Reprinted from

Proc. Natl. Acad. Sci. USA

Vol. 73, No. 11, pp. 4230-4234, November 1976
Neurobiology

Metabolic mapping of the primary visual system of the monkey by
means of the autoradiographic [!4C ]deoxyglucose technique _

(ocular dominance columns/blind spot/cerebral glucose utilization/cerebral energy metabolism/brain)

C. KENNEDY*, M. H. DEs RosiErst, O. SAKURADAT, M. SHINOHARAT, M. REIVICH?, J. W. jenn’ AND

L. SOKOLOFF*S

* Department of Pediatrics, Georgetown University School of Medicine, Washington, D.C. 20007; * Laboratory of Cerebral Metabolism, National Institute of
Mental Health, National Institutes of Health, Bethesda, Maryland 20014; and + Department of Neurology, University of Pennsylvania School of Medicine,

Philadelphia, Pa. 19174

Cc icated by Sey S. Kety, September 9, 1976

ABSTRACT An autoradiographic technique that employs
2-[!4C]deoxyglucose to measure the local rates of glucose utili-
zation within the brain has been a plied to the binocular visual

stem of the Macaque monkey. This method, which pictorially
di isplays the relative rates of glucose consumption in the com-

nent structures of the brain, delineates the regions of altered
functional activity because of the close relationship between
functional activity and energy metabolism. Bilateral retinal
stimulation results in the delineation of different rates of glucose
consumption in at least four cytoarchitectural layers of the
striate cortex. The most intense metabolic activity appears to
be in Layer IV, the locus of the termination of the geniculo-
cortical pathway. Bilateral visual occlusion lowers the rates of
glucose consumption in the striate cortex and markedly reduces
the metabolic di ifferentiation of the various layers. Unilateral
visual deprivation delineates the laminae of the lateral genic-
ulate body and the ocular dominance columns of the striate
cortex, It also results in the autoradiographic visualization of
regions with normally monocular input in the striate cortex, such
as the rostral portions of the mushroom-like configurations in
the calcarine cortex, which represent the extreme temporal
crescents of the visual fields, and small regions in the most
caudal part of the mushroom configurations, which are believed
: represent the cortical loci of the blind spots of the visual

ields,

 

Previous studies of the binocular visual system in the Macaque
monkey have demonstrated the persistence of monocular
representation of retinal units of the two eyes throughout the
visual pathways from the lateral geniculate ganglia to the striate
cortex (1-4). Although each retina projects more or less equally
to both hemispheres, their projections remain segregated and
terminate in six well-defined laminae in the lateral geniculates,
three each for the ipsilateral and contralateral eyes (2-4). This
segregation is preserved in the optic radiations which project
the monocular representations of the two.eyes for any segment
of the visual field to adjacent regions of Layer IV of the striate
cortex (1, 2). The cells responding to the input of each monoc-
ular terminal zone are distributed transversely through the
thickness of the striate cortex resulting in a mosaic of columns,
0.3-0.5 mm in width, alternately representing the monocular
inputs of the two eyes. The nature and distribution of these
ocular dominance columns have previously been characterized
by electrophysiological techniques (1), Nauta degeneration
methods (2), and by autoradiographic visualization of axonal
and transneuronal transport of [*H]proline- and [®H]fucose-
labeled protein and/or glycoprotein (3, 4).

We recently described a method for the simultaneous mea-
surement of the rates of glucose consumption in most of the
macroscopic structural components of the brain (5-7). The
method is based on the accumulation of 2-deoxy-D-[!4C]glu-

 

* To whom correspondence should be addressed.

4230

cose-6-phosphate in the tissues after an intravenous pulse of
2-deoxy-D-[!4C] glucose. Deoxyglucose is transported across the
blood-brain barrier by the same carrier that transports glucose,
and it competes with glucose for hexokinase, the‘enzyme that
phosphorylates both. The rate of [!4C]deoxyglucose phospho-
rylation is, therefore, quantitatively related to the rate of glucose
phosphorylation and depends on the relative concentrations of
both in the precursor pools and the kinetic constants of hexo-
kinase for the two substrates. In a steady state of glucose me-
tabolism, the net rate of glucose phosphorylation equals the rate
of glucose utilization. Glucose consumption can then be cal-
culated by means of an equation that defines it in terms of the
time course of the relative concentrations of [!4C]deoxyglucose
and glucose in the plasma, the final tissue concentration of
[!4C]deoxyglucose-6-phosphate, the fractional turnover rate
of the free [}4C]deoxyglucose pool in the tissue, and the ratios
of the Michaelis-Menten kinetic constants of hexokinase for
deoxyglucose and glucose (6). The key to the localization pos-
sible with the method is a quantitative autoradiographic tech-
nique for the measurement of local tissue concentrations of
[!4C]deoxyglucose-6-phosphate. Even without the quantifi-
cation, however, the autoradiographs are themselves pictorial
representations of the local rates of glucose consumption
throughout the brain. Inasmuch as local energy metabolism in
the brain, as in other tissues, is closely coupled to local functional
activity, the method has proved effective for the delineation
of local cerebral regions with altered functional activity in re-
sponse to experimentally modified physiological states (7).

The binocular visual system of the monkey appeared to offer -
an appropriately intricate and complex pattern of functional
organization to test the capacity of the [!4C]deoxyglucose
method to define cerebral regions of altered functional activity.
We have, therefore, applied the [!4C]deoxyglucose technique
to map and demonstrate pictorially on the basis of local meta-
bolic rate the distribution of monocular representation in the
monkey brain.

METHODS

Rhesus monkeys of either sex weighing 2.5-3.5 kg were used
in these studies. Polyethylene catheters were inserted in a
femoral artery and vein after light halothane and nitrous oxide
anesthesia, and the animals were then placed in a restraining
chair and allowed to recover from the anesthesia for at least 4
hr. The experimental period was initiated by the intravenous
injection of a pulse of 100 wCi/kg of [}4C]deoxyglucose (specific
activity, 50-55 mCi/mmol; New England Nuclear Corp.,
Boston, Mass.) contained in 3 ml of normal saline. In most of the
animals, timed arterial blood samples were drawn during the
ensuing 45 min period for the measurement of the arterial
Neurobiology: Kennedy et al.

plasma [!4C]deoxyglucose and glucose concentrations required
for eventual quantitation of local cerebral glucose utilization.
Forty-five minutes after the pulse, the animal was killed by the
intravenous injection of a large dose of sodium thiopental fol-
lowed by a saturated solution of KCl sufficient to stop the heart.
The animal was then decapitated, the extracranial soft tissues
and lower mandible were removed as rapidly as possible, and
the head was frozen in Freon XII chilled to —60° with liquid
nitrogen. With careful attention to keeping the tissues as cold
as possible by repeated chilling in dry ice, the frozen head was
cut into several pieces with a band saw, the brain portions were
dissected free of bone, and the brain pieces were coated with
embedding medium (Lipshaw Manufacturing Co., Detroit,
Mich.), and fixed to object-holders appropriate to the micro-
tome to be used. Brain pieces so prepared were stored in a
freezer at ~70° until sectioned.

Brain sections, 20 wm in thickness, were prepared in an
American Optical Co. (Buffalo, N.Y.) cryostat maintained at
—21° to —22°. The brain sections were picked up on glass cover
slips, dried on a hot plate at 60°-70° for at least 5 min, and
placed sequentially in an x-ray cassette. Autoradiographs were
prepared from these sections directly in the x-ray cassette with
Kodak single-coated blue-sensitive, Medical X-ray Film, Type
SB-54 (Eastman Kodak Co., Rochester, N.Y.). The exposure
time was generally about 5-6 days, and the exposed films were
developed according to the instructions supplied with the
film.

Three groups of animals with different conditions of visual
input were studied: (i) intact binocular vision; (#) bilateral visual
occlusion; (i#/) monocular visual occlusion. Visual occlusions
were achieved either by enucleation or by insertion of opaque
plastic discs in the appropriate eyes at the time of the surgical
procedure for the insertion of the catheters. Animals with intact
vision in one or both eyes were either exposed to the ambient
light of the laboratory or surrounded by a black-white complex
geometric pattern that was illuminated and rotated during the
experimental procedure. The results were qualitatively similar
with either method of visual occlusion and with or without the
rotating geometric pattern.

RESULTS

The autoradiographs obtained with the procedure used in these
studies represent the distribution of {!4C]deoxyglucose-6-
phosphate in the tissues, and the concentration of this com-
pound in a tissue reflects closely that tissue’s rate of glucose
utilization or energy metabolism (6, 7). The autoradiographs
are, therefore, essentially pictorial representations of the relative
rates of glucose consumption in the various anatomical com-
ponents of the brain; the darker the region, the greater the rate
of metabolism. Experimental modification of the visual input
produced consistent changes in the pattern of distribution of
the rates of energy metabolism which coincided closely with
the changes in functional activity expected from known
physiological and anatomical properties of the binocular visual
system.

Normal binocular vision

In animals with intact binocular vision no bilateral asymmetry
was seen in the autoradiographs of the structures of the visual
system (Figs. 1A and 2A). The lateral geniculate bodies and
oculomotor nt lei appeared to be of fairly uniform density and
essentially the same on both sides (Fig. 1A). Although not il-
lustrated in this report, the optic nerves and tracts were clearly
discernible in other autoradiographic sections; they exhibited
the low densities characteristic of white matter and appeared

Proc. Natl. Acad. Sci. USA 78 (1976) 4231

 

5.0mm

Fic. 1. Autoradiography of coronal brain sections at the level of
the lateral geniculate bodies. Large arrows point to the lateral ge-
niculate bodies; smal] arrows point to oculomotor nuclear complex.
(A) Animal with intact binocular vision. Note the bilateral symmetry
and relative homogeneity of the lateral geniculate bodies and oculo-
motor nuclei. (B) Animal with bilateral visual occlusion. Note the
reduced relative densities, the relative homogeneity, and the bilateral
symmetry of the lateral geniculate bodies and oculomotor nuclei. (C)
Animal with right eye occluded. The left side of the brain is on the left
side of the photograph. Note the laminae and the inverse order of the
dark and light bands in the two lateral geniculate bodies. Note also
the lesser density of the oculomotor nuclear complex on the side
contralateral to the occluded eye.

to be bilaterally symmetrical. The visual cortex was essentially
the same on both sides (Fig. 2A), but throughout all of Area 17
there was heterogeneous density distributed in a characteristic
laminar pattern that distinguished it from the adjacent Area
18 and other regions of the cerebral cortex (Figs. 2A and 3). At
least four different levels of density could be readily discerned
which could be correlated with the known cytoarchitectural
layers of the striate cortex apparent in myelin- and thionin-
stained sections (Fig. 4). A wide band of relatively low but
uniform density in the autoradiograph encompassed Layers I,
IL, and III which could not be distinguished further on the basis
of [!4C]deoxyglucose uptake (Fig. 4). Layers IVa, IVb, and IVc
were clearly associated with high rates of glucose utilization as
indicated by the density of the autoradiographic regions cor-
responding to them; Layers IVb and IVc were the most active,
and exhibited the densest patterns of all layers seen in the au-
toradiograph (Fig. 4). When the autoradiograph is compared
with the section stained for myelin, it appears that the line of
Gennari lies within the layer with greatest metabolic activity
(Fig. 4). In Layer V, there appears to be a fairly abrupt decline
in density, but the activity in Layer VI is intermediate between
those of Layers IV and V (Fig. 4). These observations indicate
4232 Neurobiology: Kennedy et al.

 

5.0mm

Fic. 2. Autoradiographs of coronal brain sections at the level of
the striate cortex. (A) Animal with normal binocular vision. Note the
laminar distribution of the density; the dark band corresponds to
Layer IV. (B) Animal with bilateral visual deprivation. Note the al-
most uniform and reduced relative density, especially the virtual
disappearance of the dark band corresponding to Layer IV. (C)Animal
with right eye occluded. The half-brain on the left side of the photo-
graph represents the left hemisphere contralateral to the occluded
eye. Note the alternate dark and light striations, each approximately
0.3-0.4 mm in width that represent the ocular dominance columns.
These columns are most apparent in the dark band corresponding to
Layer IV, but extend through the entire thickness of the cortex. The
arrows point to regions of bilateral asymmetry where the ocular
dominance columns are absent. These are presumably areas with
normally only monocular input. The one on the left, contralateral to
occluded eye, has a continuous dark lamina corresponding to Layer
IV which is completely absent on the side ipsilateral to the occluded
eye. These regions are believed to be the loci of the cortical repre-
sentations of the blind spots (see text).

that in animals with binocular visual input the rates of glucose
consumption in the visual pathways are essentially equal on
both sides of the brain and relatively uniform throughout the
optic nerves, offtic tracts, oculomotor nuclei, and lateral ge-
niculate bodies but markedly different in the various layers of

Proc. Natl. Acad. Sci. USA 73 (1976)

 

AUTORADIOGRAPH
-——

1.0mm

THIONIN STAIN

Fic. 3. Comparison of autoradiograph and thionin stain of a
section of visual cortex at junction of Areas 17 and 18. The autoradi-
ograph was made first from the frozen but undried brain section; the
exposure of the film was carried out at —30°. The section was then
fixed and stained with thionin. The arrows point to the abrupt al-
teration of the cortical layers in the stained section and their auto-
radiographic representations in the autoradiographs at the junction
of Areas 17 and 18.

the striate cortex. The most intense metabolic activity appears
to be in sub-layers of Layer IV (Figs. 2A and 4) which coincide
.with the sites of termination of the geniculocortical projections
on spine-bearing stellate cells (2, 8). Within each layer or sub-
layer, however, the metabolic activity appears to be relatively
uniform throughout the extent of the cortex representing the
various portions of the visual fields.

Bilateral visual deprivation

Autoradiographs from the animals with both eyes occluded
exhibited generally decreased labeling of all components of the
visual system, but the bilateral symmetry was generally retained
(Figs. 1B and 2B), and the density within each lateral geniculate
body was for the most part fairly uniform (Fig. 1B). In the
striate cortex, however, the marked differences in the densities
of the various layers seen in the animals with intact bilateral
vision (Fig. 2A) were virtually absent so that, except for a faint
delineation of a band within Layer IV, the concentration of the
label was essentially homogeneous throughout the striate cortex
(Fig, 2B).

Unilateral visual deprivation

Autoradiographs from monkeys with only monocular input
exhibited markedly different patterns from those described
above. The optic nerve ipsilateral to the occluded eye showed
less labeling than the contralateral nerve; the optic tracts ex-
hibited bilateral symmetry, but both tracts were nonuniform
with regions of higher density in the dorsolateral portions
(autoradiographs not shown). Both lateral geniculate bodies
exhibited exactly inverse patterns of alternating dark and light
bands corresponding to the known laminae representing the
Neurobiology: Kennedy et al.

1]
I
pif
Wa
|
We}
y|
m1 |
THIONIN STAIN AUTO = . SPIELMEYER’S
RADIOGRAPH STAIN
ee“
1.0mm

Fic. 4. Comparison of autoradiograph, thionin stain, and myelin
stain of a section of striate cortex from a monkey with intact binocular
vision. The autoradiograph was made from the same section subse-
quently stained with thionin by the procedure described in the legend
to Fig. 3. An immediately adjacent section was stained for myelin with
Spielmeyer’s stain. The thionin stain distinguishes the cell-sparse
Layers I, IVb, and V and the three cell-dense regions, Layers II, II
and IVa (considered as one), Layer IVc, and Layer VI, according to
Brodman’s system. The dense zone in the autoradiograph traverses
all of Layer IV and encompasses the stria of Gennari which is de-
marcated by the myelin stain. In some animals a thin dark band
separate from Layer IV, possibly Layer IVa, could be discerned in the
autoradiograph.

regions receiving the different inputs from the retinae of the
intact and occluded eyes (Fig. 1C). Bilateral asymmetry was
also seen in the oculomotor nuclear complex; a lower density
was apparent in the nuclei contralateral to the occluded eye
(Fig. 1C). In the striate cortex the pattern of distribution of the
(?4C]deoxyglucose-6-phosphate appeared to be a composite of
the patterns seen in the animals with intact and bilaterally oc-
cluded visual input. The pattern found in the former regularly
alternates with that of the latter in columns oriented perpen-
dicularly to the cortical surface (Figs. 2C and 5). The dimen-
sions, arrangement, and distribution of these columns are
identical to those of the ocular dominance columns described
by Hubel and Wiesel (1-3). In a tangential section at the oc-
cipital pole, the columns appeared in cross section as dots ar-
ranged in parallel rows which seem to merge in Layer IV,
where the labeling is most dense, giving the picture of a long
palisade (Fig. 5B). The apparent increase in width of the col-
umns in a few selected regions may reflect this merging as the
plane of sectioning becomes parallel with the palisade (Fig. 5).
The autoradiographs also confirm the conclusion reached from
single cell recording techniques that the ocular dominance
columns, although most prominent in Layer IV, also involve
the entire cortical thickness (1); anatomic and [!4C]proline
and/or [!4C}fucose autoradiographic techniques have dem-
onstrated their distribution almost entirely in Layer IV
(2-4).

There are two regions of the striate cortex where the ocular
dominance columns are absent. These are regions known to
have only monocular input even in animals with normal bi-
lateral vision. One region is the rostral portion of the mush-
room-like area of the calcarine cortex (Fig. 5A). This region
receives input only from the most nasal crescent portion of the
contralateral retina. Monocular occlusion removes all input and
lowers [!4C]deoxyglucose uptake in this region only on the
contralateral side, but leaves the corresponding ipsilateral region

   

Proc. Natl. Acad. Sci. USA 73 (1976) 4238

 

+
5.0mm

Fic. 5. Autoradiographs of a parasagittal section (A) and two
coronal sections (B and C) of the striate cortex of an animal with the
left eye occluded. The parasagittal section was made in the left
hemisphere of the brain (ipsilateral to occluded eye) 10 mm from the
mid-line. The lines marked B and C in the left panel indicate the
planes of the coronal sections in panels (B) and (C) made through the
right hemisphere of the brain. The ocular dominance columns are
clearly visible in all the sections and are of generally the same di-
mensions and arrangement in the sagittal (A) and coronal (C) sections.
The section near the occipital tip (B) transects the long axes of the
columns, and they, therefore, appear as dots arranged in rows or
stripes; some appear to be connected by a line as though they were
slab-like in three dimensions which confirms previous observations
obtained with electrophysiological and anatomical techniques (1, 2,
12). The cortical loci of the blind-spots are indicated by the arrows,
low in density: ipsilateral to the occluded eye and dark on the con-
tralateral side, but devoid of columns in both. Note also the absence
of columns in the rostral portion of the mushroom-like configuration.
These regions without ocular dominance columns are areas with
normally only monocular input.

unaffected (7). The other normally monocular regions are the
loci of cortical representation of the blind spots of the visual
fields. The area of the optic disc in the nasal half of each retina
cannot transmit to this region of the contralateral striate cortex
which, therefore, receives its sole input from an area in the
temporal half of the ipsilateral retina. Occlusion of one eye
deprives this region of the ipsilateral striate cortex of all input
while the corresponding region of the contralateral striate cortex
retains uninterrupted input from the intact eye. The metabolic
reflection of this ipsilateral monocular input is seen in the
autoradiographs in Fig. 2C and Fig. 5. The regions that we
believe to be the loci of the representations of the blind-spots
are located in the tops of the mushroom-like configurations of
the folded cortex in the calcarine fissures. They are about 3 mm
deep to the surface and involve a strip of cortex about 4 mm in
length. They are devoid of ocular dominance columns. On the
side contralateral to the occluded eye, this region gives the
autoradiographic pattern of striate cortex with normal bilateral
visual input; on the ipsilateral side, this region looks like striate
cortex from an animal with totally deprived vision (Fig. 2C).
We presume that these areas, which are so prominent in the
autoradiographs, are the loci of the representations of the
blind-spots because of their appropriate responses to the ex-
perimental conditions and their locations consistent with present
knowledge of the cortical representation of the visual fields in
the monkey (9).

DISCUSSION

The results of these experiments not only confirm the structural
and functional organization of the binocular visual system of
the monkey established by painstaking electrophysiological and
anatomic studies (1-3) but also provide a direct pictorial visu-
alization of the distribution, nature, and extent of the regions
of monocular representation throughout the brain. They also
4234 Neurobiology: Kennedy et al.

demonstrate the effectiveness and potential usefulness of
metabolic mapping by means of the ['4C]deoxyglucose tech-
nique in the localization and delineation of brain loci with ex-
perimentally altered functional activity.

The binocular visual system has previously been visualized
autoradiographically (3, 4), but by a method based on an en-
tirely different principle from that underlying the ['*Cjde-
oxyglucose technique. The method used was the one based on
the axoplasmic and trans-synaptic transport of [[H]proline-
and/or [3H]fucose-labeled protein and glycoprotein (10, 11).
This method is dependent on anatomical continuity or conti-
guity and maps continuously only the anatomical pathway la-
beled from the site of injection of the tracer to its termination.
Although trans-synaptic transport does occur, there is marked
attenuation of the transfer of label at the synapse (10), and
terminal segments of multi-synaptic pathways may, therefore,
not be visualized. In contrast, the [!4C]deoxyglucose technique
maps all areas of the brain simultaneously on the basis of their
local rates of glucose consumption and levels of functional ac-
tivity. The method can detect regions of altered functional
activity but provides no information about the anatomical
pathways involved. Attenuation does not occur with this tech-
nique, and that is probably the reason why the [!4C]deoxyglu-
cose technique, in contrast to the axonal transport method, could
confirm the findings of electrophysiological studies (1) that the
ocular dominance columns in the striate cortex are not confined
only to Layer IV but extend throughout the thickness of the
cortex (Fig. 2C).

Like most autoradiographic methods both the axonal trans-
port and the (!4C|deoxyglucose techniques are subject to the
problem of diffusion or migration of the labeled tracer from
its original position in situ. Because its trapped labeled com-
pound, {!4C]deoxyglucose-6-phosphate, is water-soluble, the
[!4C]deoxyglucose method is particularly vulnerable to this
problem. We have recently noted that diffusion of the label can
occur in brain maintained at —15° to —18°. Ina previous report
(7) which contained an autoradiograph illustrating the cortical
locus of the blind spot, this locus appeared as an ovoid structure
extending through the entire thickness of the cortex, and the
ocular dominance columns were poorly defined. There was
clearly distortion due to diffusion. By meticulous attention to
the storage of the frozen brain at —70° and to its sectioning as
rapidly as possible at —21° to —22°, we have been able to re-

Proc. Natl. Acad. Sci. USA 78 (1976)

duce the diffusion artifact and obtain autoradiographs with the
resolution seen in this report. It is likely that even better reso-
lution can be achieved by further refinements of the autora-
diographic procedures.

Dr. M. H. Des Rosiers is a Fellow of Le Conseil des Recherches
Médicales du Canada.

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2. Hubel, D. H. & Wiesel, T. N. (1972) “Laminar and columnar
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3. Wiesel, T. N., Hubel, D. H. & Lam, D. M. K. (1974) “Auto-
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4. Rakic, P. (1976) “Prenatal genesis of connections subserving
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5. Sokoloff, L., Reivich, M., Patlak, C. S., Pettigrew, K. D., Des
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7. Kennedy, C., Des Rosiers, M., Jehle, J. W., Reivich, M., Sharp,
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8. Garey, L. J. & Powell, T. P. S. (1971) “An experimental study of
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Reprinted by the
U.S. DEPARTMENT OF HEALTH, EDUCATION, ANO WELFARE
National Institutes of Health