Biol., 33 (1967) 53-60.
57 POMERAT, C. M., HENDEL~AN, W. J., RAIBORN, C. W., AND MASSEY, J. F., Dynamic aspects of
nervous tissue in vitro. In H. HYD~N (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 119-178.
58 PRICE, J. L., AND POWELL, T. P. S., An experimental study of the origin and course of the centri-
fugal fibres to the olfactory bulb in the rat, J. Anat. (Lond.), 107 (1970) 215-237.
59 PRICE, J. L., AND POWELL, T. P. S., Certain observations on the olfactory pathway, J. Anat.
(Lond.), 110 (1971) 105-126.
60 PRICE, J. L., AND WOOLSEY, T. A., Autoradiographic demonstration of pathways in the mammalian
CNS, Anat. Ree., 169 (1971) 406.
61 RAISMAN, G., COWAN, W. M., AND POWELL, T. P. S., The extrinsic afferent, commissural and
association fibres of the hippocampus, Brain, 88 (1965) 963-996.
62 ROGERS, A. W., Techniques in Autoradiography, Elsevier, New York, 1967.
63 SALPETER, M. M., AND BACI-IMANN,L., Autoradiography with the electron microscope. A proce-
dure for improving resolution, sensitivity and control, J. Cell Biol., 22 (1964) 469-477.
64 SALPE'rER, M. M., BACHMANN, L., AND SALPETER, E. E., Resolution in electron microscope auto-
radiography, J. Cell Biol., 41 (1969) 1-20.
65 SCHONBACH, J., AND CU~NOD, M,, Axoplasmic migration of protein. A light microscopic auto-
radiographic study in the avian retino-tectal pathway, Exp. Brain Res., 12 (1971) 275-282.
66 SCHONBACH, J., SCIJONBACH, C., AND CU~NOD, M., Rapid phase of axoplasmic flow and synaptic
proteins: An electron microscopical autoradiographic study, J. comp. Neurol., 14l (1971) 485-498.
67 SIDMAN, R. L., Autoradiographic methods and principles for study of the nervous system with
thymidine-3H. In W. J. H. NAUTA AND S. O. E. EBBESSON (Eds.), Contemporary Research
Methods' in Neuroanatomy, Springer, New York, 1970, pp. 252-274.
68 TAYLOR, A. C., AND WEISS, P., Demonstration of axonal flow by the movement of tritium-
labeled protein in mature optic nerve fibers, Proc. nat. Acad. Sci. (Wash.), 54 (1965) 1521-1527.
69 THURSTON, J. M., AND JOFTES, D. L., Stains compatible with dipping radioautography, Stain
Technol., 38 (1963) 231-235.
70 WEtNGROD, D., FOLKMAN, J., AND SADE, R. M., A method for rapid autoradiography, Exp. Cell
Res., in press.
71 WEISS, P., A~D HOLLAND, Y., Neuronal dynamics and axonal flow. II. The olfactory nerve as
model test object, Proc. nat. Acad. Sci. (Wash.), 57 (1967) 258-264.
72 WOOLSEY, T. A., Somatosensory, auditory and visual cortical areas of the mouse, Johns Hopk.
Med. J., 121 (1967) 91-112.
73 WOOLSEY,T. A., AND PRIC~, J. L.: A n experimental autoradiographic study of the cortico-thalamic
connections in the mouse, in preparation.
74 WOOLSEY, T. A., AND VAN DER LOOS, H., The structural organization of layer IV in the somato-
sensory region (SI) of mouse cerebral cortex: The description of a cortical field composed of
discrete cytoarchitectonic units, Brain Research, 17 (1970) 205-242.

THE AUTORADIOGRAPHIC DEMONSTRATION OF AXONAL CONNECTIONS, Cowan, 1972

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THE AUTORADIOGRAPHIC DEMONSTRATION OF AXONAL CONNECTIONS, Cowan, 1972

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BRAIN RESEARCH 21

W. M. COWAN, D. I. GOTTLIEB, ANITA E. HENDRICKSON, J. L. PRICE AND

Most of the published work on the phenomenon of axoplasmic transport has

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

(1) Preparation and delivery of the radioactively labeled precursor

We have used tritiated leucine (L-[4,5-aH]leucine) in all of our experiments;

(2) Postinjection survival times

As with degeneration methods, it is important to determine the optimum sur-

Brain Research, 37 (1972) 21-51

(3) Fixation and histological processing

We have used a variety of routine histological fixatives for light microscopic

Brain Research, 37 (1972) 21-51

A wide variety of standard histological stains are applicable to autoradio-

(6) Electron microscopic applications

One of the principal advantages of the autoradiographic method for tracing

Brain Research, 37 (1972) 21-51

We have used the autoradiographic method for tracing neuronal connections

(1) The injection site

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

(2) A utoradiographic study of known projection systems

Brain Research, 37 (1972) 21-51

.f.'.., ~ .:,~..;,,,:., ,~:, .: :

q'L -I ., "~L" ¢" "~, "r~ ~.

Brain Research, 37 (1972) 21-51

'- . "-7 " " "Z, ~% "" ":''

lateral p a r t o f the olfactory tubercle, b u t if mitral cells are labeled t h r o u g h o u t the

Brain Research, 37 (1972) 21-51

Gralns / 3600p2 ~ _ _ _ _ ~ ~ ~ Grains / 3600p2

o ~ / ; o 7 . ~.;<U . . . . . •, ,..~ ....... I

. . . . . . '. •. . . . . . o ~.a~-o~o '

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

(3) The localization of the rapidly and slowly transported materials

Several electron microscopic autoradiographic studies have served to establish

Brain Research, 37 (1972) 21-51

phase has been studied with the light m i c r o s c o p e is the c o m m i s s u r a l p r o j e c t i o n o f the

Brain Research, 37 (1972) 21-51

-atD molecu la re &

l o 0 j~ I~ • al veus ,~_ q ~ ; z . ' r ~ L ~ ,j

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

(4) Evidence for the absence of transport by fibers of passage

A n i m p o r t a n t question which must be considered is whether or not fibers passing

~dh,.,~"~'z" O ..~ .2 ~ "wwret

Brain Research, 37 (1972) 21-5l

t e r m i n a l p r o j e c t i o n sites o f the bulb. The absence o f labeling over the p i r i f o r m cortex

(5) The absence of retrograde transport of labeled materials

It r e m a i n s to be shown t h a t w h a t e v e r i n c o r p o r a t i o n o f a m i n o acids m a y occur

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

Our observations, together with those of previous workers, demonstrate that

(1) The physiological basis of the method

Brain Research, 37 (1972) 21-51

Although dendrites contain free ribosomes and rough endoplasmic reticulum,

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

Brain Research, 37 (1972) 21-51

(2) Practical considerations

Pragmatic advantages and disadvantages of an experimental method often

Brain Research, 37 (1972) 21-51