Proc. Natl. Acad. Sci.
USA
Vol. 91, pp. 8408-8412, August 1994
Neurobiology
Nerve growth factor-induced ocular dominance plasticity in adult
cat visual cortex
(neurotrophic factor/striate cortex/monocular deprivation)
QIANG Gu, YULIN Liu, AND MAX S. CYNADER
Department of Ophthalmology, University of British Columbia, 2550 Willow Street, Vancouver, BC Canada V5Z 3N9
Communicated by Richard Held, April 25, 1994
ABSTRACT Activity-dependent modifiability of cortical
ocular dominanc occurs only during early postnatal life,
within the so-called "critical period," but not thereafter in
adult visual cortex. To examine the role of neurotrophins in the
activity- and age-dependent imulation-induced moifib
of visual cortex, we tested whether intracortical inion of
nerve growth factor could induce ocular dominance plasticity
in adult visual cortex. Nerve growth factor was continuously
infused, by means of osmotic minipumps, into striate cortex of
adult cats for 2 weeks. At the time of minipump implantation,
one eyelid of the experimental animals was sutured closed.
After 3 weeks of monocular deprivation, the ocular dominance
distribution of neurons in the striate cortex was a sd uing
single unit recording. We found that monocular deprivation
imposed on adult animals in conjunction with nerve growth
factor infusion causes an ocular dominance shift toward the
deprived eye. Although the underlying mechanisms remain
uncertain, the results indicate that nerve growth factor can
enhance activity-dependent synaptic modification and remod-
eling in adult visual cortex.
Over 30 years ago Wiesel and Hubel (1) showed that depriving
one eye of visual input during early postnatal development
caused that eye to lose functional connections with the visual
cortex and to lose visual capabilities as well. Since then,
intense investigations of this ocular dominance (OD) plasticity
have identified neuronal activity (2), several neurotransmitters
(3-5) and receptors (6-10), as well as hormones (11) and
growth factors (12-14) as being involved in this process.
Normally, cortical OD plasticity occurs only within a transient
"critical period" during early postnatal development, but not
thereafter in adult animals (15-17). To examine the hypothesis
that intracortical infusion of nerve growth factor (NGF) may
enhance functional modifications in mature visual cortex, we
studied OD plasticity in adult cat visual cortex treated with
NGF. We report here that monocular deprivation imposed on
adult animals causes an OD shift toward the deprived eye. The
results show that substantial synaptic modification can be
reinstated even in the mature visual cortex.
MATERIALS AND METHODS
A total of 12 adult cats was used in this study. They were
between 2 and 3 years of age, long after the end of the critical
period (15-17). The monocular eyelid suture and osmotic
minipump implantation were performed in the same surgical
intervention under i.v. Brietal (methohexital sodium) anesthe-
sia supplemented with Halothane inhalation. The cats were
placed in a stereotaxic frame. Two osmotic minipumps (Alzet,
model 2002), connected with polyethylene tubing to stainless
steel cannulae, were implanted bilaterally beneath the skin of
the neck of each animal. One minipump contained the 2.5S
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.
8408
subunit of NGF (human recombinant; Genentech), while the
other minipump contained only artificial cerebrospinal fluid
and served as a control. Previous studies have shown that
NGF is stable at 370C for 2 weeks and is released and diffused
from the minipump for this 2-week period (18). The NGF was
continuously infused at the rate of 0.1 jug/hr through the
infusion cannulae, whose tips were inserted 2 mm below the
dura and secured to the skull with dental cement, at Horsley-
Clarke coordinates P-5, L+2. The OD distributions of neu-
rons in both hemispheres were assessed in 11 cats 3 weeks
after monocular deprivation. During the first 2 weeks of this
deprivation period, NGF was continuously infused into the
visual cortex. The dosage of NGF used in this study was
chosen on the basis of an earlier report (19), which showed that
application of the same rate and duration of NGF infusion
promoted transplant survival and fiber ingrowth within the
striatum ofadult rats. For electrophysiology, the animals were
anesthetized, paralyzed, and prepared for routine single unit
recording with care taken to sample a sufficient volume of
cortex in each animal (10, 16, 20, 21). Stimuli were presented
and receptive properties were analyzed quantitatively with
VAST, a Macintosh-based computer program for electrophys-
iology (22). OD classes were formed using a modification of
the Hubel-Wiesel seven-point scale (23). Cells of group 1 were
driven only by the deprived eye; for cells of group 2 there was
marked dominance of the deprived eye; for group 3, slight
dominance. For cells in group 4 there was no obvious differ-
ence between the two eyes. In group 5 the nondeprived eye
dominated slightly; in group 6, markedly; and in group 7 the
cells were driven only by the nondeprived eye. Neurons that
had spontaneous activity but did not show any responses to
visual stimuli were rated "U." For comparison, the sampled
cells in OD classes of each individual hemisphere were first
normalized. Group data of each OD class are shown in OD
histograms (see Figs. 1-3) with means and standard error bars.
The distributions of OD classes observed in NGF-treated and
control hemispheres were statistically evaluated using a x2
test. To avoid subjective bias of sampling data, we performed
blind experiments in four of the recorded cats. During both the
minipump implantation and recording sessions, the experi-
menters did not know which minipumps contained NGF or
control solution. To examine any apparent pharmacological
effects of NGF on neuronal responses, monocular deprivation
and NGF infusion were imposed on another animal for only 12
days, and cortical recordings were performed while the mini-
pump was still delivering NGF. After electrophysiological
recording sessions, all animals were perfused transcardially
and the brains were fixed and preserved for further anatomical
analyses.
RESULTS
Results for all 11 animals studied are shown in Fig. 1 A and
B. In the control hemispheres (N = 11), a total of 354 neurons
Abbreviations: NGF, nerve growth factor; OD, ocular dominance.
 Neurobiology: Gu et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8409

U,
-

0)
Qo

1 2 3 4 5 6 7 U 1 2 3 4 5 6 7 U
0 0 0 0

(A
U

e0)

1 2 3 4 5 6 7 U 1 2 3 4 5 6 7 U
0 0 0 0

FIG. 1. OD distribution of neurons compiled from striate cortices of both control and NGF-treated hemispheres. (A) Control hemispheres
(N = 11). (B) NGF-treated hemispheres (N = 11). (C) Data from blind experiments sampled from control hemispheres (N = 4). (D) Data from
blind experiments sampled from NGF-treated hemispheres (N = 4). Cells of group 1 are driven only by the deprived eye; for cells of group 2
there is marked dominance of the deprived eye; for group 3, slight dominance. For cells in group 4 there is no obvious difference between the
two eyes. In group 5 the nondeprived eye dominates slightly; in group 6, markedly; and in group 7 the cells are driven only by the nondeprived
eye. Class U, neurons that have spontaneous activity but do not show any responses to visual stimuli; n, total number of recorded neurons.

was recorded and most neurons were responsive to visual
stimuli presented via either eye as in normal animals. In the
NGF-treated hemispheres (N = 11), however, about one-
third of the 670 encountered neurons were responsive to
visual stimuli presented only via one eye. The more effective
route for visual stimulation was the eye that had been sutured
shut during the NGF infusion period (Fig. 1B). The results
from blind experiments were similar to those of other exper-
iments: in the control hemispheres most neurons remained
binocularly driven (Fig. 1C), while in the NGF-treated hemi-
spheres most neurons responded more vigorously via the
deprived eye and many neurons were responsive solely to the
deprived eye (Fig. 1D).
We analyzed our data separately concerning possible dif-
ferences between ipsilateral and contralateral hemispheres
(Fig. 2). The OD shift was observed regardless of whether the
NGF-treated hemisphere was ipsilateral or contralateral to
the deprived eye. The differences between OD distributions
representing NGF-treated and control hemispheres were
statistically significant at the P < 0.01 level (X2 test).
In most penetrations, neurons were recorded in the vicinity
of the infusion cannula (<3 mm). To determine whether NGF
infusion also affected neurons far away from the infusion
cannula, recordings were performed at different distances
from the infusion site in two NGF-treated hemispheres. The
OD distributions are shown in Fig. 3. While the OD distri-
bution of neurons at distances >3 mm remained similar to
that found in the control hemispheres (Fig. 3A), the OD
distribution of neurons within 3 mm of the infusion cannula
was strongly shifted toward the deprived eye (Fig. 3B).
Despite the change of ocularity in the NGF-treated hemi-
spheres, other receptive field properties of cortical neurons
such as orientation selectivity, direction selectivity, optimal
stimulation velocity, and spontaneous activity in the NGF-
treated hemispheres appeared to be the same as in normal
adult visual cortex and did not differ from those recorded in
the control hemispheres.
To examine any apparent pharmacological effects of NGF
on cortical neurons, we recorded one adult cat 12 days after
minipump implantation and monocular deprivation, keeping
the minipump in place during the entire recording session.
Both the response vigor and orientation tuning curves of
neurons recorded near the NGF infusion cannula appeared
the same as those recorded in the control hemisphere (P >>
8410 Neurobiology: Gu et al. Proc. Natl. Acad. Sci. USA 91 (1994)
possible reasons for the shift in cortical OD to the deprived
A Control B NGF-treated eye.
40 n = 62 n = 267 Functional connections between the eyes and cortical cells

I
(A
30 -
can be changed either by alterations in the number of synaptic
U
4-
u contacts or by modification of existing synapses' efficacy.
20 - TT We obtained preliminary results (24) indicating that the
NGF-treated hemispheres in these animals exhibit an in-

U,
10
0-
-
Lx creased level of immunoreactivity of phosphorylated GAP-
43, which is localized selectively in active growth cones (25,
26), and synaptophysin, a presynaptic marker. This suggests
(A
0 1 2 34 6 7I .U 1234567 U that NGF infusion induces local axonal sprouting and for-
0 0 0 0
0 mation of new synapses, which in turn contribute to the
increased plasticity reported here.
Why should the distribution of cortical OD shift toward the
us
deprived eye? Reiter and Stryker (8) found a similar para-
4-
doxical OD shift in kittens when they perfused a zone of
0f cortex with muscimol, a y-aminobutyric acid type A
(GABAA) receptor agonist. The perfusion blocked postsyn-
aptic responsiveness in the zone of cortex, and they inter-
preted their results in terms of a covariance rule for synaptic
modification, which predicts that decorrelation between syn-
aptic input and postsynaptic response should lead to selective
50 weakening of inputs associated with the more active eye. One
Control F NGF-treated interpretation of the results reported here is that NGF has
40 n = 50 n = 160 two effects: first, a trophic effect, which increases plasticity,
t 30
IT and second, a pharmacologic effect similar to muscimol,
which results in decreased responsivity of cortical neurons
'4-4
0
20
and hence the same sort of shift toward the deprived eye that
was observed by Reiter and Stryker. Recordings during
10- which NGF was continuously infused into the visual cortex
suggest, however, that NGF does not appear to have phar-
01
1 2 34 5 6 7 U
_~~~~lU
1 2 34 5 6 7
macological muscimol-like effects on cortical neurons. We
therefore postulate another mechanism, based on the evi-
* 0 * 0
dence for local sprouting mentioned above, to explain our
observations. (i) NGF stimulates sprouting of cholinergic
axons via NGF receptors. While intrinsic cortical fibers, both
U, GABAergic and non-GABAergic, and also geniculocortical
O
a0
fibers, are all candidates for sprouting, the most likely fiber
-
population to be sprouting is the cholinergic pathway origi-
eo nating from the basal forebrain. These fibers are known to
have receptors for NGF (27-29), and earlier studies have
shown that NGF can facilitate sprouting of cholinergic fibers
in the adult central nervous system (30-32). (ii) Since ace-
1 2 34 5 6 7
0 0
U 123 4 5 6 7
0 0
U tylcholine facilitates N-methyl-D-aspartate (NMDA)-
mediated responses (33-35), increased cholinergic input
FIG. 2. OD distribution of neurons compiled from striate cortex would increase responsivity of cortical neurons to the excit-
either ipsilateral (A, B, E, and F) or contralateral (C, D, G, and H) atory lateral geniculate nucleus input, which uses NMDA
to the deprived eye. (A) Control hemispheres, ipsilateral to the receptors as a major postsynaptic target (36, 37). Recordings
deprived eye, recorded nonblind (N = 3). (B) NGF-treated hemi- performed during and after NGF infusion did not seem at first
spheres, ipsilateral to the deprived eye, recorded nonblind (N = 4). glance to indicate any greater responsivity in NGF-treated
(C) Control hemispheres, contralateral to the deprived eye, recorded visual cortex that might be expected from this hypothesis.
nonblind (N = 4). (D) NGF-treated hemispheres, contralateral to the However, since recordings were performed under anesthe-
deprived eye, recorded nonblind (N = 3). (E) Control hemisphere,
ipsilateral to the deprived eye, recorded blind (N = 1). (F) NGF- sia, which is generally considered to turn off central cholin-
treated hemispheres, ipsilateral to the deprived eye, recorded blind ergic transmission, any increased responsivity caused by
(N = 3). (G) Control hemispheres, contralateral to the deprived eye, cholinergic input may be observable only in NGF-treated
recorded blind (N = 3). (H) NGF-treated hemisphere, contralateral visual cortex of alert animals. (iii) Long-lasting increased
to the deprived eye, blind recorded (N = 1). Conventions are the responsivity decreases synaptic efficacy either by reduction
same as in Fig. 1. of postsynaptic receptor sensitivity or by reduction of trans-
mitter release, possibly through phosphorylation/dephos-
0.05, Student's t test). In the area close to the NGF-infusion phorylation processes or retrograde messengers. Evidence
cannula there was already a substantial OD shift toward the supporting this last point comes from long-term potentiation
deprived eye after only 12 days of monocular deprivation (LTP) experiments: when stimulus trains, either at high or
(data not shown). low frequency, were continuously given after tetanic stimu-
lation, which normally induces LTP, long-term depression
DISCUSSION was observed instead of LTP (38-40). (iv) These processes
Our results demonstrate that intracortical infusion of NGF lead to a selective weakening of the active input connections,
into adult visual cortex induces OD plasticity. However, while input connections from the deprived eye remain unaf-
monocular deprivation causes an OD shift toward the de- fected. This mechanism is speculative, but it provides a
prived eye. We consider, first, the issue of how NGF framework within which further experiments to define the
increases plasticity in adult visual cortex and second, the cellular mechanisms involved can be conducted.
 Neurobiology: Gu et A
50
40 -
." 30-
0
e 20-
10 -
0-
1 2 3 4 5 6 7
0 0
FIG. 3. OD distribution of neurons recorded at different distances from the infusion cannula. (A) Distances >3 mm from the infusion cannula.
(B) Within a distance of 3 mm from the infusion cannula. All cells were recorded in NGF-treated hemispheres ipsilateral to the deprived eye.
Conventions are the same as in Fig. 1.
Our results showing that NGF effects are restricted to the
area of infusion despite evidence that NGF can be retro-
gradely transported to the basal forebrain (41) suggest that
NGF effects were primarily due to interaction between NGF
and NGF receptors in the immediate surroundings. How-
ever, since there is cross-reactivity between the various
members of the neurotrophin family and their receptors,
NGF, in the relatively high concentrations present at the
infusion site, may exert its effects not only through interac-
tions with its specific receptors but also through interactions
with receptors for other neurotrophins. Experiments involv-
ing infusion of other neurotrophins at varied concentrations
would be important in addressing this point.
Recently it has been reported that intraventricular injection
of NGF prevents the OD shift that normally occurs in young
rat and kitten visual cortex: neurons in the NGF-infused
hemispheres remained binocular despite monocular depriva-
tion (12-14). A competition hypothesis was proposed (13),
suggesting that afferents from the two eyes compete for
trophic substances released by activated postsynaptic cells in
the visual cortex and that application of saturating doses of
NGF would render competition ineffective. This competition
mechanism, however, cannot explain the OD shift toward the
deprived eye in adult visual cortex described here. There are
many differences between young and adult animals that may
account for these results. First, the level of NGF and NGF
receptors is different between young and adult visual cortex
(ref. 42; unpublished data) and so are many constituents of
the neural environment (state of myelination, adhesion mol-
ecules, distribution of receptors, etc.). In addition, there is
evidence that NGF affects choline acetyltransferase activity
differentially in young and adult neocortex (43). Neverthe-
less, our finding is consistent with the results obtained from
young animals, if one considers that two processes may
operate in parallel in young visual cortex following monoc-
ular deprivation: one is a normal activity- and age-dependent
OD shift toward the nondeprived eye, and the other is an OD
shift toward the deprived eye resulting from the NGF treat-
ment. The combination of both processes may have allowed
neurons to remain binocular in the young animal.
Regardless of the mechanisms involved, our results pro-
vide evidence that NGF can induce OD plasticity in adult
visual cortex. The ability to reinduce cortical OD plasticity in
adult animals offers hope for new therapeutic approaches to
amblyopia based on neurotrophin therapy.
We thank Dr. N. Swindale for comments on the manuscript and V.
Proc. Natl. Acad. Sci. USA 91 (1994) 8411
U 1 2 3 4 5 6 7 U
0 0
Booth for technical assistance. The generous supply of NGF from
Genentech is gratefully acknowledged. This work was supported by
the International Human Frontier Science Program, the Canadian
Medical Research Council, and the Network of Centers of Excel-
lence for Neural Regeneration and Functional Recovery.
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