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The Journal of Neuroscience, December 15, 1998, 18(24):10735-10748
Perinatal Gonadectomy Exerts Regionally Selective, Lateralized
Effects on the Density of Axons Immunoreactive for Tyrosine Hydroxylase
in the Cerebral Cortex of Adult Male Rats
M. F.
Kritzer
Department of Neurobiology and Behavior, State University of New
York at Stony Brook, Stony Brook, New York 11794-5230
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ABSTRACT |
The catecholamine innervation of the cerebral cortex is essential
for its normal operations and is implicated in cortical dysfunction in
mental illness. Previous studies in rats have shown that the
maturational tempo of these afferents is highly responsive to changes
in gonadal hormones. The present findings show that perinatal hormone
manipulation also has striking, region- and hemisphere-specific
consequences for cortical catecholamines in adulthood.
The effect of perinatal gonadectomy on catecholamines was examined in
representative sensory, motor, and association cortices of adult male
rats by combining hormone manipulation with immunocytochemistry for
tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis. Qualitative and quantitative comparison of
immunoreactivity in rats perinatally gonadectomized or sham-operated
revealed complex changes in gonadectomized subjects; in cingulate
cortex, TH immunoreactivity was strongly and bilaterally diminished, in
sensory and motor cortices, axon density was decreased in left
hemispheres, but was minimally affected on the right, and in a premotor
cortex, gonadectomy was without significant effect in either
hemisphere. Corresponding analyses in gonadectomized rats supplemented
with testosterone revealed a protective influence, albeit one in which TH immunoreactivity so showed regional and hemispheric variability in
responsiveness to hormone replacement. These complex patterns of TH
sensitivity suggest highly asymmetric hormone stimulation of cortical
catecholamines. Such discriminative action may contribute to sex
differences in the functional maturation and lateralization of the
cortex and may also have bearing on disorders such as dyslexia, which
show sexual dimorphisms, and in which functional laterality of the
cortex may be particularly at issue.
Key words:
dopamine; neocortex; estrogen; androgen; tyrosine
hydroxylase; cerebral hemisphere
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INTRODUCTION |
Gonadal hormones exert important
influence on developing cortical functions (Beatty, 1979 ; Bachevalier
and Hagger, 1991). Interestingly, however, hormones show a remarkable
degree of regional and hemispheric selectivity in their actions. In
infant monkeys (Clark and Goldman-Rakic, 1989; Bachevalier et al.,
1990) for example, testicular hormones speed maturation of working
memory skills computed within orbital prefrontal cortex, but retard
acquisition of habit formation: a functional province of inferotemporal
cortex. Sexual dimorphisms in developmental disorders such as dyslexia and schizophrenia also suggest roles for hormones in the lateralization of cortical functions (Gur, 1979; Geschwind and Behan, 1982; Tallal, 1991), a possibility consistent with hormone effects on tail posture in
rats, a function bearing homology to handedness in man (Rosen et al.,
1983), and in the sex-specific patterns of functional lateralization
that normally distinguish the cortices of men and women (McGlone,
1980).
Regional stimulation of cortical structures are likely substrates for
such patterned hormone influence over cortical functioning. Hormone
stimulation of cortical thickness, for example, differentially affects
the left and right hemispheres and further discriminates among
functionally specialized cytoarchitectonic subdivisions (for review,
see Diamond, 1991). Other examples of regional and hemispheric
anisotropies in structural endpoints include hormone stimulation of
cortical neuron dendrite structure (Kolb and Stewart, 1991), an
important component of cortical circuit structure. However, catecholamine afferents are also essential for cortical operations (Brozoski et al., 1979; Stam et al., 1989; Wilcott and Xuemei, 1990)
and are repeatedly implicated in the cortical dysfunction of disorders
that also display sexual dimorphisms (Davis et al., 1991). The present
study explored whether regionally selective stimulation of these
afferent systems may be an additional means by which gonadal steroids
differentially influence cortical function and/or functional laterality.
Previous studies in laboratory animals have shown that gonadal steroids
influence catecholamine innervation in juveniles (Stewart et al., 1991;
Stewart and Rajabi, 1994) and in adults (Battaner et al., 1987; Adler
et al., 1998). It is uncertain, however, whether changes in
gonadal hormones early in life have long-term consequences for cortical
neurochemistry. Furthermore, regional specificity in hormone
stimulation of cortical catecholamines has never been rigorously
examined. Resolving these issues has relevance both for the development
of normal cortical functions, some of which mature only after puberty
(Goldman-Rakic, 1987), and for sexually dimorphic cortical deficits in
developmental disorders such schizophrenia and Tourette's syndrome,
which can dramatically change, e.g., stabilize or worsen, at similar
life stages (DeLisi et al., 1989; Kurlan, 1992). Accordingly,
catecholamine innervation, identified immunohistochemically with
antibodies recognizing the synthetic enzyme tyrosine hydroxylase, was
qualitatively and quantitatively compared in representative sensory,
motor, and association cortices of adult male rats that had been
gonadectomized, gonadectomized and supplemented with testosterone, or
sham-operated at birth. The region-specific consequences of gonadectomy
that were revealed suggests that highly asymmetric hormone influences
normally stimulate cortical catecholamine innervation. Such
differential influence over these functionally critical afferents could
be relevant for the sex differences that characterize the maturation,
functional lateralization, and disease-related vulnerability of
specific subsets of cortical functions.
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MATERIALS AND METHODS |
Animals
Eighteen adult male Sprague Dawley rats were used. All
procedures involving animals have approval from the Institutional
Animal Care and Use Committee of State University of New York at Stony Brook and have been refined to minimize the use of animals and their
discomfort. All rats used were bred from dams that had resided in the
Stony Brook colony for at least 3 weeks before mating.
Each of the males used in this study was either gonadectomized or
sham-operated (see below) within 8 hr of birth. Gonadectomized animals
were divided into two groups: those injected daily with vehicle (sesame
oil, GDX-SO), and those injected daily with 2 µg/kg testosterone
proprionate (Sigma, St Louis, MO; adjusted weekly for body weight)
suspended in sesame oil (GDX-TP). Sham-operated control animals (CTRL)
were injected with sesame oil vehicle only. All animals were maintained
in litters that included one or two female siblings until weaning at
21 d of age. Thereafter, animals were housed in cages containing
two or three males, with food and water available ad
libitum under a constant 12 hr light/dark cycle.
Surgeries
Gonadectomies were performed on rat pups within 8 hr of birth.
Neonates were anesthetized using a combination of intraperitoneal injection of buprenorphin (0.0045 mg in sterile H2O) and
ether inhalation. Lidocaine solution was then applied to the abdomen, and a midline incision was made. The testes were removed bilaterally, and the incision was sutured. Sham operations consisted of
administration of anesthesia, a midline incision, and suturing of the skin.
Euthanasia
After an 8 week survival, rats were deeply anesthetized with a
mixture of ketamine (0.09 ml/100 gm) and xylazine (0.05 ml/100 gm)
injected intramuscularly. After deep reflexes had disappeared, animals
were transcardially perfused first with 50-100 ml of 0.1 M
phosphate buffer (PB) and then with a series of two fixative solutions:
4% paraformaldehyde in 0.1 M PB, pH 6.0 (flow rate 30 ml/min, duration 5 min), followed by 4% paraformaldehyde, in 0.1 M borate buffer, pH 9.5 (flow rate 35 ml/min, duration 20 min). Brains were then removed, blocked, and cryoprotected in 0.1 M PB containing 30% sucrose before rapid freezing in
powdered dry ice and storage at  80°C.
The androgen-sensitive medial, ventral, and lateral bulbocavernosus
muscles were dissected out immediately after euthanasia and weighed.
Mean muscle weights, whole body weights, and percent of whole body
weight represented in the dissected muscle mass are presented in Table
1 for the three animal groups.
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Table 1.
Mean weight (grams ± SD) of whole body,
bulbocavernosus muscles, and percent body weight represented by
bulbocavernosus muscle mass
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Immunocytochemistry
Blocked brains were secured to the stage of a freezing microtome
and serially sectioned at a thickness of 40 µm in the coronal plane.
Left hemispheres were marked with subcortically placed sectioning
artifacts made with a 16 gauge hypodermic needle. Sections were
immunoreacted according to standard procedures. Initially, sections
were rinsed in 0.1 M PB and then incubated in 1%
H2O2 for 30 min. After further rinses in PB,
sections were placed in 1% sodium borohydride in PB for an additional
30 min, and then rinsed in 50 mM Tris-buffered saline
(TBS). Sections were next placed in a blocking solution [50
mM TBS containing 10% normal swine serum (NSS)] for 2 hr,
and then incubated in one of two commercially available primary
antibodies (2-3 d, diluted in TBS containing 1% NSS, 4°C).
Anti-tyrosine hydroxylase antibodies (Chemicon, Temecula, CA; Eugene
Tech International, Ridgefield Park, NJ) were used at working dilutions
of 1:2000 and 1:500, respectively. After exposure to primary
antibodies, the tissue sections were rinsed in TBS, incubated in
biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA;
2 hr, room temperature, working dilution 1:100), rinsed again in TBS,
and then incubated in avidin-biotin-complexed horseradish peroxidase
(ABC, Vector; 2 hr, room temperature). Sections were then rinsed
thoroughly in Tris buffer, pH 7.6, and reacted using 0.07%
3,3'-diaminobenzidine (DAB) as chromagen.
Silver-gold intensification
The DAB reaction product was intensified according to methods of
Kitt et al. (1988). Briefly, DAB-reacted, slide-mounted sections were
incubated in 1% silver nitrate (pH 7.0, 45 min, 55°C), briefly rinsed in running distilled H2O, and then incubated in
0.1% gold chloride (10 min, room temperature). Sections were then
rinsed again in distilled H20, fixed in 5% sodium
thiosulfate (10 min, room temperature), counterstained with 2% cresyl
violet, and placed under coverslips.
Control experiments
The immunocytochemical labeling procedures above were performed
on representative sections from hormonally intact and manipulated animals with omission of primary antiserum or secondary antibodies. Treated control sections were silver-gold-intensified side-by-side with normally immunoreacted slides.
Qualitative evaluation
Detailed examination of the laminar distribution, the course,
approximate density, and the morphology of TH-immunoreactive axons was
performed in representative sections throughout the rostrocaudal extent
of dorsal anterior cingulate cortex (area Cg1), primary motor cortex
(area AgL), primary somatosensory cortex (area Par1), and premotor
cortex (area AgM) in each of the three groups of animals (Donoghue and
Wise, 1982; Zilles, 1990; Fig. 1). Two
antibodies directed against TH were used in initial studies. At least
two series of sections, immunoreacted on different days, was obtained
from each animal used in this study.
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Figure 1.
Schematic diagram showing a lateral view of the
rat cerebral cortex (top) and a representative cross
section (bottom) taken at the level of approximately the
anteroposterior midpoint of the septal nucleus. In the coronal section,
the locations of area Cg1 and Par 1 of Zilles (1990) and of areas AgM
and AgL (Donoghue and Wise, 1982) are shown. Qualitative analyses were
performed at representative rostrocaudal levels within each of these
four areas. Quantitative measures were derived from sections matching
the cross section depicted. In each section used for quantitative
study, camera lucida drawings of tyrosine hydroxylase-immunoreactive
fibers were obtained from layer II/III and from layer V of all four
areas. In total, these drawings subtended virtually the entire span of
areas Cg1, AgM, and AgL present within the section; the stippled
area offset with dashed lines within area Par1
illustrates the approximate location of the subportion of this region
from which quantitative measures were obtained. olf,
Olfactory bulb; cc, corpus callosum; cd,
caudate nucleus.
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Quantitative evaluation
A superior signal and signal-to-noise ratio was consistently
obtained using the antibody from Chemicon, in the
paraformaldehyde-fixed tissue. This prompted the decision to use this
antibody exclusively for quantitative studies. Because subtle gradients
in the density of catecholamine innervation in the rat cerebral cortex
have been identified along major axes (anteroposterior, mediolateral;
Van Eden et al., 1987), the decision was made to focus on a single anteroposterior level for quantitative evaluation. The level selected, lying at roughly the midseptal nucleus (Fig. 1), was chosen for its
optimal representation of the four regions examined. For these studies,
two to three tissue sections from this level were cut for all animals
on the same day and immunoreacted as a group. Slides were coded before
quantitative analyses, and all analyses were performed by a single
observer (M.F.K.).
Quantitative analyses of fiber density and orientation were performed
on camera lucida drawings made of immunoreactive fibers visualized
under bright-field illumination using a 63× oil immersion objective.
In each of the four areas examined, layers II/II and V were analyzed in
left and right hemifields. Cresyl violet counterstaining was used to
identify cortical regions and layers. Section thickness within the
region to be evaluated was measured beforehand by roll-focusing from
section surface-to-surface using the calibrated fine-focus of the
microscope (Zeiss Axioskop).
Innervation density. Individual camera lucida drawings subtended
widths of ~100-300 µm measured parallel to the cortical surface, a
height dictated by the thickness of the layer, and a depth spanning the
thickness of the tissue section. Three nonoverlapping drawings were
obtained from each cortical layer, from each area of interest, from
each animal. In total, the drawings from areas Cg1, AgM, and AgL
subtended nearly the entire cytoarchitectonic field present in tissue
sections at the level selected for analysis (Fig. 1). In area Par1, a
specific subregion was defined for analysis by (1) an approximate 3 o'clock position within the hemisphere and (2) its radial alignment
with a local maxima in the width and cell-packing density of layer IV.
Other than these constraints, no attempts were made to preselect the
location of drawings.
Camera lucida drawings were digitized as black and white photograph
quality images (Hewlett Packard 4C Scanner, Deskscan 2.0 software; Palo
Alto, CA), and imported into NIH Image 5.8 software. Fiber density
estimates were obtained from these imported images by first
skelatinizing images to replace lines of varying width with ones of
uniform thickness and then measuring mean pixel density (NIH Image
5.8). This automated measure is directly proportional to
two-dimensional fiber length (Kritzer and Kohama, 1998).
Fiber orientation/trajectory. Fiber orientation
relative to the cut surfaces of the tissue sections was quantitatively
evaluated in the same sections used to analyze fiber density. For these analyses, separate sets of camera lucida drawings were made tracing portions of fiber segments that were in focus within single focal planes. At least 100 fiber segments were traced from each area, from
each hemisphere, from each of two layers, in each individual animal.
The two dimensional lengths of these segments were then measured using
calibrated stereological software (MacStereology, Runturly
Microsystems). Populations of length measurements were statistically
compared among animal groups (Kolmogorov-Smirnoff, nonparametric
analysis). Mean lengths and the percentage of fibers <15 µm in
length in individual animals were also compared.
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RESULTS |
Efficacy of hormone treatments
The surgical procedures and injection schedules used
in this study are identical to methods previously used to deplete and maintain physiological levels of gonadal hormones in rats (Sodersten, 1984). Their effectiveness was confirmed in this study by weighing the
androgen-sensitive bulbocavernosus muscles, which are sensitive index
to circulating testicular hormones (Wainman and Shipounoff, 1941;
Collins et al., 1992). As anticipated (Collins et al., 1992), bulbocavernosus muscle mass in GDX-SO animals dropped to ~17% of
controls (Table 1). Although total body weights were also lower than
normal (~82% of normal), when expressed as a percent of total body
mass, an 80% was still evident in bulbocavernosus mass in the
gonadectomized group (Table 1). In contrast, total body weight,
bulbocavernosus weight, and percent body weight represented in
bulbocavernosus muscle was 98, 105, and 106% of normal, respectively, in gonadectomized animals supplemented with testosterone proprionate (GDX-TP, Table 1). Statistical evaluation (ANOVA followed by Dunnett
two-tailed post hoc comparisons; p < 0.05) revealed that all of the weights in GDX-SO animals and none of
the weights in GDX-TP animals were significantly different from controls.
Specificity of immunostaining
The morphology, distribution, and the density of TH-immunoreactive
axons was examined at representative rostrocaudal levels in the dorsal
anterior cingulate cortex (area Cg1): an association area, the primary
motor cortex (area AgL), a premotor cortical region (area AgM), and the
primary somatosensory cortex (area Par1; Donoghue and Wise, 1982;
Zilles, 1990; Fig. 1). Two commercially available anti-TH antisera were
used, and both produced patterns of immunostaining in control animals
(Fig. 2; see Figs. 6, 8, 10) that
were similar to previous immunocytochemical (Berger et al., 1985a; Van
Eden et al., 1987; Papadapoulos et al., 1989) and radioautographic
(Descarries et al., 1987) studies of cortical TH- and
dopamine-containing axons in rats. Immunoreactive axons also displayed
morphologies and orientations anticipated in previous studies (Berger
et al., 1985a; Febvret et al., 1991; see Fig. 4A,D). These qualitative parallels
support the specificity of immunostaining in this study for
TH-containing fibers. This specificity was further supported by weak,
nonpatterned staining in control experiments in which primary or
secondary antibodies were omitted. Although it has been argued that TH
immunoreactivity has some selectivity for dopamine axons in rodent
cortex (Berger et al., 1985b), this question of selectivity was not
addressed in this study. Thus, although dopaminergic axons were
undoubtedly in the majority in cingulate cortex (Lewis et al., 1979),
the relative contributions of dopaminergic and noradrenergic axons to
TH immunoreactivity was uncertain, particularly in lateral cortices
where these inputs are more similarly dense (Levitt and Moore, 1978;
Morrison et al., 1978).
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Figure 2.
Representative camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers within the dorsal anterior cingulate
cortex (area Cg1). The left (L)
and right (R) hemispheres of a control
(CTRL) animal, an animal gonadectomized on the day of
birth (GDX-SO), and an animal
gonadectomized at birth and supplemented with testosterone proprionate
(GDX-TP) are shown. The approximate
borders of cortical layers are marked by the roman numerals
appearing on the left. The immunostaining
represented was obtained using a commercially available antibody
purchased from Chemicon. Comparison across the animal groups
represented illustrates the stark loss of axon density in both
hemifields of the cingulate cortex of gonadectomized animals and the
appearance of more normal levels of innervation in gonadectomized
animals that received injections of testosterone proprionate. In all
animal groups, seemingly normal, layer-specific patterns of tyrosine
hydroxylase-immunoreactive fiber orientation are preserved.
wm, White matter.
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TH immunocytochemistry in gonadectomized animals
Areas Cg1, AgM, AgL, and Par 1 each displayed expected patterns of
dense to moderate TH immunoreactivity in control animals. In
age-matched rats gonadectomized as neonates (GDX-SO), however, visual
inspection alone revealed markedly abnormal patterns of catecholamine
innervation. It was also evident that there were regional differences
in the effects of gonadectomy on cortical catecholamine innervation
among sensory, motor, and association areas. Gonadectomy either
profoundly and symmetrically diminished TH innervation in the left and
right hemispheres, markedly decreased immunoreactivity in one cerebral
hemifield but not the other, or was without obvious effect in either
hemisphere. These three outcomes, described separately below, occurred
side-by-side in adjacent cytoarchitectonic cortical fields and were
discernible in sections immunoreacted with each of the two anti-TH
antisera used; supporting quantitative data were obtained using the
antibody from Chemicon exclusively. A final subsection describes TH
immunoreactivity in corresponding regions of neonatally gonadectomized
animals that were supplemented with testosterone proprionate
(GDX-TP).
Hemispherically symmetric depletion of TH immunoreactivity: dorsal
anterior cingulate cortex
Gonadectomy-induced decreases in catecholamine innervation were
most striking in the dorsal anterior cingulate cortex (area Cg1; Fig.
2). In control animals, this region was densely labeled, with prominent
bands of immunoreactivity standing out over layers Ia and II (Berger et
al., 1985a, 1988; Febvret et al., 1991). In GDX-SO animals, however, TH
immunoreactivity was clearly diminished in the left and right
hemifields (Fig. 2). The dense tangle of immunoreactive fibers that
normally mark the supragranular layers (see Fig. 4A),
for example, was replaced in gonadectomized animals by a loose mesh of
TH-immunoreactive processes (see Fig. 4B). Innervation was also diminished in the infragranular layers, where immunoreactivity dropped from the moderate levels in intact animals to
sparse innervation in GDX-SO rats (Fig. 2). Quantitative evaluation of
TH-immunoreactive axon density in representative supragranular (layer
II) and infragranular (layer V) layers more precisely defined these
effects (Fig. 3). First, in control
animals baseline immunoreactivity was approximately twofold greater in
supragranular compared with infragranular layers. Corresponding
measures in GDX-SO rats revealed that effects of hormone manipulation
were proportionate in these two strata. In the left hemisphere, mean TH
axon density was 28.8 and 27.7% of normal in the supragranular and
infragranular layers, respectively, and on the right, mean TH axon
density dropped to 32.8% of normal in layer II and 38.2% of normal in
layer V (Fig. 3).
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Figure 3.
Scatterplots of mean pixel density measures
derived from camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of
the left and right anterior dorsal cingulate hemifields of control
(CTRL), gonadectomized
(GDX-SO), and gonadectomized,
testosterone proprionate-supplemented
(GDX-TP) animals. The points plotted
(triangles) correspond to raw data points that are
inclusive of all measures obtained from each of the six individual
animals comprising each of the three experimental groups.
Horizontal bars mark the group means of the pixel
density measures. The decreases in axon density in GDX-SO animals are
proportionately similar across layers and hemispheres, and values in
both are significantly less then controls. Treatment of gonadectomized
animals with testosterone proprionate yields statistically normal
innervation density in layer II, but produces axon density levels that
fall between and are significantly different from GDX-SO and control
values in layer V.
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The reliability of density estimates was verified in an ANOVA with a
repeated measures design. This analysis (which included GDX-TP
animals), identified significant main effects of treatment (F(2,15) = 31.36; p < 0.0001)
and layer (F(1,15) = 110.47; p < 0.0001), the latter consistent with near the twofold differences in
axon density in the supragranular and infragranular layers. However,
individual animals were excluded (p > 1.0) as
significant sources of variance in the data. Effects of hemisphere
(F(1,15) = 0.24; p < 0.88) and
hemisphere-by-treatment interactions (F(2,15) = 2.29; p < 0.13) were also nonsignificant, reinforcing
the symmetry of the response of axons to hormone manipulation.
Subsequent post hoc comparisons (Dunnett two-tailed;
p < 0.05), confirmed that differences in axon density
between GDX-SO and control animals were significant in both layers and
hemispheres evaluated (Fig. 3).
The quantitative effects of neonatal gonadectomy took place in the
absence of obvious morphological disturbance. Although clearly reduced
in number, the TH-immunopositive axons of GDX-SO animals displayed to
expected morphologies (Fig.
4B). Furthermore, TH-positive axons displayed normal patterns of layer-specific orientations, e.g., a predominance of horizontally running fibers in
layer I and more randomly oriented, short axon segments in layers II/VI
(Figs. 2, 4A,B). This second point
of similarity was relevant for quantitative changes in axon density
because the units for comparison, two-dimensional fiber length, are
sensitive to fiber orientation. For example, an increase in the
percentage of fibers coursing at steep angles with respect to the
tissue section surface, which would be most attenuated in
two-dimensional drawings, could lead to underestimation of fiber
content. Although the qualitative similarities noted in fiber
orientation among GDX-SO and control rats argue against such a confound
(Figs. 2, 3A-C) for further confirmation, fiber
orientation was quantitatively evaluated (see Materials and Methods).
Although these analyses indicated that axon arbors sampled were not
statistically invariant (Kolmogorov-Smirnoff nonparametric comparison;
p < 0.01), mean fiber lengths were similar among
animal groups (Table 2). Equally important, the percentages of arbor sampled that corresponded to
shortest axon segments (<15 mm) were overlapping for control and
GDX-SO animals (Fig. 5). These findings
argue against method-introduced foreshortening of axon measures in
GDX-SO rats.
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Figure 4.
Representative bright-field photomicrographs
illustrating the morphology and orientation of axons immunoreactive for
tyrosine hydroxylase in layer II of the anterior dorsal cingulate
cortex (area Cg1, A-C)
and layer III of the primary motor cortex (area AgL,
D-F) in controls
(CTRL), gonadectomized rats
(GDX-SO), and rats gonadectomized and
supplemented with testosterone proprionate
(GDX-TP). All sections were immunoreacted
for tyrosine hydroxylase (antibody from Chemicon) and are
counterstained for Nissl substance with 1% cresyl violet. None of the
cellular staining depicted in any of the panels corresponds to
tyrosine-immunopositive somata. In both regions represented, the same
features of axon morphology and orientation that characterize control
animals are also found in the immunoreactivity of gonadectomized and
gonadectomized, testosterone-supplemented animals; distinctive features
such as the short, tortuous axons segments in layer II of the cingulate
cortex (A-C), and the long radial fibers
that are prominent in layer III of the primary motor cortex
(D-F) are preserved across
experimental groups. Clear decreases in the number of immunoreactive
axon segments, however, readily distinguish the GDX-SO animal from
control and GDX-TP cases. Scale bars:
A-C, 100 µm; E,
F, 500 µm.
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Table 2.
Fiber orientation was quantitatively evaluated by measuring
two-dimensional fiber lengths within a single focal plane
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Figure 5.
Scatterplots of the percentages of axon arbor
measured at a single focal plane that are <15 µm in two-dimensional
length in the supragranular and infragranular layers of cingulate
(Cg1), premotor (AgM), primary
motor (AgL), and primary somatosensory
(Par1) cortex. For each plot, points represent raw data
inclusive of all measures obtained from each of the six control animals
(large black squares), the six gonadectomized animals
(small open squares), and the six gonadectomized animals
supplemented with testosterone proprionate (small open
circles) analyzed in this study. The relative high percentage
of short fiber segments in layer II of the cingulate cortex is
consistent with its signature pattern of innervation by short, highly
branched, randomly oriented axon segments. In all other regions and
layers, shortest axon segments, which could include segments oriented
steeply with respect to the plane of the tissue section and, thus, most
significantly foreshortened in two-dimensional drawings, comprised
~10% or less of the total axon arbor sampled. Data collected for all
three animal groups is largely overlapping. Area AgM represents an
exception in which a disproportionate number of GDX-SO animals had more
short axon segments than GDX-TP or control animals.
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Unilateral depletion of TH immunoreactivity: primary motor and
somatosensory cortices
The distribution and density of TH-immunoreactive axons in the
primary motor (AgL) and somatosensory (Par1) cortices of control rats
were similar to previous descriptions of cortical catecholamine innervation (Berger et al., 1985a; Febvret et al., 1991; Fig. 6; see Fig. 8). Both regions showed a
moderate innervation, characterized by horizontally running processes
in layer I, long, radially oriented fibers in the supragranular layers,
and shorter, more randomly oriented axons in infragranular layers. In
GDX-SO rats, this innervation was markedly reduced, but in a
hemisphere-specific manner. Whereas axon density in the left
hemispheres of GDX-SO animals was well below normal in the right motor
and sensory hemifields, gross inspection suggested no obvious decrement
in immunoreactivity (Fig. 6; see Fig. 8). However, quantitative
evaluation revealed small but consistent decreases in the density of TH
immunoreactivity; in the right area AgL, axon density measures were
consistently ~80% of normal (Fig.
7),
and in area Par1, axon density in layers II/III and V represented 63 and 67% of normal, respectively (Figs. 8,
9). These declines were small compared
with effects in the left hemisphere, where in the motor cortex, for
example, TH innervation dropped on average to 38.8 and 30.9% of normal
in the supragranular and infragranular layers, respectively (Fig. 7).
Similarly, in the left somatosensory area, mean axon density in GDX-SO
rats was 22.6 and 30.5% of normal in corresponding strata (Fig.
9). Thus, in sensory and motor areas, the effects of gonadectomy within a given hemisphere were proportionate for supragranular and
infragranular layers but were remarkably different across
hemispheres.
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Figure 6.
Representative camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers within the primary motor cortex (area
AgL). The left (L) and right
(R) hemispheres of a control
(CTRL) animal, an animal gonadectomized on the day of
birth (GDX-SO), and an animal
gonadectomized at birth and supplemented with testosterone proprionate
(GDX-TP) are shown. The approximate
borders of cortical layers are marked by the roman
numerals appearing on the left. The
immunostaining represented was obtained using a commercially available
antibody purchased from Chemicon. Comparison across the animal groups
represented illustrates the pronounced reduction in axon density in the
left primary motor hemifield in gonadectomized animals, and the
appearance of more normal levels of innervation in the right hemifield
of gonadectomized animals, and in both hemispheres of gonadectomized
animals that received injections of testosterone proprionate. In all
animal groups, seemingly normal, layer-specific patterns of tyrosine
hydroxylase-immunoreactive fiber orientation are preserved in both
hemispheres. wm, White matter.
|
|
View larger version (14K):
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[in a new window]
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Figure 7.
Scatterplots of mean pixel density measures
derived from camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of
the left and right primary motor hemifields of control
(CTRL), gonadectomized
(GDX-SO), and gonadectomized,
testosterone proprionate-supplemented
(GDX-TP) animals. The points plotted
(triangles) correspond to raw data points that are
inclusive of all measures obtained from each of six individual animals
comprising the three experimental groups. Horizontal
bars mark numerical group means of the pixel density measures.
The large decreases in axon density in left motor hemifields of GDX-SO
animals are proportionately similar across layers and are significantly
less than controls in both layers II/III and V. More modest, albeit
statistically significant, decreases in density mark the supragranular
and infragranular layers of the right motor hemifield of GDX-SO
animals. Treatment of gonadectomized animals with testosterone
proprionate yields statistically normal innervation density in layers
II/III and V of the left primary motor cortex but has seemingly no
effect on TH axon density in these layers on the right.
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|
View larger version (86K):
[in this window]
[in a new window]
|
Figure 8.
Representative camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers within the primary somatosensory
cortex (Par1). The left (L) and
right (R) hemispheres of a control
(CTRL) animal, an animal gonadectomized on the day of
birth (GDX-SO), and an animal
gonadectomized at birth and supplemented with testosterone proprionate
(GDX-TP) are shown. The approximate
borders of cortical layers are marked by the roman
numerals appearing on the left. The
immunostaining represented was obtained using a commercially available
antibody purchased from Chemicon. Comparison across the animal groups
represented illustrates a striking decrease in tyrosine
hydroxylase-immunoreactive axon density in the left somatosensory
hemifield in gonadectomized animals and normal appearing innervation in
the right hemifield of gonadectomized animals, and in both hemispheres
of gonadectomized animals that received testosterone proprionate
treatment. In all animal groups, qualitatively normal, layer-specific
patterns of tyrosine hydroxylase-immunoreactive fiber orientation are
preserved. wm, White matter.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
Figure 9.
Scatterplots of mean pixel density measures
obtained from camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of
the left and right primary somatosensory hemifields of control
(CTRL), gonadectomized
(GDX-SO), and gonadectomized,
testosterone proprionate-supplemented
(GDX-TP) animals. The points plotted
(triangles) correspond to raw data points that are
inclusive of all measures obtained from each of six individual animals
from each of the three experimental groups. Horizontal
bars mark the numerical group means of the pixel density
measures. The large decreases in axon density in left somatosensory
cortex in GDX-SO animals are proportionately similar in supragranular
and infragranular layers, with both values being significantly
different from controls. More modest, yet statistically significant,
decreases in density also mark the supragranular and infragranular
layers of the right somatosensory cortex in GDX-SO animals. Treatment
of gonadectomized animals with testosterone proprionate yields
statistically normal innervation density in layers II/III and V of the
left primary somatosensory cortex but has minimal effect on TH axon
density in corresponding layers of the right somatosensory field.
|
|
Axon density measures were statistically analyzed in ANOVAs; each
identified main factors of treatment (motor:
F(2,15) = 26.62, p < 0.0001;
sensory: F(2,15) = 46.04, p < 0.0001), and hemisphere (motor: F(1,15) = 98.44, p < 0.0001; sensory: F(1,15) = 16.89, p < 0.0001) as significant contributors to
variance in the data. Significant treatment-by-hemisphere interactions
(motor: F(2,15) = 96.16, p < 0.0001; sensory: F(2,15) = 16.07, p < 0.0001) were also identified, consonant with the
difference in the degree to which immunoreactivity was affected in left
and right hemifields. Variation among individual animals, however, was
not a significant source (p > 1.0) of variance
in density measures. Subsequent post hoc comparisons
(Dunnett two-tailed; p < 0.05) confirmed that large
gonadectomy-induced axon density reductions on the left, as well as the
more modest decreases in the right sensory and motor hemifields were
significant in upper and lower cortical layers (Figs. 7, 9).
Despite quantitative differences, axon morphology and orientation were
preserved in GDX-SO rats (Figs. 4E, 6, 8). For
example, the supragranular layers of the left and right sensory and
motor hemifields were innervated mainly by long, radially oriented, moderately varicose axons (Figs.
4D,E, 6, 8). The similarities in
orientation in particular suggested that gonadectomy-induced shifts in
fiber orientation were unlikely to account for group differences in
axon density. Quantitative analyses of fiber orientation reinforced
this conclusion; axon arbor was either statistically indistinguishable
from controls (Table 2), or frequency analysis of axon segment length
revealed extensive overlap in the percentage of fibers corresponding to
shortest axon segments among gonadectomized and control animals (Fig.
5). Finally, although one individual GDX-SO animal had some of the
highest percentages of fibers measuring 15 µm or less, in no cases
did shortest segments account for more than ~10% of total axon
arbor. Thus, bias introduced in measurements as a function of fiber
orientation could make at most a fractional contribution to the group
differences in TH innervation observed.
Minimal effects on TH innervation: premotor cortex
In contrast to the other areas examined, TH immunoreactivity in
the premotor cortex (area AgM) appeared normal in both hemispheres of
GDX-SO animals (Fig. 10). Quantitative
analyses also revealed extensive overlap in measures of axon density
obtained in GDX-SO rats and controls (Fig.
11). However, a small but consistent
decrease in TH innervation in GDX-SO animals relative to controls was
revealed in which axon density varied between 84 (layer V, right
hemisphere) and 88% (layer II/III, right hemisphere) of normal (Fig.
11). An initial ANOVA revealed a weak main effect of treatment
(F(2,15) = 3.937; p < 0.0422)
but unambiguously excluded individual animals (p > 1.0) and hemispheres (F(2,15) = 2.09;
p < 0.1687) as significant sources of variance in the
data. Subsequent post hoc comparisons revealed that
axon density differences between control and GDX-SO rats were not
significant (Dunnett two-tailed, p < 0.05; Fig. 11).
View larger version (107K):
[in this window]
[in a new window]
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Figure 10.
Representative camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers within the premotor cortex (area
AgM). The left (L) and
right (R) hemispheres of a control
(CTRL) animal, an animal gonadectomized on the day of
birth (GDX-SO), and an animal
gonadectomized at birth and supplemented with testosterone proprionate
(GDX-TP) are shown. The approximate
borders of cortical layers are marked by the roman
numerals appearing on the left. The
immunostaining represented was obtained using a commercially available
antibody purchased from Chemicon. Comparison across the animal groups
represented illustrates the insensitivity of tyrosine
hydroxylase-immunoreactive axons to gonadectomy, with and without
testosterone proprionate supplementation, in both hemifields. In all
animal groups normal, layer-specific patterns of tyrosine
hydroxylase-immunoreactive fiber density, distribution, and orientation
are preserved. wm, White matter.
|
|
View larger version (15K):
[in this window]
[in a new window]
|
Figure 11.
Scatterplots of mean pixel density measures
derived from camera lucida drawings of tyrosine
hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of
the left and right premotor hemifields of control
(CTRL), gonadectomized
(GDX-SO), and gonadectomized,
testosterone proprionate-supplemented
(GDX-TP) animals. The points plotted
(triangles) correspond to raw data points that are
inclusive of all measures obtained from each of six individual animals
comprising the three experimental groups. Horizontal
bars mark the numerical group means of the pixel density
measures. Overall, measures of axon density in control, GDX-SO, and
GDX-TP animals are overlapping, and none of the differences in group
mean values among the groups are statistically significant.
|
|
Quantitative analysis of fiber orientation revealed layers in which
fiber populations in GDX-SO rats were statistically indistinguishable from controls (Table 2). For the others, however, frequency analysis indicated that individual GDX-SO animals had among the highest proportions of short axon segments (Fig. 5). Although tempered by fact
that shortest axon segments accounted for a small percentage of axon
arbor overall, even a potentially modest method-related foreshortening
of axon segments in GDX-SO cases should be kept in mind when
considering the small, nonsignificant differences in axon density
measures in these animals.
TH immunocytochemistry in gonadectomized animals after
hormone replacement
To determine whether gonadectomy-induced changes in cortical TH
innervation were sensitive to hormone replacement, corresponding analyses of immunoreactivity were performed in gonadectomized animals
that were supplemented with testosterone proprionate. In most cases,
hormone replacement provided obvious protective effects against
gonadectomy-induced decreases in TH immunoreactivity (Figs. 2,
4C,F, 6, 8, 10). Quantitative analyses, however,
suggested subtle regional differences in the effectiveness of hormone
replacement. In area Cg1, for example, axon densities in GDX-TP animals
closely approximated control levels in layer II (99 and 93% of normal in the left and right hemifields, respectively), but in layer V axon
density only reached ~65% of normal (Fig. 3). These intermediate values were significantly different (ANOVA, Dunnett two-tailed post hoc comparison; p < 0.05) from
both control and GDX-SO densities (Fig. 3).
In areas Par1 and AgL, differences in the effects of TP were also
observed. In left hemifields, TH innervation in GDX-TP rats was between
78% of normal (layer II/III of area Par 1) and 93% of normal (layer V
of area AgL) and statistically indistinguishable from controls (ANOVA,
Dunnett two-tailed post hoc comparison; p < 0.05; Figs. 7, 9). On the right, however, axon
densities in both sensory and motor cortices were overlapping with and
statistically indistinguishable from measures obtained in GDX-SO
animals (Figs. 7, 9). Testosterone treatment also showed minimal effect
on TH immunoreactivity in area AgM, where axon densities of control, GDX-SO, and GDX-TP animals were all overlapping and statistically indistinguishable (ANOVA, Dunnett two-tailed post
hoc comparison; p < 0.05; Fig.
11).
| |
DISCUSSION |
Sex differences and/or hormone modifiability in the maturation and
lateralization of cortical functions (McGlone, 1980; Rosen et al.,
1983; Clark and Goldman-Rakic, 1989; Bachevalier et al., 1990) and in
the incidence of specific constellations of cortical deficits in
sexually dimorphic developmental disorders, e.g., schizophrenia and
dyslexia (Geschwind and Behan, 1982; Seeman and Lang, 1991; Tallal,
1991) indicate functionally relevant, selective patterns of hormone
influence within the maturing cerebrum. This study revealed highly
differentiated effects of perinatal gonadectomy on cortical
catecholamine innervation, an innervation important for both cortical
function and dysfunction (Stam et al., 1989; Wilcott and Xuemei, 1990;
Davis et al., 1991). That these effects were attenuated by treatment
with testosterone proprionate suggested that changes in hormone levels
were primarily responsible for the complex series of changes observed.
This causal implication was strengthened by the fact that technical
factors that could yield variability in axon quantification were
minimized. For example, consistent preservation of TH antigens was
maximized by holding parameters of fixation constant, consistency in
immunostaining was optimized by reacting tissue across subjects in
parallel, and regional sampling strategies were strictly defined to
ensure comparison of corresponding cortical regions, subregions, and layers. Steps were also taken to control for error introduced in the
method of axon quantification. First, section thickness was measured to
verify that sampled areas were of comparable breadth. Furthermore,
quantitative analysis of fiber orientation was performed to determine
whether compression of information in two-dimensional drawings along
the z-axis biased measures across groups; these studies
revealed little evidence for treatment-induced shifts in the percentage
of fibers that would be most foreshortened in two-dimensional
representations (Fig. 5). These elements of experimental design and
analysis thus support the reliability of quantitative evaluation of TH
immunoreactivity and reinforce conclusions that catecholamine axons in
the adult rat cerebrum are profoundly sensitive to changes induced in
the perinatal hormone environment. Comparative analyses further
revealed that across cortical regions, catecholamine axons are
differentially sensitive to hormone stimulation.
Of the four regions examined, TH innervation was most affected in the
cingulate cortex, where axon density was strikingly and bilaterally
diminished. In primary somatosensory and motor cortices, TH
immunoreactivity was significantly depleted in the left hemisphere but
was only modestly reduced on the right, the latter effects only being
brought to light in quantitative analyses of axon density. In the
premotor area, however, catecholamine afferents appeared qualitatively
and quantitatively normal in gonadectomized animals, thus providing
these studies with a fortuitous internal control.
There was also regional variation in the effectiveness of hormone
replacement. Overall, those regions that were most affected by
gonadectomy tended to be those most responsive to hormone replacement. In primary somatosensory and motor cortices, for example, testosterone treatment yielded values of axon density that were indistinguishable from normal in left hemifields but that were largely overlapping with
those of gonadectomized animals on the right. This variable effectiveness may be related to differences between experimentally maintained and native hormone environments; whereas testosterone replacement yielded physiological levels of gonadal steroids, the
sustained pulse delivered differed markedly from the stereotyped peaks
and valleys in circulating hormone levels that normally bathe the
postnatal male brain (Resko et al., 1968; Weisz and Ward, 1980).
Accordingly, although hormone exposure alone seemed a sufficient
stimulus in many regions, in others, e.g., layer V of cingulate cortex,
the right sensory and motor hemifields, precise temporal patterning of
hormone exposure may also be part of an adequate stimulus for
catecholamine innervation. It is also possible, however, that some of
the decline in TH innervation observed in GDX-SO animals occurred
secondarily to removal of the testes but independently of gonadal
steroid influence.
Possible mechanisms
Mechanisms governing the complex patterns of hormone stimulation
observed in this study were not directly explored. However, some
inferences can be drawn from the patterns of hormone sensitivity observed. For example, regional hormone sensitivity does not appear readily explained by factors such as the maturational state, functional specialization, or axon density of target cortices. Whereas
maturational gradients for most features of cortical development occur
along mediolateral or rostrocaudal planes (Smart, 1984), there were no
systematic changes in hormone sensitivity along either of these major
axes. Functional specialization was also not consistently related to
hormone sensitivity. The functionally disparate sensory and motor
cortices, for example, showed similar responses to gonadectomy, whereas
two more closely related regions, motor and premotor cortex, responded
very differently to hormone manipulation. Finally, gonadectomy-induced changes in axon density were not simply more or less detectable in
densely or lightly innervated regions, because the two most heavily
innervated cortices examined, areas Cg1 and AgM, were most and least
affected by gonadectomy, respectively.
The distribution of intracellular hormone receptors in the midbrain and
brainstem, however, suggests links between genomic hormone action
levied at the cells of origin of cortical catecholamine afferents and
their regional patterns of hormone sensitivity. Not only do subsets of
catecholamine neurons in the substantia nigra, ventral tegmentum, and
locus coeruleus contain intracellular estrogen (ER- and ER- )
and/or androgen receptors (AR) (Heritage et al., 1981; Kritzer, 1997a;
Shughrue et al., 1997) but, among midbrain neurons, ER-, ER-, and
AR are most abundant in the ventral tegmental area (Kritzer, 1997a;
Shughrue et al., 1997), the major source of catecholamine input to the
highly hormone-sensitive cingulate cortex. The distribution of cortical
ER-, on the other hand, may provide more intriguing parallels with
the lateralized consequences of hormone manipulation. Thus, whereas
levels of ER- peak during the first week of postnatal life (Shughrue
et al., 1990; Miranda and Toran-Allerand, 1992), in lateral cortex (including sensory and motor areas) this transient peak coincides with
a near twofold right over left difference in the number of intracellular binding sites (Sandhu et al., 1986). Interestingly, a
decline in receptor levels and concomitant disappearance of hemispheric
differences in maximal binding during the first two weeks of life
(Sandhu et al., 1986) subtends the timeline emerging in preliminary
studies in rats for the effectiveness of perinatal gonadectomy to
stimulate lateralized deficits in TH innervation in motor cortex
(Kritzer, 1997b). However, in addition to the possibilities above,
cortical androgen receptors, which are highly lateralized in the cortex
of fetal monkeys (Scholl and Kim, 1990), cortical ER-, which seems
to remain elevated within the cerebrum throughout the lifespan
(Shughrue et al., 1997), as well as nongenomic and even transneuronal
routes of hormone stimulation must also be considered among candidate
endocrine signaling pathways in the hormone sensitivity of cortical
catecholamine innervation.
Comparison with previous studies
Few studies have examined hormone effects on cortical
catecholamine systems. To date the only previous study pairing
perinatal gonadectomy with neurotransmitter analysis in adulthood
revealed no changes in dopamine, noradrenalin, or serotonin levels in
HPLC analyses of cortical homogenates (Siddiqui and Shah, 1997). These findings seem incongruous with changes reported here in
immunoreactivity for catecholamine-synthesizing enzymes. However, among
methodological differences separating these studies, one that may be
relevant to these seeming discrepancies is that cortical homogenates
comprised virtually the entire cortical mantle (Siddiqui and Shah,
1997). In light of present findings suggesting regional hormone
sensitivity, analyses conducted on pooled cortical regions may be an
inappropriate comparison.
Previous studies have also examined hormone effects on the maturation
of catecholamine innervation in juvenile animals (Stewart et al., 1991;
Stewart and Rajabi, 1994) and on transmitter levels or enzyme
immunoreactivity in adults (DuPont et al., 1981; Battaner et al., 1987;
Adler et al., 1998; Kritzer and Kohama, 1998). Interestingly, gonadectomy in adult males produces an increase either in transmitter levels (Battaner et al., 1987) or immunoreactivity for related synthetic enzymes (Adler et al., 1998), an effect opposite to the present findings. These findings, coupled with preliminary investigations in this laboratory of the early postnatal period (Kritzer, 1997b) suggest that the outcomes of gonadectomy on cortical catecholamines can be markedly different at different stages of the
lifespan. This, in turn, suggests that features such as critical periods and differential activational and organizational effects that
are tenets of hormone influence in reproductive and neuroendocrine brain centers (for review, see Breedlove and Arnold, 1983) may also
apply to gonadal steroid stimulation of cortical catecholamine innervation. It is possible that the highly discriminating actions of
gonadal steroids identified in this study on cortical catecholamine innervation hold relevance not only for normal cortical maturation and
lateralization, but also for disorders such as schizophrenia and
dyslexia that show sexual dimorphisms, and in which functional laterality of the cortex is particularly vulnerable. Accordingly, it
may be especially pressing to establish details of the timing as well
as the relative permanence or plasticity of the effects of gonadal
hormone stimulation on cortical catecholamines in future studies.
| |
FOOTNOTES |
Received July 21, 1998; revised Sept. 28, 1998; accepted Oct. 6, 1998.
This work was supported by a FIRST Award, R29NS35422 to M.F.K. I
thank Ms. Charu Venkatesan and Mr. Alex Adler for expert technical
assistance with animal surgeries and histology.
Correspondence should be addressed to Mary F. Kritzer, Department of
Neurobiology and Behavior, State University of New York at Stony Brook,
Stony Brook, NY 11794-5230.
| |
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Abstract
Introduction
