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The Journal of Neuroscience, November 1, 1998, 18(21):8826-8838
Establishment of Patterned Thalamocortical Connections Does Not
Require Nitric Oxide Synthase
Eva M.
Finney and
Carla J.
Shatz
Department of Molecular and Cell Biology and Howard Hughes Medical
Institute, University of California, Berkeley, California 94720
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ABSTRACT |
Subplate neurons are early-generated neurons that project into the
overlying neocortex and are required for the formation of ocular
dominance columns. A subset of subplate neurons express nitric oxide
synthase (NOS) and produce nitric oxide (NO), a neuronal messenger
thought to be involved in adult hippocampal synaptic plasticity and
also in the establishment of certain specific connections during visual
system development. Here, we examine whether the NOS-containing
subplate neurons are involved in ocular dominance column formation in
the ferret visual system. Ocular dominance columns form in ferrets
between postnatal day 35 (P35) and P60. NOS expression in the visual
subplate is low at birth, increases to a maximum at the onset of ocular
dominance column formation, and falls thereafter. Nevertheless,
blockade of NOS with daily injections of nitroarginine from P14 to P56
fails to prevent the formation of ocular dominance columns, although
NOS activity is reduced by >98%. To test further a requirement for
NOS in the patterning of connections during CNS development, we
examined the cortical barrels in the somatosensory system of mice
carrying targeted disruptions of NOS that also received injections of
nitroarginine; cortical barrels formed normally in these animals. In
addition, barrel field plasticity induced by whisker ablation at birth
was normal in nitroarginine-injected NOS knock-out mice. Thus, despite the dynamic regulation of NOS in subplate neurons, NO is unlikely to be
essential for the patterning of thalamocortical connections either in
visual or somatosensory systems.
Key words:
ocular dominance columns; nitric oxide synthase; visual
system; barrels; knock-out; nitroarginine; synaptic plasticity
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INTRODUCTION |
A fundamental question in
developmental neurobiology is how specific sets of patterned
connections form during development. Ocular dominance columns, the
alternating pattern of left- and right-eye inputs to layer 4 of primary
visual cortex in binocular mammals, are a well-characterized system in
which to study establishment of specific neuronal connections. Early in
development, inputs from the right and left eyes are intermingled
(LeVay et al., 1978
, 1980). Later, via an activity driven, competitive
process, the final adult pattern is established (Wiesel and Hubel,
1965; Hubel et al., 1977; LeVay et al., 1980; Stryker and Strickland,
1984; Stryker and Harris, 1986; for review, see Shatz, 1990; Katz and Shatz, 1996; Crair et al., 1998). Recently, subplate neurons have also
been shown to be required for the establishment of ocular dominance
columns; early ablation of subplate neurons in cats prevents the
segregation of right- and left-eye thalamocortical inputs (Ghosh and
Shatz, 1992, 1994; Ghosh, 1995). To learn more about how subplate
neurons influence ocular dominance column formation, here we have
investigated a possible role for those subplate neurons that are
NADPHdiaphorase-positive (Sandell, 1986; Mizukawa et al., 1988;
Derer and Derer, 1993; Luth et al., 1995; Yan et al., 1996; Yan and
Ribak, 1997; Finney et al., 1998).
NADPH-diaphorase (hereafter called diaphorase)-positive subplate
neurons are of special interest because diaphorase has been identified
with nitric oxide synthase (NOS), the synthesizing enzyme for nitric
oxide (NO) (Bredt et al., 1991; Dawson et al., 1991; Hope et al.,
1991). NO has been implicated in the refinement of retinogeniculate
connections into the ON-OFF sublaminae of the lateral geniculate
nucleus (LGN) during development. Cramer et al. (1995b) demonstrated a
developmental regulation of diaphorase-expressing cells in the LGN of
the ferret; blockade of NOS activity with the competitive inhibitor
nitroarginine reduced the degree of segregation of retinal axons into
ON-OFF sublaminae (Cramer et al., 1996). Similarly, Williams et al.
(1994) demonstrated a transient expression of diaphorase in the chick
tectum; when NOS activity was blocked, the refinement of the
retinotectal map was impaired (Wu et al., 1994, 1996). These
observations suggest that NO may be involved in the selective
retainment or elimination of immature synaptic connections early in
development (for review, see Williams, 1996).
NO is also thought to be crucial for some forms of adult synaptic
plasticity, such as long-term potentiation (LTP) in the hippocampus (O'Dell et al., 1991; Schuman and Madison, 1991; Zhuo et
al., 1993; Kantor et al., 1996; Son et al., 1996) or long-term depression (LTD) in the cerebellum (Crepel and Jaillard, 1990; East and Garthwaite, 1990; Lev-Ram et al., 1995, 1997; for recent reviews, see Holscher, 1997; Huang, 1997; Wang et al., 1997). NO can
also influence the strength of synaptic connections in the cortex
(Nowicky and Bindman, 1993; Friedlander et al., 1996a; Harsanyi and
Friedlander, 1997a) and at the developing neuromuscular junction (Wang
et al., 1995). LTP and LTD can be demonstrated also at developing
synapses (Bear et al., 1992; Kirkwood et al., 1993; Mooney et al.,
1993; Harsanyi and Friedlander, 1997b), including the thalamocortical
synapses in the rodent (Crair and Malenka, 1995; Kirkwood et al.,
1995). Thus, it is reasonable to ask whether there might be a
requirement for NO not only in adult synaptic plasticity but during
development as well, particularly in view of the presence of
diaphorase-positive subplate neurons. Perhaps the subplate neurons are
necessary for the segregation of LGN axons into ocular dominance
columns because subplate neurons supply the cortex with NO. Thus,
subplate neuron ablations might prevent ocular dominance column
formation by removing NO, a critical intercellular messenger needed for
synaptic strengthening or weakening.
Here, we have tested this hypothesis in several ways. First, we
examined the time course of expression of NOS by subplate neurons in
the ferret visual system. Then, NOS activity was blocked during the
period of ocular dominance column formation using the NOS inhibitor
nitroarginine. We also examined mice carrying targeted mutations in two
forms of NOS: neuronal NOS (nNOS) or endothelial NOS (eNOS) knock-out
mice (Huang et al., 1993, 1995). Mice deficient in both the endothelial
and neuronal isoforms of NOS have deficits in hippocampal LTP (Son et
al., 1996). Abnormal cortical connectivity or plasticity in nNOS
/
and eNOS/ mice was assessed by examining barrel field formation or
response to peripheral follicle removal (Van der Loos and Woolsey,
1973; Woolsey and Wann, 1976). Because multiple isoforms of nNOS exist
in addition to the genetically disrupted isoform (Brenman et al.,
1996), we also assessed barrel field plasticity in nNOS and eNOS
knock-out mice given daily injections of nitroarginine to eliminate
virtually all NOS activity. Although onset of NOS expression in the
visual subplate and cortex of ferrets correlates well with ocular
dominance column formation, we found that ocular dominance columns form
normally in nitroarginine-treated ferrets. In addition, barrel field
formation and plasticity are unaffected in NOS knock-out mice even when
given nitroarginine. Thus, NO is not likely to be involved in the
formation or plasticity of these major thalamocortical connections.
Moreover, subplate neurons are likely to exert their effects on ocular
dominance column formation via an NO-independent mechanism.
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MATERIALS AND METHODS |
A total of 41 ferrets and 60 mice were studied. All surgical
procedures were performed according to a protocol approved by the
Animal Care and Use Committee of the University of California at
Berkeley and Public Health Service policy.
Tissue preparation. At the completion of an experiment,
animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbitol (6 grains/ml; 1 ml/10 lb; Anthony Products, Arcadia, CA) and perfused transcardially with cold 0.1 M
sodium phosphate buffer and then 4% paraformaldehyde in 0.1 M sodium phosphate buffer.
NADPH-diaphorase histochemistry. Perfused ferret brains were
post-fixed 1 hr in 4% paraformaldehyde and sunk in 25% sucrose. Visual cortex sections were cut sagittally at 25 µm on a
sliding freezing microtome. Free-floating sections were incubated in
0.1 mg/ml nitroblue tetrazolium (NBT) (N5514; Sigma, St. Louis,
MO), 1.0 mg/ml 
-NADPH (Sigma), and 0.3% Triton X-100 (T9284, Sigma) at 37°C for 45 min to several hours according to the method of Vincent and Kimura (1992). The reaction was stopped by washing in
sodium PBS (8 gm/l NaCl, 2.16 gm/l Na2HPO4·7
H2O, 0.2 gm/l KCl, and 0.2 gm/l
KH2PO4) for 20 min. Sections were
mounted onto subbed slides, dried overnight, counterstained with
neutral red (50 040; Chroma-Gesellschaft Schmid and Company,
distributed by Roboz Surgical Instrument Company, Washington, DC),
dehydrated briefly through graded alcohols, dipped in xylene for 1 min,
and coverslipped with Krystalon (EM Diagnostic Systems,
Gibbstown, NJ). Coverslips were sealed with clear nail polish.
Quantification of diaphorase-positive cells. Three
representative sections from medial to lateral in the visual cortex of postnatal day 5 (P5), P15, P25, P35, P45, and adult ferrets
(n = 2 each) were reacted as described above for
NADPH-diaphorase histochemistry. Cortical layers and
diaphorase-positive neurons were drawn using a camera lucida device.
For criteria used to determine layer 6/subplate boundaries, see Chun
and Shatz (1989a,b) or Luskin and Shatz (1985). Camera lucida drawings
were digitized (Apple Color One Scanner; Apple Computer, Cupertino,
CA), and the area of each layer was calculated by determining the
pixels contained within each layer (circumscribed manually in
Metamorph; Universal Imaging Corporation, West Chester, PA) and
dividing by the number of pixels in one square millimeter. The number
of diaphorase-positive cells was counted manually for each layer. Cells
falling on the borders between layers were counted separately; this
value was divided by two, with one-half the border cells attributed to
the overlying layer and one-half to the underlying layer. The density
of diaphorase-positive cells per layer was derived by dividing the
total number of diaphorase-positive neurons in each layer by the area
of that layer. Final density values for each layer were averaged for
each case and then combined to obtain average values for all cases at
each age.
NOS immunohistochemistry and colocalization with
NADPH-diaphorase. Some sections from ferret brains used for
NADPH-diaphorase histochemistry were also used for NOS
immunohistochemistry. Tissue prepared for diaphorase histochemistry was
then incubated according to the method of Bredt et al. (1990) for 1 hr
at room temperature in blocking solution containing 1 mg/ml bovine
serum albumin (A-7888, Fraction V; Sigma), 0.025% sodium azide
(26628-22-8; Mallinckrodt, Paris, KY), 0.3% Triton X-100 (789704;
Boehringer Mannheim, Indianapolis, IN), and 1% normal goat serum
(Vector Laboratories, Burlingame, CA) in PBS. Tissue was incubated in
rabbit anti-NOS primary antibody (diluted in blocker at 1:70; a gift
from Dr. David Bredt, University of California, San Francisco, CA) at
4°C overnight, washed three times in PBS, incubated in goat
anti-rabbit secondary antibody (1:200; Vector laboratories), washed
three times in PBS, and developed using the Vectastain Elite ABC kit
and 0.05% diaminobenzidine (DAB) (D5905; Sigma) in PBS with 0.01%
hydrogen peroxide. For sections used for both diaphorase and NOS
immunohistochemistry, diaphorase was performed first, with incubation
times shortened to facilitate visualization of both blue and brown
products within the same cells. Longer incubation times tended to stain
cells intensely, preventing discrimination of blue from brown reaction products.
Nitroarginine and verapamil injections (see Table 1).
Nitroarginine (N5501; Sigma) (10 mg/kg per day) and verapamil
(verapamil hydrochloride injection, USP; American Regent Laboratories,
Shirley, NY) (5 mg/kg per day) were injected daily intraperitoneally
into ferrets from P14 to P56 (Table 1).
[In earlier experiments, ferrets were injected from P20 to P56 with
nitroarginine alone (Table 1).] Verapamil, a calcium channel blocker,
is an anti-hypertensive agent and prevents the high blood pressure
effects of nitroarginine at these dosages (Cramer et al., 1996). At
P56, ferrets were perfused for [3H]proline
autoradiography. Nitroarginine was injected intraperitoneally at 5 mg/kg per day into P0 mice within 12 hr of birth and daily thereafter
until P7-P17, when they were perfused for cytochrome oxidase or wheat
germ agglutinin (WGA)-HRP histochemistry. Control animals were
age-matched and either injected with 0.9% saline (ferrets) or
unmanipulated (mice).
NOS enzymatic activity assay (See Figs. 6, 8). The protocol
for this NOS assay was that of Huang et al. (1993). Visual cortices from ferrets (contralateral to the eye injection from the same animals
in which transneuronal autoradiography was used to examine the ocular
dominance columns) or whole brains from 129 SV wild-type (Jackson Laboratories, Bar Harbor, Maine) or NOS knock-out (a gift from
Drs. Mark Fishman and David Bredt) mice were suspended in 10 vol of
ice-cold buffer [50 mM Tris-HCl, pH 7.4 (T-3253; Sigma),
with 1 mM EDTA (E5134; Sigma) and 1 mM
EGTA (E4378; Sigma)] and homogenized. One milliliter of
homogenized tissue was put into eppendorf tubes and centrifuged at
10,000 × g for 15 min at 4°C. Twenty-five
microliters of supernatant from each sample were incubated for 15 min
at room temperature in duplicate (with or without 10 µl of 6 mM CaCl2) with 100 µl of reaction mix.
The reaction mix contained 1 mg/ml NADPH (N-6504; Sigma), 1 µl/ml 3H-arginine (30-60 Ci/mmol, A-3680; Sigma), 2 mM flavin adenine dinucleotide (F-6625; Sigma), 2 mM flavin mononucleotide (F-2253; Sigma), 2.5 × 10
3 µg/ml calmodulin (P1915; Sigma), and 0.05 mM tetrahydrobiopteran (CN-250; Biomol, Plymouth Meeting,
PA). The reaction was stopped with 3 ml of deionized water. The
reaction solution was loaded onto a column (2083366050; Evergreen
Scientific, Los Angeles, CA) of 0.5 ml of Dowex-50W resin (sodium form)
(50X8-400; mesh size 200-400; Sigma) and allowed to drip through into
glass scintillation vials. (To generate the sodium form of Dowex, the
resin was suspended in water and stirred with NaOH pellets until pH 12. The resin was allowed to settle, and water was decanted. Rinses were
repeated until pH 7 was reached.) Sixteen milliliters of scintillation cocktail were added to each sample. Samples were counted with 1-5 min
sampling times on a Beckman Instrument (Palo Alto, CA) LS5000 TD
scintillation counter. NOS activity was calculated as counts in the
presence of calcium chloride minus counts without calcium chloride.
Because NOS is calcium-dependent (Bredt and Snyder, 1990), counts
without calcium chloride constituted nonspecific background. Activity
was expressed as a percent of control levels (age-matched unmanipulated
or saline-injected animals).
Eye injections. The method of transneuronal transport after
intraocular injection of [3H]proline was used to
label ocular dominance columns in layer 4 of the visual cortex
(LeVay et al., 1978). [3H]proline (TRK.534;
Amersham Life Science, Little Chalfont, Buckinghamshire, England) was
concentrated in a speed vacuum and reconstituted in sterile 0.9%
saline. Ferrets were anesthetized with isoflurane (3%) and oxygen (1 l/min), and the area around one eye was shaved and sterilized with
nolvalsan and alcohol. [3H]proline was injected
into the posterior chamber of one eye, both nasally and temporally (6 and 6.5 ml, respectively; 2 mCi total) using a Unimetrics (Anaheim, CA)
or Hamilton (Reno, NV) 50 µl syringe with a 33 gauge needle. Survival
time after injection was 7-10 d.
Autoradiography. Perfused ferret brains were post-fixed
overnight in 4% paraformaldehyde and then sunk in 25% sucrose in 0.1 M sodium phosphate buffer. Visual cortex sections were cut
sagittally at 25 µm on a sliding freezing microtome, mounted onto
subbed slides, defatted through graded alcohols and xylenes, and dipped for autoradiography as described previously (LeVay et al., 1978; Cabelli et al., 1997). After 3-6 weeks, the slides were developed and
coverslipped using Permount (Fisher Scientific, Houston, TX).
Quantification of ocular dominance columns. Images of
representative sections processed for autoradiography from the visual cortex of saline (n = 4) or nitroarginine-treated
(n = 9) animals (5-18 sections/animal) were acquired
with a CCD-72 video camera (Dage MTI, Michigan City, IN) using National
Institutes of Health Image software, and ocular dominance columns were
quantified as described previously (Cabelli et al., 1995). Briefly,
images were spatially smoothed (5 × 5 pixel average), and a
brightness profile was derived along a line drawn through the middle of
layer 4 (parallel to the pial surface) on each section. The brightness
profile was additionally low-pass filtered (HAM;
fc = 20%) and analyzed with Superscope software
(GW Instruments, Somerville, MA), using the first derivative, for
(1) periodicity (midpoint-to-midpoint of injected-eye columns),
(2) column width (width of individual injected-eye columns), and (3)
peak-to-trough amplitude (brightest point of injected-eye columns to
darkest point of adjacent uninjected-eye columns, in units of 8-bit
pixel gray scale values). To account for possible differences in column
parameters across medial to lateral visual cortex, we divided the
mediolateral extent of the visual cortex into equivalent thirds
(medial, intermediate, and lateral) and analyzed each third separately.
Because we found the variability for medial and lateral groups to be
much greater than that for the intermediate group, only data from the
intermediate group were used in our statistical comparisons. Parameter
values for sections within an animal were averaged to acquire a single value per animal. Parameter values for all the saline animals were
averaged and compared with the average of all the nitroarginine animals
to determine whether significant differences exist. Saline and
nitroarginine data were compared using a Student's t test, two-tailed assuming equal variances, a = 0.05.
Visualization of barrel fields with cytochrome oxidase.
Neocortex from perfused mouse brains (see Table 1) was removed and flattened between glass slides overnight in 4% paraformaldehyde. Flattened cortices were sectioned at 50 µm on a sliding freezing microtome. Free-floating sections were reacted for cytochrome oxidase
histochemistry (130 ml of 0.1 M sodium phosphate buffer, 6 gm of sucrose, 75 mg of DAB, and 35 mg of cytochrome C) (cytochrome C
from Sigma, C2506) in the dark at 37°C according to the method of
Chiaia et al. (1992). Reactions were stopped with three rinses in 0.1 M sodium phosphate buffer. Sections were mounted onto
subbed slides, dehydrated through graded alcohols and xylene, and
coverslipped with Permount.
Whisker ablations. Within 12 hr of birth, mice were
anesthetized on ice, an incision was made just ventral to the C row of whisker follicles with a Moria knife (10315-12; Fine Science Tools, Foster City, CA), and follicles 1-4 were removed with a fine forceps (Jeanmonod et al., 1981). The incision was closed using histoacryl glue. Survival times after ablation were 6-14 d.
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RESULTS |
The results are presented in two parts. First, we describe in the
ferret visual cortex (1) the time course of ocular dominance column
formation, (2) the time course of development of diaphorase-positive subplate neurons, and (3) the consequences of NOS activity blockade on
the formation of ocular dominance columns. Second, we present the
results of studies of NOS knock-out mice that examine (1) barrel field
formation in somatosensory cortex, (2) the efficacy of genetic and
pharmacological NOS activity blockade, and (3) the consequences for the
patterning of thalamocortical connections and plasticity after NOS
blockade.
Ocular dominance column formation in ferrets
To determine whether expression of NOS-positive subplate neurons
is developmentally regulated in a way that corresponds to the formation
of patterned connections in the visual cortex of ferrets, we first
established when ocular dominance columns form in ferrets. To visualize
ocular dominance columns in ferrets, we used the technique of
transneuronal transport after monocular injection of
[3H]proline (Fig. 1,
Table 1). At the earliest ages studied, P25 and P28 (1 week before eye
opening), ocular dominance columns are not yet present;
autoradiographic labeling is continuous in layer 4 of the visual
cortex, indicating that LGN axons corresponding to the injected eye
have not yet clustered into patches corresponding to the columns. By
P36, the first indications of segregation are evident from the presence
of periodic variations in the density of silver grains. By P46 and P56,
the periodicity is pronounced, and ocular dominance columns are clearly
evident in layer 4. By P67, the pattern of labeling is essentially
adult-like, with thalamic axons from the injected eye clustered densely
to define columns. In the ferret, as in the cat (LeVay et al.,
1978), some labeling is evident in layer 4 in territory belonging to
the uninjected eye even in the adult. The results of this experiment
indicate that ocular dominance columns form between P35 and P60 in the ferret (see also Ruthazer et al., 1995).
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Figure 1.
Time course of ocular dominance column formation
in ferret visual cortex. Ocular dominance columns are revealed by
autoradiography after transneuronal transport of
[3H]proline injected into one eye (dark-field
photographs; silver grains appear white).
A, At P28, transported label is
continuous in layer 4. Arrowheads in A
delineate layer 4. B, At P36, patches of
label can be detected in layer 4 (arrowheads).
C, D, At P42
(C) and P46
(D), segregation progresses.
Arrowheads in B and C
indicate ocular dominance columns belonging to the injected eye.
E-G, Segregation is pronounced by
P56 (E) and P61
(F) and appears adult-like by P67
(G). Anterior is to the left;
dorsal is to the top in these sagittal sections. All
sections are ipsilateral to the injected eye. The
asterisk in A marks labeled
geniculocortical axons running in the white matter. Scale bar, 1 mm.
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Ferret ocular dominance columns, as visualized here in sagittal
sections, are less sharp and more irregular than are those in the cat.
However, irregular periodicity may be a reflection of sectioning
through some columns obliquely rather than perpendicularly. The pattern
of ocular dominance columns in the adult ferret has been described
previously by Law et al. (1988). They found that ferret columns run in
rough stripes that sometimes intersect and vary in width from 200 to
1500 µm as viewed from the horizontal plane. Their observations
suggest that any single section through the visual cortex of ferrets
will produce ocular dominance columns of irregular width. In addition,
they reported that ~77% of the most binocular region contralateral
to the injected eye was occupied by label, whereas 49% was occupied
ipsilaterally, suggesting a substantial overlap between the two eyes.
This observation agrees with ours that some autoradiographic label
representing the injected eye persists in columns dominated by the
noninjected eye.
Developmental regulation of NOS in the visual subplate
We next investigated the time course of expression of NOS in the
visual cortex, including the visual subplate, to examine whether there
is a correlation in the timing of development of NOS-expressing
subplate neurons and formation of ocular dominance columns.
NADPH-diaphorase histochemistry [which colocalizes with NOS (Dawson et
al., 1991; Hope et al., 1991)] was used to examine the distribution of
diaphorase-positive neurons in each cortical layer from P5 to adult.
Examples of diaphorase-stained or NOS-immunopositive neurons in the
visual cortex of a P36 ferret are shown in Figure 2. Both methods reveal essentially
identical patterns of staining, with the majority of neurons at this
age located in the subplate. The diaphorase reaction fills neuronal
somata and dendrites intensely; the location and morphology of these
neurons indicate that they are subplate neurons (Luskin and Shatz,
1985; Chun et al., 1987; Antonini and Shatz, 1990; Allendoerfer and
Shatz, 1994; Finney et al., 1998). Diaphorase-positive subplate neurons
are multipolar or bipolar, with their long axes usually running
parallel to the pial surface. Diaphorase-positive, inverted pyramidal
subplate neurons were not observed. Sections reacted for both NOS
immunohistochemistry and diaphorase histochemistry reveal neurons
labeled with both the blue NBT product of the diaphorase reaction and
the brown DAB product of the NOS immunohistochemistry, confirming as in other systems where this has been examined (Dawson et al., 1991; Hope
et al., 1991) that both methods stain identical neurons in the visual
cortex (data not shown).
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Figure 2.
NOS-immunoreactive and
NADPH-diaphorase-stained adjacent sections from P36 ferret visual
cortex. A, B, NOS immunohistochemistry
(A) stains cells in a pattern similar to that of
diaphorase histochemistry (B). C,
Higher magnification view of area boxed in
B shows several diaphorase-positive subplate neurons
(arrows). Note the typical horizontally oriented
processes of these neurons. B and C are
counterstained with neutral red to reveal cortical layers.
CP, Cortical plate; SP, subplate. Scale bars, 100 µm.
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The time course of development and the pattern of distribution of
diaphorase-positive neurons in ferret visual cortex are shown in the
camera lucida drawings of Figure 3.
Diaphorase-positive neurons are present at all ages examined from P5 to
adulthood (n = 2 animals per time point) and in all
layers but are especially prevalent in the subplate and layer 6 and are
sparse in layers 2-4. These observations are presented quantitatively
in Figure 4. The density of
diaphorase-positive neurons in the subplate (Fig. 4A)
is low at P5 (5.4 ± 1.9 per mm2) but increases
dramatically by P25 (37.2 ± 16.9 per mm2) and
P35 (26.6 ± 10.0 per mm2), just before the
onset of ocular dominance column formation (see above). By P45, when
ocular dominance columns are well on their way to forming, the density
of diaphorase-positive subplate neurons has decreased to close to adult
levels (P45, 17.8 ± 3.7 per mm2; adult,
15.7 ± 3.2 per mm2). This fall in the density
of labeled subplate neurons between P25 and adulthood is not caused by
an increase in the overall area of the subplate. Measurements of
subplate area (Fig. 4B) show that there is a >50%
decrease in the area of the subplate between P5 and adulthood,
consistent with previous observations in other species [cat (Chun and
Shatz, 1989b); monkey (Kostovic and Rakic, 1990); human (Kostovic and
Rakic, 1990)]. In fact, between P25 and adulthood, the subplate area
falls modestly, yet the density of subplate neurons falls dramatically.
These observations indicate that the absolute numbers of
diaphorase-positive subplate neurons peak between P25 and P35, followed
by a decline at subsequent ages. Thus, the onset of ocular dominance
column formation coincides with a dramatic fall in the numbers of
diaphorase-positive subplate neurons.
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Figure 3.
Time course of diaphorase expression in the
cortex. Individual camera lucida drawings at various ages of
representative diaphorase-stained and neutral red-counterstained ferret
visual cortices, similar to the section in Figure
2B, are shown. Diaphorase-positive neurons are
represented by black dots. Cortical layers are indicated
by lines. In the adult, the subplate is labeled
WM [white matter (former subplate)].
Diaphorase-positive neurons are present in all cortical layers, most
especially in the subplate and deep cortical layers at
P25 and P35. All sections are cut in the
sagittal plane. Anterior is to the left, and dorsal is
to the top. CP, Cortical plate;
SP, subplate; 1-6, the cortical
layers, which are only labeled at P25 (although all
layers are delineated at each time point except for P5,
when cortical layers are ill-defined). Scale bar, 1 mm.
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Figure 4.
Changes in the density of diaphorase-positive
neurons and the area of the subplate during development of ferret
visual cortex. A, Histogram showing the density of
diaphorase-positive neurons in superficial, infragranular, and subplate
layers from P5 to adult. At P5, cortical layers consist primarily of
layers 5 and 6. At each age, the mean density of diaphorase-positive
cells in the subplate and deep cortical layers is greater than that in
cortical layers 1-4. Error bars for this (and all following figures)
represent SDs. The differences in the mean subplate values at
each age are greater than would be expected by chance (ANOVA;
a = 0.05; p = 0.0001). The
density of diaphorase-positive neurons in the subplate at P25 is
significantly different from that at all other ages
(Student-Newman-Keuls test; p < 0.05); in
addition, P35 is significantly different from P5
(p < 0.05). B, Changes in
the area of the subplate underlying the visual cortex with age. The
subplate as a percent of the total area of the visual cortex decreases
steadily from ~55% at P5 to ~10% by adulthood.
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Blockade of NOS activity during ocular dominance
column formation
Results described above demonstrate that just before the beginning
of ocular dominance column formation in layer 4, many subplate neurons
and also neurons in layers 5 and 6 are diaphorase positive. It is
possible that the rise and subsequent fall in diaphorase-positive subplate neurons are required to trigger the onset of geniculocortical segregation. To test this hypothesis, we blocked NOS activity by making
daily intraperitoneal injections of the NOS competitive antagonist
nitroarginine (East and Garthwaite, 1990; Dwyer et al., 1991; Cramer et
al., 1996; Moore and Handy, 1997). Two sets of experiments were
conducted (Table 1). In the first, nitroarginine alone was injected
from P20, before the onset of column formation, to P56. In the second
set, the injections were started even earlier, at P14, to cover the
entire period during which subplate neurons undergo changes in NOS
levels. In this second set of experiments, verapamil was also given to
prevent blood pressure fluctuations caused by injections of
nitroarginine alone. Coinjection of verapamil and nitroarginine
successfully prevents hypertension in ferrets and also reduces the
extent of segregation of retinal ganglion cell axons into the ON-OFF
sublaminae within the LGN (Cramer et al., 1996).
Nitroarginine injections beginning either at P14 or P20 through P56 had
no detectable effect on the segregation of LGN axons into ocular
dominance columns, as assessed by means of transneuronal transport of
[3H]proline after an eye injection (Fig.
5A,B). Because segregation is
normally more evident in the hemisphere ipsilateral to the eye
injection [because of spillover of label in the contralateral hemisphere into LGN layers receiving input from the uninjected eye
(LeVay et al., 1978)], the autoradiographic labeling pattern was compared in the ipsilateral hemisphere of nitroarginine-
(n = 9) and saline-treated (n = 4)
animals. Ocular dominance columns in layer 4 of nitroarginine-treated
animals were statistically indistinguishable from normal ones
(p > 0.05) in periodicity, width, and
peak-to-trough amplitude as shown quantitatively in the histograms of
Figure 5C.
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Figure 5.
Ocular dominance columns are normal after
nitroarginine treatment from P14 to P56. Transneuronal transport after
monocular injection of [3H]proline was used to
reveal the pattern of geniculocortical terminals (dark-field
autoradiographs). A, Sagittal section through the
primary visual cortex of a ferret injected with saline from P14 to P56.
B,Sagittal section through the visual cortex of a ferret
receiving nitroarginine from P14 to P56. A and
B are both from cortex ipsilateral to the eye injection,
where segregation into columns is clearest (LeVay et al., 1978).
Anterior is to the right, and dorsal is to the
top. Scale bar, 500 µm. C,
Quantification of ocular dominance column features (periodicity, column
width, and peak-to-trough amplitude) showing that all are
indistinguishable from normal features in the nitroarginine-treated
animals. See Materials and Methods for quantification details.
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The interpretation of this result is critically dependent on the
effectiveness of the NOS blockade. NOS produces NO and citrulline in
stoichiometric amounts from arginine (Bredt and Snyder, 1990). Because
the half-life of NO is in the order of seconds, it is impractical to
assay NO production directly. Thus, we assessed NOS activity after
nitroarginine treatment from P14 to P56 using a protocol that
measures the conversion of [3H]arginine to
[3H]citrulline (Huang et al., 1993). NOS activity
was measured in the visual cortex contralateral to the eye injection in
the same animals in which transneuronal autoradiography had been used
to examine the ocular dominance columns. Despite the presence of normal
ocular dominance columns, NOS activity in all cases (Fig. 6; n = 5) had been
reduced by >95% compared with that in saline or uninjected controls,
and in one case, NOS activity was almost undetectable from background
(see Materials and Methods). It is conceivable that the nitroarginine
blockade took effect too slowly to prevent the onset of segregation.
Thus, six additional ferrets (1 at P14, 2 at P22, 2 at P24, and 1 at
P37) were injected with nitroarginine and then assayed for NOS activity
24-48 hr later. NOS activity was in all cases reduced by 90% or
greater, with an average blockade of 95% (Fig. 6). Once again, one
animal had levels of NOS activity actually indistinguishable from
background. Consequently, we believe that the treatment produced a
rapid, lasting, and effective (up to 95-100%) blockade before and
during the entire period of ocular dominance formation. Because columns form normally, we conclude that, unless the extremely low residual NOS
activity is sufficient, NO is not required.
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Figure 6.
NOS catalytic activity in the visual cortex is
reduced to ~2% of control levels in ferrets treated with
nitroarginine from P14 to P56 (left column).
(Cortex is from the same animals whose opposite hemisphere was examined
for formation of ocular dominance columns.) NOS blockade took effect
within 24-48 hr (right column), because NOS activity
was reduced by >90% in six ferrets (1 at P14, 2 at P22, 2 at P24, and
1 at P37) given nitroarginine for 1-2 d. NOS activity was assessed as
described in Materials and Methods and is expressed as a percent of
that in age-matched control (saline-injected or unmanipulated) ferrets.
NA, Nitroarginine
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Formation of barrels in the somatosensory cortex of
NOS/ mice
Because it is still conceivable that small residual NOS activity
could be sufficient to permit the segregation of geniculocortical axons
into ocular dominance columns, we next examined the formation of
thalamocortical connections in the somatosensory cortex of NOS
knock-out mice (Huang et al., 1993, 1995). Because multiple isoforms of
NOS exist (Lowenstein et al., 1992; Bredt, 1995; Silvagno et al., 1996;
Xia and Bredt, 1996; Brenman et al., 1997), we obtained mice deficient
in either the neuronal (nNOS) (Huang et al., 1993) or the endothelial
(eNOS) (Huang et al., 1995) isoforms of NOS. The development of barrels
in the somatosensory cortex was examined by cytochrome oxidase
histochemistry (Chiaia et al., 1992). Figure 7 shows that nNOS (n = 6)
or eNOS (n = 2) homozygous knock-outs have barrels
present at P12 (see also Table 1), the earliest age that barrels can be
normally visualized by this histochemical method. The pattern of
barrels seems well defined and of normal size and shape in nNOS or eNOS
knock-out mice. Thus, neither the rate of development nor the pattern
of barrels is perturbed in these animals.
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Figure 7.
Barrels in the somatosensory cortex of nNOS or
eNOS knock-out mice are indistinguishable from those in normal
mice. Tangential sections through flattened somatosensory cortex were
stained with cytochrome oxidase histochemistry. A,
Wild-type (129 SV) mouse barrel field at P6. B, nNOS
knock-out mouse barrel field at P3. C, eNOS knock-out
mouse barrel field at P12. Scale bars, 250 µm.
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Barrels might have formed normally in neuronal or endothelial
knock-outs because alternate, intact isoforms might have compensated for the missing enzyme activity. At least six different splice variants
of nNOS are known, including a form of nNOS not disrupted in the nNOS
knock-out (Brenman et al., 1996, 1997); there are also inducible
isoforms of NOS found in macrophages (Lowenstein et al., 1992, 1993;
Reiling et al., 1994). Thus, we decided to block all NOS activity by
injecting nNOS or eNOS knock-out mice with nitroarginine rather than by
breeding double n/eNOS knock-out animals. nNOS (Fig.
8A) and eNOS (Fig.
8B) knock-out mice received daily injections (within
12 hr of birth) of nitroarginine for 1-7 d. NOS activity in whole
brain homogenates of unmanipulated nNOS knock-out mice at P7 was
reduced to ~3% of wild-type levels (Fig. 8A,
P7, 3.0 ± 0.2%; n = 2). This reduction agrees quite well with previous
reports in which a similar arginine-to-citrulline enzymatic assay
protocol was used to show that unmanipulated nNOS knock-outs have nNOS
catalytic activity ~0.2-7% of that in control brains (Huang et al.,
1993). Moreover, nNOS knock-outs given daily nitroarginine from P0 to
P7 had NOS activity reduced to ~1% (P0-P7 NA, 1.0 ± 1.4%; n = 4) of wild-type activity. After 7 d
of treatment, one nNOS/ mouse had undetectable levels of NOS
activity. Even as early as 24 hr after the beginning of nitroarginine
injections, NOS activity had fallen to ~1% of wild-type levels
(P0 + 24 hr NA, 0.8 ± 0.7%; n = 5).
One nitroarginine-treated nNOS knock-out mouse had NOS activity levels
indistinguishable from background after just 24 hr of blockade. Thus,
combining nitroarginine treatment with the nNOS homozygous animals
reduced NOS activity to ~1% of that in wild-type littermates.
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Figure 8.
NOS activity is greatly reduced in brain
homogenates from nNOS (A) or eNOS
(B) knock-out mice given daily nitroarginine
injections from the day of birth (P0). NOS activities
for age-matched uninjected and nitroarginine-injected NOS knock-out
animals are presented as pairs. As expected, NOS activity persists in
single knock-out animals, especially in eNOS/ mice. (nNOS/ mice
were examined at P7; eNOS/ mice were examined at
P2 or P7.) However, NOS activity is
greatly reduced in nitroarginine-injected nNOS or eNOS homozygous
knock-out mice. Nitroarginine blockade was rapid and took effect within
24 hr of birth. Activity is expressed as a percent of that in
age-matched, wild-type (129 SV) mice. P0-P7 NA, Mice
received nitroarginine daily from P0 to
P7; P0 or P1 + 24 hr NA, mice received
nitroarginine at P0 or P1 and were
assayed for NOS activity 24 hr later; P0 + 48 hr NA,
mice received nitroarginine daily from birth to P2 (48 hr total).
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Again, as expected from previous studies (Son et al., 1996), there is
significant residual NOS activity in eNOS homozygous knock-out mice
(Fig. 8B, P7, 67.0 ± 13.9% of wild
type; n = 3). After nitroarginine injections between P0
and P7, NOS activity is reduced to ~10% of that in wild-type mice
(P0-P7 NA, 9.8 ± 3.0%; n = 4).
Activity blockade in eNOS mice given nitroarginine is effective within
24 hr; eNOS mice examined 24 hr after the onset of nitroarginine
treatment had ~6% residual NOS activity (P0 or P1 + 24 hr
NA, 5.7 ± 0.8%; n = 3) compared with
~60% (P2, 58.4 ± 7.7%; n = 2)
residual activity in uninjected P2 eNOS/ animals. These
observations indicate that NOS activity is greatly reduced in nNOS/
and eNOS/ mice given nitroarginine from birth (in some cases to
levels indistinguishable from background) and that this blockade can be
fully effective within 24 hr of birth.
Formation and plasticity of barrels in NOS homozygous knock-out
mice treated with nitroarginine
Given the fact that nitroarginine treatment from birth can
rapidly reduce NOS activity to near baseline values, especially in the
nNOS/ mice, we next examined whether barrels can form in these
animals after nitroarginine administration. We designed an experiment
in which initial barrel formation and subsequent barrel plasticity
could be monitored simultaneously by ablating one row of whiskers at P0
in nNOS or eNOS knock-out mice given nitroarginine daily from birth to
P7 (Table 1). When a row of whiskers in the periphery is ablated in
wild-type mice at P0, animals develop a dramatically altered barrel
field pattern in the cortex in which the barrels corresponding to the
ablated row of whiskers shrink, fuse, and sometimes even disappear, and
the adjacent barrels enlarge (Fig.
9A) (Van der Loos and Woolsey, 1973; Weller and Johnson, 1975; Woolsey and Wann, 1976; Jeanmonod et
al., 1977). Whisker ablation can thus be used as an assay for the
ability of central projections to reorganize in response to changes at
the periphery. Within the somatosensory cortex of NOS/ mice treated
with nitroarginine, barrels formed normally and exhibited normal barrel
field plasticity in response to whisker ablation (Fig.
9B,C). As in wild-type animals (Fig. 9A), whisker
rows adjacent to the ablated whiskers (rows B,
D) expanded to occupy the space abandoned by the ablated
row. Because NOS catalytic activity in these nitroarginine-injected NOS
homozygous knock-out mice is, in some cases, indistinguishable from
background, NO is not likely to be required either for normal barrel
formation or for barrel field plasticity.
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Figure 9.
Initial barrel formation and barrel field
plasticity persist after nitroarginine treatment in nNOS and eNOS
homozygous knock-outs. C-row peripheral whisker
follicles were removed at P0. A, Barrel fields in a
wild-type mouse at P8. B, Barrel fields in an nNOS/
mouse given nitroarginine from P0 to P6. C, Barrel
fields in an eNOS/ mouse given nitroarginine from P0 to P12. In
each animal, arrows point to fused C-row
barrel representations. Barrels have formed normally in the
unmanipulated A and E rows (top
rows), whereas the neighboring B and D
rows have expanded to occupy cortical territory originally
designated for the shrunken and fused C-row barrels.
Cytochrome oxidase histochemistry of tangential sections through
flattened cortex is shown. Scale bars, 500 µm.
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DISCUSSION |
In this study, we have examined a possible role for NO in
the formation of patterned connections between thalamus and cortex in
the ferret visual system and in the somatosensory system of mice
carrying targeted mutations in NOS. Our study was motivated by many
previous observations (see introductory remarks) suggesting that NO can
be involved in a variety of aspects of activity-dependent synaptic
plasticity both during development and in adulthood. Nevertheless, the
results of our study demonstrate that the initial segregation of
geniculocortical axons into ocular dominance columns is not likely to
require NO. Moreover, the emergence of the adult pattern and plasticity
of thalamocortical connections in the somatosensory system of the mouse
is also unlikely to require NO. In both species, we were able to
decrease the activity of NOS rapidly (within 24 hr of treatment) by
>95% and sometimes by >99%, either by pharmacological blockade with
nitroarginine or by examining mice lacking eNOS or nNOS that were also
treated with nitroarginine. Unless the residual NOS activity is
sufficient, we are forced to conclude that NO is not essential for the
development of the adult pattern of thalamocortical connections in
either visual or somatosensory systems or for the plasticity induced by
whisker ablation in the somatosensory system.
Previous studies have also failed to reveal a role for NO in the
development of other sets of connections in the CNS. The development of
the cholinergic fiber patch system in the rat superior colliculus (Mize
et al., 1997) is normal after nitroarginine treatment. Retinal
projections to the LGN are normal in unmanipulated nNOS knock-out mice
or pharmacologically NOS-blocked hamsters (Frost et al., 1994), and
pharmacological blockade of NOS does not affect formation of
eye-specific layers in the ferret LGN (Cramer et al., 1995a). We also
observed normally segregated retinogeniculate projections to the LGN in
nitroarginine-treated eNOS or nNOS mice (data not shown). Consequently,
NO would seem to be involved in only a subset of the synaptic
remodeling that takes place in the developing brain as the adult
pattern of connectivity emerges.
NO in ocular dominance column formation
Two previous studies have also examined the requirement for NO in
experience-dependent modification of ocular dominance in cat visual
cortex. In these experiments, Ruthazer et al. (1996) and Reid et al.
(1996) tested ocular dominance column plasticity in cats after
nitroarginine infusion by minipump. Both studies assessed the effects
of NOS blockade on the physiologically measured shift in ocular
dominance produced by monocular visual deprivation. Both found normal
ocular dominance shifts and concluded that NO was not likely to be
involved in ocular dominance plasticity in cat visual cortex. Despite
these previous findings, we nevertheless thought it worthwhile to
examine the role of NO in the "initial" segregation of LGN axons
into the ocular dominance columns in layer 4 as opposed to ocular
dominance plasticity induced by altering the balance of visual activity
between the two eyes. We reasoned that the initial steps in pattern
formation might use mechanisms independent from those needed to respond
subsequently to alterations of sensory inputs. Second, we assessed the
effect of NO blockade anatomically by examining the pattern of
segregation of eye input within layer 4 of the visual cortex rather
than by examining the ocular dominance of cortical neurons
physiologically. Third, we began the NO blockade developmentally much
earlier than did these other studies, long before eye opening and 3 weeks before the onset of ocular dominance column formation in ferrets
(eye opening is at approximately P32). The previous studies began NOS
blockade in cats no earlier than P24 (Ruthazer et al., 1996) and as
late at P46 (Reid et al., 1996), after the onset of ocular dominance column formation in cats [approximately P21 (LeVay et al., 1978)] and
long after eye opening in kittens (eye opening is approximately P8).
Nevertheless, blockade of NOS beginning much earlier than in these
previous studies and well before eye opening has no discernible effect
on the formation of ocular dominance columns in layer 4 of ferret
visual cortex.
NOS expression in neurons of the subplate and deep cortical plate
is dynamically regulated
Results described here demonstrate that the density of
NADPH-diaphorase-positive neurons in ferret visual subplate is low just after birth but increases dramatically at P25 and P35, just before
ocular dominance columns become visible and precisely when thalamic
axons are first beginning to segregate. As segregation proceeds, the
number of NADPH-diaphorase-positive neurons in the visual cortex and
subplate then falls to reach adult levels at approximately the time
that column formation in layer 4 is complete. This correlation in the
rise in diaphorase expression in visual cortex with the onset of ocular
dominance column formation in layer 4 is in accord with observations
that NOS expression is developmentally regulated in other structures.
NOS expression also correlates with the period of refinement of retinal
projections in the chick optic tectum (Williams et al., 1994),
mammalian superior colliculus (Mize et al., 1996), and visual thalamus
(Cramer et al., 1995b; Guido et al., 1997) and during cerebellar
development (Schilling et al., 1994; Li et al., 1997). If NO is not
involved directly in the patterning of thalamocortical connectivity,
what, then, might the rather dynamic developmental regulation of NOS signify? One known function of NO is to regulate blood flow in capillaries (Goadsby et al., 1992; Lowenstein et al., 1994; Huang et
al., 1995). It is conceivable that at these particular times in
development, cortical blood flow somehow requires special control or
instruction from cortical neurons. In this context it is worth noting
that because we administered verapamil to counteract the hypertensive
effects of NOS inhibition, cortical vasculature appeared normal in the
treated ferrets. (Even in those initial animals that did not receive
verapamil, however, ocular dominance columns formed normally and
cortical vasculature appeared unaltered.) It is also possible that NO
might be involved in establishing aspects of cortical connectivity that
we did not examine, such as perhaps the intrinsic horizontal
connections that form earlier than the ocular dominance columns (Dalva
and Katz, 1994; Weliky and Katz, 1994).
NOS knock-out mice and neuronal pattern formation
To test the role of NO in the formation of patterned
thalamocortical connections in another primary sensory area, we also examined barrel field formation and barrel field plasticity in the
somatosensory cortex of mice carrying targeted mutations in nNOS or
eNOS. As in ferret visual cortex, so too in mouse neocortex, NADPH-diaphorase is developmentally regulated, including in the subplate (Derer and Derer, 1993) and barrel field (Mitrovic and Schachner, 1996). Within somatosensory cortex, diaphorase expression becomes evident in the neuropil in barrel hollows by P3 and in peaks at
P6 and disappears by the end of the second postnatal week (Mitrovic and
Schachner, 1996). Thus, in the mouse, diaphorase expression correlates
with the critical period for barrel field formation and plasticity
(P0-P7) and can even be used to visualize barrels (Franca and Volchan,
1995).
In the rodent barrel cortex, the mechanism of initial pattern formation
may not be activity-dependent (Chiaia et al., 1992; Henderson et al.,
1992), whereas barrel field plasticity most likely is (Schlaggar et
al., 1993) because application of the NMDA receptor blocker APV
significantly reduces reorganization of thalamocortical afferent input
in response to peripheral insult. Consequently, both barrel field
formation and plasticity were tested here. Even though we blocked the
activity of the remaining isoforms of NOS in nNOS or eNOS homozygous
knock-out mice by making daily nitroarginine injections, we found that
both barrel field formation and barrel field plasticity were normal in
whisker-ablated mice. It should be noted that the mice used for NOS
activity assays were not the same mice as those used for barrel field
assessment. Gathering both types of information from the identical
animal was technically impossible because of the tissue requirements of
the NOS enzymatic activity assay. Nevertheless, given the profound blockade and small variability in NOS activity in the brains that were
assayed for NOS activity after nitroarginine treatment (Fig. 8), we
think it unlikely that any of the animals examined for barrel formation
or plasticity escaped complete blockade. Thus again, as with the
formation of ocular dominance columns, we are forced to conclude that
NO is unlikely to play a role in the formation of the adult pattern of
connections in the mouse somatosensory system.
The preservation of neuronal patterning events in NOS-blocked mice is
also consistent with a preservation of some forms of hippocampal LTP in
double endothelial and neuronal knock-out mice (Son et al., 1996).
Although LTP is normal in single knock-out animals, LTP is reduced in
the stratum radiatum of double knock-out animals (O'Dell et al., 1994;
Son et al., 1996). However, LTP in the stratum oriens is preserved and
is resistant to NOS block. Our observations and those of Ruthazer et
al. (1996) and Reid et al. (1996) would imply that synaptic
strengthening in the cortex during development may also involve an
NO-independent form of LTP. Indeed, Friedlander and colleagues have
shown that synaptic potentiation in cat visual cortex is NO-dependent
in adults (Harsanyi and Friedlander, 1997a) but not during development
(Harsanyi and Friedlander, 1997b). Thus, the nervous system may use
differing strategies at different times and places to regulate and
maintain synaptic connectivity.
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FOOTNOTES |
Received May 13, 1998; revised Aug. 4, 1998; accepted Aug. 10, 1998.
This work was supported by the National Eye Institute Grant EY02858 to
C.J.S. and by a Howard Hughes Medical Institute (HHMI) predoctoral
fellowship to E.M.F. C.J.S. is an investigator of the HHMI. We
would like to thank Drs. Mark Fishman and Paul Huang for the eNOS and
nNOS knock-out mice. We are also indebted to Dr. David Bredt for the
gift of the NOS antibody, the NOS enzymatic activity assay protocol,
and additional nNOS knock-out animals and to Dr. Nick Chiaia for the
mouse barrel field/cytochrome oxidase protocol. Dr. Daniel Feldman
generously assisted with statistical analysis of the diaphorase data.
Holly Aaron performed the statistical analysis of ocular dominance
columns. We thank also Denise Escontrias for her assistance with
surgical procedures and animal husbandry.
Correspondence should be addressed to Dr. Carla J. Shatz, Department of
Molecular and Cell Biology and Howard Hughes Medical Institute, 221 Life Sciences Addition, University of California, Berkeley, CA 94720.
Dr. Finney's present address: Department of Psychology 0109, University of California-San Diego, La Jolla, CA 92093.
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Top
Abstract
Introduction
