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Activity-dependent regulation of calcium-binding proteins in the developing rat olfactory bulb

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THE JOURNAL OF COMPARATIVE NEUROLOGY 387:12–26 (1997)
Activity-Dependent Regulation of
Calcium-Binding Proteins in the
Developing Rat Olfactory Bulb
BENJAMIN D. PHILPOT, JAE H. LIM, AND PETER C. BRUNJES*
University of Virginia, Department of Psychology, Charlottesville, Virginia 22903
ABSTRACT
Intracellular calcium, important in a variety of second messenger cascades, is regulated
in part by calcium-binding proteins such as calretinin, parvalbumin, and calbindin. These
proteins are highly concentrated in the rat main olfactory bulb and are localized in distinct
neuronal populations. In the present study, postnatal expression was characterized immunohistochemically in normal rats and in rats with functional olfactory deprivation caused by
unilateral naris closure, a manipulation that attenuates electrical activity in the bulb. Bulbs
were examined from rats that had undergone naris closure or sham surgery on either
postnatal day 1 (P1) or P30 and were allowed varying subsequent survival times.
Each of the calcium-binding proteins showed both distinct patterns of early expression
and differential susceptibility to olfactory restriction. For example, at P10, the densest
immunoreactivity was observed for calretinin, a protein whose expression was the least
affected by naris closure. After occlusion from P1-P30, there was a 30% reduction in the
density of calbindin-immunoreactive profiles in the glomerular layer, and parvalbuminimmunoreactive profiles were reduced by 64% in the external plexiform layer. Unlike many
other changes induced by deprivation, the effects of olfactory restriction on calbindin and
parvalbumin expression were not age dependent: naris closure from P30–P60 caused similar
substantial decreases in calbindin and parvalbumin immunoreactivities.
These data demonstrate that the expression of calbindin and parvalbumin in rat bulb is
regulated, in part, by afferent activity that is associated with full sensory experiences. The
reductions of these calcium-binding proteins following olfactory deprivation are likely to be
commensurate with altered control of intracellular calcium. J. Comp. Neurol. 387:12–26,
1997. r 1997 Wiley-Liss, Inc.
Indexing terms: calbindin; calretinin; parvalbumin; naris closure; sensory deprivation
Intracellular free calcium affects a variety of cellular
responses through its actions as a second messenger. For
example, calcium-mediated signaling cascades activate
immediate early genes (Sheng et al., 1990), affects protein
expression (Ghosh and Greenberg, 1995), and alters neuronal survival (Choi, 1988; Collins et al., 1991). Calciumbinding proteins are one means to control intracellular
free calcium levels, either as regulators of calciummediated responses and/or as local buffers of cytoplasmic
calcium (Baimbridge et al., 1992; Clapham, 1995). Moreover, increasing evidence suggests that calcium-binding
proteins dramatically shape neural activity. For example,
supraoptic neurons switch from phasic to continuous firing
modes when levels of the calcium-binding protein calbindinD28k are experimentally manipulated (Li et al., 1995). As
calcium-binding proteins are potent regulators of diverse
cellular events, their characterization will lend insights
into cellular function.
r 1997 WILEY-LISS, INC.
Immunocytochemistry for calcium-binding proteins has
been used successfully to identify several specific neuronal
populations (e.g., Celio, 1990; Celio and Heizmann, 1991;
Rogers and Résibois, 1992). Interestingly, striking immunolabeling has been observed for these proteins in the rat
olfactory bulb (Résibois and Rogers, 1992; Lipp et al.,
1993) where calretinin, calbindin, and parvalbumin, members of the EF-hand calcium-binding protein family, occupy distinct and non-overlapping neuronal populations.
Grant sponsor: National Institutes of Health; Grant number: DC-00338;
Grant sponsor: National Research Service Award; Grant number: MH11068.
*Correspondence to: Dr. Peter C. Brunjes, 102 Gilmer Hall, Department
of Psychology, University of Virginia, Charlottesville, VA 22903. E-mail:
brunjes@virginia.edu
Received 11 November 1996; Revised 12 May 1997; Accepted 12 May
1997
CALCIUM-BINDING PROTEINS IN DEVELOPING OLFACTORY BULB
Calbindin- and calretinin-immunoreactive neurons are
most dense in the glomerular region (Baimbridge and
Miller, 1982; Halász et al., 1985; Celio, 1990; Jacobowitz
and Winsky, 1991; Briñón et al., 1992; Rogers, 1992;
Rogers and Résibois, 1992; Kosaka et al., 1995), whereas
most parvalbumin-immunoreactive neurons are shortaxon cells restricted to the external plexiform layer (Kosaka et al., 1987b; Celio, 1990; Kosaka et al., 1994a,b;
Toida et al., 1994, 1996).
As a primary sensory relay, the olfactory bulb is a
propitious area to examine activity-dependent regulation
of calcium-binding proteins. Activity is easily manipulated
by occluding an external naris and thus blocking airflow
through one side of the nasal cavity. This manipulation has
a number of dramatic effects. For example, when naris
closure is performed on the day after birth (P1) and pups
are examined at P30, there is a profound (25%) reduction
in the volume of the ipsilateral bulb (Brunjes, 1994) that is
at least partially due to decreased granule and periglomerular cell numbers, perhaps via apoptosis (Frazier and
Brunjes, 1988; Frazier-Cierpial and Brunjes, 1989;
Najbauer and Leon, 1995). Shortly after naris closure,
experimental bulbs exhibit decreased protein synthesis
(Korol and Brunjes, 1990), metabolism (Cullinan and
Brunjes, 1987) and expression of the protein product of the
immediate early gene, c-fos (Klintsova et al., 1995), all of
which have been shown to be calcium-mediated events in
other regions of the brain.
Naris occlusion also has rapid physiological consequences (Iwahara et al., 1973; Gray and Skinner, 1988;
Philpot et al., 1997). Normal bulb function is characterized
by high-frequency, rhythmic activity that is correlated
with the respiratory cycle. The repetitive bursting activity
is similar to the well-documented phenomenon of longterm potentiation in the hippocampus and visual system.
After naris blockade, low-frequency tonic activity is observed, a situation more similar to conditions necessary to
induce long-term depression (Philpot et al., 1997). Interestingly, long-term depression/potentiation are both calciumdependent mechanisms (Bear and Malenka, 1994), suggesting that a shift in free calcium levels may account for the
widespread effects of naris closure. The high-frequency
phasic activity seen in the normal condition may activate
N-methyl-D-aspartate (NMDA) type receptors and cause
rapid calcium fluctuations, whereas the low-frequency
tonic activity seen after naris closure may cause a slight,
yet sustained, increase in calcium levels. Owing to the
types of anatomical, biochemical, and physiological consequences observed after naris closure, changes in calcium
regulation may contribute to the cascade of alterations
associated with odor deprivation (Brunjes, 1994).
The present study uses immunocytochemistry to examine developmental changes in the distribution of calretinin, calbindin, and parvalbumin in bulbs from normal and
naris-occluded rats in order to characterize further their
normal developmental expression and to examine if their
expression is altered by the activity-dependent changes
that follow naris closure.
MATERIALS AND METHODS
Subjects
Offspring of Long-Evans hooded rats purchased from the
Charles River Breeding Laboratories (Wilmington, MD)
were used. Rats were housed in polypropylene cages (40 3
13
25 3 16 cm) and given food and water ad libitum. The
colony room was maintained on a 16/8 hour light/dark
cycle. Litters were culled to 10 on Postnatal Day 1 (P1, the
day after the day of birth). P1 subjects were coldanesthetized and received either unilateral occlusion of
the right external naris via cautery or a sham manipulation consisting of cautery of the dorsal surface of the nose
(Meisami, 1976). Pups were then warmed and returned to
their mothers. Naris-occluded and sham-operated rat pups
were examined at P10, P20, and P30. In addition, a second
group received sham or naris cautery on P30 and were
killed on P60. At least four animals were examined for
each age, experimental condition, and antibody treatment.
The experimental procedures used in these studies were
performed under animal care protocols approved by the
University of Virginia’s Institutional Animal Care and Use
Committee.
Tissue preparation and immunostaining
Subjects were given an overdose of barbiturates and
perfused intracardially with a 0.1 M phosphate-buffered
saline (PBS), pH 7.4, followed by Bouin’s fixative. Brains
were carefully dissected and post-fixed overnight in the
Bouin’s fixative. Brains were then dehydrated for 24 hours
in 70% ethyl alcohol, run through an ascending alcohol
series, cleared in toluene, and embedded in paraffin.
Twelve micrometer sections, cut in either the coronal or
horizontal plane, were mounted onto gelatin-coated slides.
Sections were de-paraffinized for 5 minutes in xylene,
rehydrated through a descending alcohol series, rinsed
three times with 0.1 M phosphate buffer (PB), pH 7.4, and
placed for 15 minutes in 3% H202 to quench endogenous
peroxidases. After three washes in PB, sections were
incubated for 1 hour in 10% normal goat serum (NGS) in
PB to prevent non-specific staining. Sections were then
incubated with a mouse monoclonal antibody to calbindin
(SWant, Bellinzona, Switzerland; 1:1,000), a rabbit polyclonal to calretinin (Chemicon, Temecula, CA; 1:5,000), or
a rabbit polyclonal to parvalbumin (SWant, Bellinzona,
Switzerland; 1:2,500). In addition, a monoclonal antibody
to tyrosine hydroxylase (Incstar, Stillwater, MN; 1:5,000)
was used as a basis for staining comparisons. All antibodies and the avidin-biotin complex (ABC) solutions were
diluted in PB with 10% NGS to reduce nonspecific binding.
Slides were incubated for 24-72 hours at 4°C, washed three
times with PB, and transferred into a solution containing
biotinylated secondary antibodies (Dako, Carpinteria, CA,
swine anti-rabbit or goat anti-mouse; 1:100) for 1 hour.
Following three rinses in PB, slides were placed into ABC
solution (Vector, Burlingame, CA; 100 µl each of solutions
A and B in 50 ml solution) for 90 minutes. After three
washes in PB, slides were treated for 15–25 minutes with
diaminobenzidine (DAB) and 0.02% H202 in Tris-buffered
saline or PB, pH 7.4. Slides were then dehydrated, cleared,
and coverslipped with DPX (BDH, Ltd., Poole, U.K.).
Control experiments, consisting of the same protocol but
omitting exposure to the primary antibodies, consistently
resulted in the absence of staining. The specificities of the
antibodies used in the present study have been previously
described (Kägi et al., 1987; Rogers, 1987; Celio et al.,
1990). Because the current study makes comparisons
across animals of different ages and experimental conditions, several precautions were taken to ensure that
histological artifacts were minimized. For example, tissue
from several groups was processed in parallel whenever
14
B.D. PHILPOT ET AL.
possible. Furthermore, sections from each animal were
subjected to several immunostaining techniques to determine which were consistent and maximal. For example,
antibody dilutions and exposure durations were optimized
for the most robust staining. Tissue samples from each
subject were pre-incubated in various concentrations of
Triton X-100 to increase antibody penetration, as well as
lower concentrations of normal goat serum, to prevent
over-blocking, in order to demonstrate that antigenicity
was maximal. Examinations of the effects of naris occlusion were facilitated by the fact that left/right comparisons
could be made within single sections.
Quantification
Both left and right bulbs in control and naris-occluded
subjects were analyzed for the density and size of immunoreactive cell profiles. Segments of 600 µm were selected for
measurement from the medial wall. The entire width of
the granule cell, external plexiform, and/or glomerular
layers was examined by using a microcomputer-based
image analysis system (MCID software, Imaging Research, St. Catharines, Ontario, Canada). Only layers with
substantial labeling were quantified. Images were consistently thresholded for each pair of bulbs such that all areas
containing DAB reactions were black and the background
white, and visual inspection verified that all labeled
profiles were detected. Threshold levels were individually
set for each pair of bulbs to ensure that all immunoreactive
profiles were identified, including cells of the lowest optical
density, and that thresholding and light levels were unchanged in pairs of left and right bulbs. After thresholding,
the number of stained profiles in the test area was
determined by setting a minimum number of congruent
supra-threshold pixels that would be considered a labeled
profile. The image analysis system was calibrated to
ensure that the smallest and/or lightest profiles were
included in counts while excluding background staining.
At least three pairs of sections from at least four subjects/
age group were measured. A repeated measures analysis of
variance (ANOVA) was performed to test if the dependent
variable (profile density) varied as a function of age group
and/or side (right versus left bulbs within subjects tested
between age groups). The data were analyzed for main
effects and for interactions. A post hoc test, Tukey’s HSD,
was used to explain univariate variations. The significance
level was placed at 0.05. For presentation purposes, the
difference in the number of immunoreactive profiles per
unit test area (profile density) for each section was determined and was calculated using the following formula:
100 3 (profile density right bulb 2 profile density left
bulb) / profile density left bulb.
Profile areas were measured for each of the calciumbinding proteins, and staining intensity was measured in
calbindin-immunoreactive profiles because there was a
large qualitative difference. Profile areas and optical densities were analyzed using semi-quantitative methods by
taking measurements from 15 somata per section (using at
least four sections per rat and at least four rats per group).
Stained profiles from each section were randomly chosen
for measurement; a grid was placed over the mid-portion of
the medial wall of each bulb and the first 15 cells encountered from the center of the grid were measured. Over 120
cells were measured for each group. Statistical analyses on
profile areas were performed as described above. For
optical densities, ANOVA’s were performed on the percent-
age difference because relative differences are a more
accurate determinant of change for this semi-quantitative
measure. The percentage difference in optical densities
was calculated with the following formula: 100 3 (optical
density right bulb 2 optical density left bulb) / optical
density left bulb.
Tyrosine hydroxylase staining was assessed for profile
densities and staining intensities in rats occluded from
P30-P60. The well-documented changes in tyrosine hydroxylase-immunoreactivity following occlusion were used
as a basis for comparison. Comparisons between the
number or optical density of profiles in right and left bulbs
were made using two-tailed t-tests.
Estimation of profile counts was chosen over more exact
stereological techniques as 1) large differences were apparent from inspection of the tissue, and therefore determination of subtle differences was deemed unnecessary, and 2)
‘‘actual’’ number was not deemed as interesting as simple
ratios of left/right differences.
Calbindin and tyrosine hydroxylase double
labeling
Subjects were given an overdose of barbiturates and
perfused intracardially with PBS, followed by 4% paraformaldehyde in PBS with 10% sucrose. Brains were then
carefully dissected and cryoprotected overnight with 20–
30% sucrose in 0.1 M PBS. The following day, 20 µm
sections were cut on a cryostat in the coronal plane.
Sections were thaw-mounted onto gelatin-coated slides,
placed in an evacuated chamber, and stored overnight at
4°C. Slides were then rinsed 3–4 times with PB and
incubated with a mouse monoclonal calbindin antibody
(SWant, Bellinzona, Switzerland; 1:1,000) and a rabbit
polyclonal tyrosine hydroxylase antibody (Chemicon, Temecula, CA; 1:5,000). All antibodies and the ABC solutions
were diluted in PB with 10% NGS to reduce nonspecific
binding. Slides were incubated for 24–72 hours at 4°C,
washed three times with PB, and prepared for one of three
double labeling procedures. 1) Sections were treated with
an anti-mouse secondary antibody for 1 hour at room
temperature and processed for ABC-DAB visualization, as
described above, to produce a brown reaction product for
calbindin-immunoreactivity. After four rinses in PB, sections were placed in anti-rabbit secondary antibody for 1
hour and prepared for ABC-DAB visualization with nickel
intensification, to produce a black reaction product for
tyrosine hydroxylase-immunoreactivity. Slides were then
dehydrated, cleared and coverslipped with DPX. 2) Slides
were first labeled for tyrosine hydroxylase-immunoreactivity with the ABC-DAB technique, and then fluorescently
labeled for calbindin-immunoreactivity by a 2 hour incubation with an 7-amino-3-[[[[succinimidyl]oxy]carbonyl]
methyl]-4-methylcoumarin-6-sulfonic acid (AMCA)-conjugated goat anti-mouse IgG (Jackson, West Grove, PA;
1:100) in PB. After four rinses in PB, slides were coverslipped with glycerol gelatin (Sigma, St. Louis, MO). 3)
Double immunofluorescence was performed by using a 2
hour incubation with AMCA-conjugated goat anti-mouse
IgG in PB, followed by a 2 hour incubation with Texas-Redconjugated goat anti-rabbit IgG (Jackson, West Grove, PA;
1:100). After four rinses in PB, slides were coverslipped
with glycerol gelatin.
Illustrations were produced with Adobe Photoshop software from images scanned from negatives. Images were
CALCIUM-BINDING PROTEINS IN DEVELOPING OLFACTORY BULB
15
Fig. 1. Photomicrographs of coronal sections depicting calbindinimmunoreactivity (ir) in the developing main olfactory bulb. At all
ages, calbindin-immunoreactivity is predominately localized to periglomerular cells in the glomerular layer (GLM), while immunoreactivity
is rare in the external plexiform layer (EPL) and absent in the mitral
cell layer (MCL). A: Postnatal Day (P) 20, calbindin-immunoreactivity
is dense in some somatic profiles in the glomerular layer. Note the
limited labeling of neuronal processes. B: P30, the number of labeled
profiles has increased, and processes are more intensely labeled. C:
P60, there are numerous, intensely labeled profiles and processes. D:
In experimental bulbs from a P60 rat following 30 days of naris
closure, there is a dramatic reduction in both the number and
intensity of stained profiles. In addition, there is only limited immunoreactivity in fibers. Scale bar 5 100 µm.
adjusted only to reflect accurately the appearance of
sections through a light microscope.
tive profiles were present in the juxtaglomerular region
(Fig. 1a). Somata were often darkly stained, while dendritic processes exhibited light labeling. Deep to the glomerular layer, labeled profiles were less frequently observed. Both the staining intensity and density of
immunoreactive profiles in all layers were less than that
seen in adult (P60) subjects (Fig. 1c). By P30, there were
numerous immunoreactive profiles along the glomerular
periphery, and a greater extent of neuronal arborizations
were immunopositive (Fig. 1b). Dark calbindin-immunoreactive profiles were numerous in the glomerular layer by
P60 (Fig. 1c). These cells often had intense labeling of
processes arborizing within glomeruli. The position, morphology, and small soma-size (5–8 µm diameter) of labeled
neurons in the glomerular layer suggested these cells were
periglomerular neurons, while stained profiles in subglomerular regions had the appearance of short-axon cells
due to their bipolar shape and slightly larger somata.
Labeled short-axon cells were most frequently observed
just deep to the mitral cell layer (Fig. 2). Occasional,
large-sized (,16 µm length) cells were observed in the
subependymal zone at all ages (Fig. 2). The flattened
morphology of these profiles resembled that of typical
migrating cells.
RESULTS
Calbindin, calretinin, and parvalbumin antisera appeared to label separate populations of cells in the bulb.
Furthermore, immunocytochemistry suggested that the
expression of calcium-binding proteins varies during development and is differentially affected by olfactory restriction. No differences in staining for any antigen were
observed between right and left bulbs in control subjects,
suggesting that there is no laterality in normal development. Likewise, staining intensity and patterning was
similar in control bulbs of unilaterally naris-occluded rats
and bulbs from sham-operated rats. The normal developmental patterns and the effects of naris closure on bulb
expression of each employed antibody will be addressed
separately below.
Calbindin
Normal development. Faint cytoplasmic calbindinimmunoreactivity was present in some cells in the glomerular layer by P10, although there was little labeling of
neuronal processes. By P20, many calbindin-immunoreac-
16
Fig. 2. Photomicrograph of calbindin-immunoreactivity in a horizontal section of a P30 olfactory bulb. Although most immunopositive
cells are localized in the glomerular layer (GLM), there is a sparse
distribution of calbindin-immunoreactive profiles in subglomerular
layers. Calbindin-immunoreactive profiles are rare in the external
plexiform layer (EPL) and granule cell layer (GCL). Immunopositive
cells are found occasionally in the internal plexiform layer (IPL), just
deep to the mitral cell layer (MCL). Large calbindin-immunoreactive
profiles are present in the subependymal zone. The black arrow points
to an immunoreactive cell in the IPL. The inset depicts a highmagnification view of an immunopositive cell in the subependymal
zone (white arrow). Scale bar 5 100 µm for the main figure and 38 µm
for the inset.
At all ages studied, there was no evidence for laterality
of calbindin expression in control animals (Fig. 3a). That
is, no significant differences were observed in the density
of immunopositive profiles between right and left bulbs
(P.0.05 for main effect of right/left bulb laterality). The
number of calbindin-immunoreactive profiles per square
millimeter increased from P20 to P60 [see Fig. 3b, control
bulbs; F(2,11)522.7, P , 0.001, main effect of age]. Furthermore, cell size was similar between right and left bulbs at
each age (P.0.05). The number of calbindin and tyrosine
hydroxylase-immunoreactive profiles in the glomerular
layer was similar in P60 control bulbs (Fig. 3b).
Effects of naris closure. Early unilateral naris closure dramatically reduced both the staining intensity and
B.D. PHILPOT ET AL.
Fig. 3. A: Mean percent difference (6 S.E.M.) in calbindinimmunoreactive profiles/mm2 between left and right bulbs from rats
receiving unilateral naris closure (nosx) or sham-surgery (sham) from
P1–P20, P1–P30, and P30–P60. The difference in calbindin-immunoreactivity is compared to the reductions seen in tyrosine hydroxylase
(TH) immunoreactivity in animals occluded from P30–P60. The number of calbindin-immunoreactive profiles is similar between left and
right bulbs of animals receiving sham-surgery. However, there is a
reduction in experimental bulbs in the density of calbindin-immunoreactivity. The reduction in stained profiles is not as dramatic as the
decrease in the number of tyrosine hydroxylase-immunoreactive
profiles. B: A comparison of the mean number (6 S.E.M.) of calbindin
or tyrosine hydroxylase immunopositive profiles/mm2 in experimental
and control bulbs from rats receiving unilateral naris closure. The
difference in calbindin-immunoreactivity is compared to the significant reductions seen in tyrosine hydroxylase (TH) in animals occluded
from P30–P60 (P,0.001 by t-test). In control bulbs, there is a
developmental increase in the density of calbindin-immunoreactive
profiles (ANOVA demonstrates a significant main effect of age,
P,0.001). The number of calbindin-immunoreactive profiles is significantly reduced in experimental bulbs (main effect of right/left bulb
laterality, P,0.005 by ANOVA). At P60, the number of calbindin and
tyrosine hydroxylase immunoreactive cells is similar in control bulbs.
the density of calbindin-immunoreactive profiles within
the glomerular layer of experimental bulbs as compared to
contralateral controls (Fig. 1d). After occlusion from P1P20, P1-P30, or P30-P60, the density of calbindinimmunoreactive profiles was reduced in experimental
bulbs as compared to contralateral controls [Fig. 3;
F(1,11)517.9, P,0.005, main effect of right/left bulb laterality]. However, there was no interaction between bulb
side and age (P.0.05). That is, the relative reductions in
profile density in experimental bulbs did not vary as a
function of age group.
CALCIUM-BINDING PROTEINS IN DEVELOPING OLFACTORY BULB
Fig. 4. High-magnification photomicrograph depicting calbindinimmunoreactivity in control (left) and experimental (right) bulbs from
a P60 rat after naris closure from P30–P60. Left: Dark somatic
labeling and extensive process labeling is apparent in the P60 control
Unlike many of the effects of naris closure in the
olfactory bulb (Brunjes, 1994), the decrease in calbindinimmunoreactivity was not age-dependent. Animals occluded from P1–P30 had a 30% reduction in calbindinimmunoreactive profiles in experimental bulbs as compared
to contralateral controls, while animals occluded from
P30–P60 had a 25% reduction (Fig. 3a). However, the
marked reduction in calbindin-immunoreactive profiles
was not as dramatic as the well-documented decrease
(,87%) in tyrosine hydroxylase-immunoreactive profile
density [Fig. 3; t(9)5245.7, P,0.001 for TH-immunoreactivity in right/left bulbs of occluded animals].
In addition to the decrease in the number of immunopositive somata, labeled profiles appeared smaller in size with
less intense staining on both their cell body and processes
(Fig. 4). Thus, these parameters were quantified.
In sham-operated rats, staining intensities of somata
were similar between right and left bulbs (Fig. 5). However, staining intensity was attenuated in experimental
bulbs as compared to contralateral controls in animals
unilaterally occluded from P1–P20, P1–P30, as well as
P30–P60 [Fig. 5; F(1,19)529.5, P,0.001 for main effect of
right/left bulb laterality]. The difference in optical densities increased with age [F(2,19)54.1, P,0.05]. The change
in calbindin immunostaining intensity between right and
left bulbs in animals occluded from P1–P30 was similar to
the significant decrease in staining intensity of tyrosine
hydroxylase-immunoreactive cells [Fig. 5; t(9)59.9, P,0.01
for the reduction in optical density of TH-immunoreactive
profiles].
In addition to the reduced intensity of calbindin immunoreactivity, the average profile area of labeled cells changed
after occlusion [Fig. 6a; F(1,13)514.3, P,0.005]. There
was a significant interaction of laterality after naris
closure with age group [F(2,13)54.5, P,0.05]. Post hoc
analyses revealed that profile areas in experimental bulbs
were significantly reduced in animals occluded from P30-60
(P,0.05). There was a uniform shift in the area of immunoreactive profiles. The histogram in Figure 6 demonstrates
that the distribution of profile areas was roughly Gaussian
for both control and experimental bulbs.
As alterations in staining of cells in the juxtaglomerular
region are similar to the well-documented changes in
17
bulb. Right: The number, staining intensity, and area of immunopositive cells is dramatically reduced in experimental bulbs. Note the
limited dendritic labeling. Scale bar 5 50 µm.
Fig. 5. Mean percent difference (6 S.E.M.) in optical densities of
calbindin and tyrosine hydroxylase (TH)-immunoreactive profiles in
naris-occluded (nosx) and sham-operated (sham) rats. In shamoperated rats, there is no difference between bulbs in staining
intensities in P20, P30, or P60 rats. However, calbindin staining
intensity is significantly reduced in animals occluded from P1–P20,
P1–P30, and P30–P60 (P,0.001, main effect of right/left bulb laterality by ANOVA). There is a marked reduction in the staining intensity
of tyrosine hydroxylase-immunoreactive profiles in rats occluded from
P30–P60 (P,0.005 by t-test).
tyrosine hydroxylase-immunoreactivity following naris closure, double-labeling was performed to test for an overlap
in the population of cells expressing the two antigens.
Three separate double-labeling techniques failed to detect
co-localization of these cells. Thus, calbindin and tyrosine
hydroxylase immunoreactivities mark distinct sub-populations of periglomerular cells.
Calretinin
Normal development. By P10, the olfactory nerve
layer exhibited variable staining. A high density of striking
calretinin-immunoreactive cells were present in the glomerular layer, and numerous labeled cells were also
observed in the granule cell layer (Fig. 7a). Relatively few
18
B.D. PHILPOT ET AL.
deeper half of the external plexiform layer had a higher
density of immunoreactive fibers than the superficial half.
At all ages studied, control subjects exhibited no evidence of laterality, as there was a negligible difference in
the density of immunopositive profiles between right and
left bulbs in the glomerular and granule cell layers (P..05).
In the glomerular layer, the number of calretinin-immunoreactive profiles per square millimeter was similar from
P10 to P60 (Table 1, control bulbs, P..05 for the main
effect of age). In the granule cell layer, the density of
immunopositive cells changed during postnatal development, reaching a highest density around P30, although the
age effect was non-significant (Table 1, control bulbs;
P..05 for main effect of age).
Effects of naris closure. Although calbindin-immunoreactivity was markedly reduced following olfactory deprivation, in the granule cell and glomerular layers there
were no changes in either the density (Table 1; P’s..05) or
area [F(1,12)51.4 for the granule cell layer; F(1,12)51.2
for the glomerular layer] of calretinin-immunoreactive
profiles for any age tested. The pattern of labeling in the
external plexiform and mitral cell layers also appeared
similar between control and experimental bulbs, although
these layers were not quantitatively analyzed.
Parvalbumin
Fig. 6. A: Mean area (6 S.E.M.) of calbindin-immunoreactive
profiles in control and experimental bulbs from rats receiving unilateral naris closure on P1–P20, P1–P30, and P30–P60. In experimental
bulbs, there is a significant reduction in the area of immunopositive
profiles following unilateral naris closure from P30–P60 (asterisk). B:
Frequency histogram of the area of calbindin-immunoreactive profiles
in experimental and control bulbs from rats receiving unilateral naris
closure from P30–P60. There is a uniform shift in the area of
calbindin-immunoreactive profiles, as the distribution is roughly
Gaussian in both control and experimental bulbs.
stained calretinin profiles were observed in the external
plexiform layer, and most mitral cells had light cytoplasmic immunoreactivity. By P20, immunoreactive profiles
were more intensely stained in all layers, and immunoreactive processes were somewhat more pronounced (Fig. 7b).
Notably, darkly labeled olfactory nerve bundles entered
some, but not all, glomeruli. The number of stained
profiles per unit area increased briefly in the granule cell
layer by P30 and then decreased slightly by P60 (Fig. 7c,d).
In both P30 and P60 rats, there was staining of variable
intensities in the olfactory nerve layer, and certain glomeruli received nerve bundles that were particularly dark
in their immunoreactivity.
Generally, the position and size of the immunopositive
cells suggested that interneuron populations were primarily immunoreactive. The faintly stained mitral cells were
the only immunopositive profiles with a large soma size.
Another consistent trait during development was that the
Normal development. Dark parvalbumin-immunostained processes were especially prevalent in the external
plexiform layer by P10, along with intense arborization in
the glomerular neuropil (Fig. 8a). Some immunopositive
fibers coursed from the external plexiform layer into the
granule cell layer. Few stained cell bodies were observed.
On P20, heavily labeled cells appeared selectively in the
external plexiform layer (Fig. 8b). In P30 rats, there was a
similar distribution of immunopositive profiles in the
external plexiform layer (Fig. 8c), although the number of
stained profiles was somewhat reduced by P60 (Fig. 8d).
At all ages, small (,8 µm diameter) soma-sized neurons
in the external plexiform layer, suggestive of short-axon
cells, were labeled. Occasional stained cells were observed
in the glomerular layer (Fig. 8c). Immunoreactive profiles
were generally located in the superficial and deep portions
of the external plexiform layer, with fewer cells present
along the mid-portion of the external plexiform layer. The
intensity of immunoreactivity often allowed fine details of
processes to be observed.
Once again, at all ages studied, there was no evidence
for laterality of parvalbumin expression in control pups;
there was a negligible difference in the density of immunopositive profiles between right and left bulbs (Fig. 9;
P..05). The number of parvalbumin-immunoreactive profiles per square millimeter decreased slightly from P20 to
P60, although the age-effect was non-significant (Fig. 9b,
controls).
Effects of naris closure. The density of parvalbuminimmunoreactive profiles was consistently reduced in experimental bulbs as compared to contralateral controls in
animals occluded from P1–P20, P1–P30, and P30–P60
[Fig. 9; F(1,9)548.9, P,0.001 for main effect of right/left
bulb laterality]. However, there was not a significant
interaction between bulb side and age group (P.0.05).
That is, the relative reduction in parvalbumin-immunostaining did not increase significantly with age. In addition to a decrease in the density of immunoreactive
profiles, dendritic labeling, although not quantified, was
Fig. 7. Photomicrographs depicting coronal sections of calretininimmunoreactivity in the developing main olfactory bulb. A: P10,
calretinin-immunoreactivity is present throughout the bulb. Variable
staining is present in the olfactory nerve layer (ONL). Many periglomerular cells in the glomerular layer (GLM) are darkly labeled. Some
short-axon cells are immunoreactive in the external plexiform layer
(EPL). In general, there are more labeled processes in the deeper half
of the EPL. Large cells in the mitral cell layer (MCL) have faint
immunoreactivity, while only some cells are labeled in the granule cell
layer (GCL). B: P20, the labeling of periglomerular cells is more
prominent. Calretinin-immunoreactivity fills some glomeruli. The
white arrowhead marks a darkly stained olfactory nerve bundle
entering a glomerulus, and the black arrowheads depict examples of
immunoreactive mitral cells. C: In this example from a P30 rat, the
olfactory nerve layer is intensely stained, and many glomeruli are
filled with dense calretinin-immunoreactive processes. D: P60, the
more numerous immunoreactive profiles in the deep portion of the
external plexiform layer is evident in this section. Black arrows mark
the MCL. Scale bar 5 200 µm.
20
B.D. PHILPOT ET AL.
TABLE 1. Mean Number (6S.E.M.) of Calretinin Immunopositive
Profiles/mm2 in Experimental and Control Bulbs from Rats Receiving
Unilateral Naris Closure
Glomerular layer
Granule cell layer
Age of
naris
occlusion
Control
bulb
Experimental
bulb
Control
bulb
Experimental
bulb
P1–P10
P1–P20
P1–P30
P30–P60
52.4 6 13.5
48.4 6 11.1
50.2 6 6.8
50.8 6 10.8
52.8 6 13.4
48.1 6 10.9
50.1 6 6.4
50.9 6 12.2
19.5 6 3.4
18.8 6 6.4
46.9 6 10.9
27.8 6 5.9
19.5 6 3.8
19.0 6 6.3
46.7 6 10.8
26.9 6 6.2
decreased in experimental bulbs (Fig. 10). The reduction in
parvalbumin labeling was not an age-dependent effect.
Occlusion from P30–P60 caused experimental bulbs to
have a similar reduction in the density of immunoreactive
profiles (64% reduction in profile density in P1–P30 subjects compared to a 43% reduction in P30–P60 subjects). In
addition to the large reduction in profile densities in each
age group, there was also a small decrease in the average
profile areas in experimental bulbs following unilateral
naris closure [F(1,9)511.56, P,.01; Fig. 11].
DISCUSSION
The present paper demonstrates that early odor restriction differentially affects cells in the olfactory bulb that
express calretinin, parvalbumin, or calbindin. While the
number of calretinin-immunopositive profiles is not affected after naris closure, parvalbumin and calbindinimmunoreactive profiles are dramatically reduced. Furthermore, observed changes in calcium-binding proteinimmunoreactivity occur even in animals occluded after the
maximal period for deprivation-dependent changes in the
bulb (Brunjes and Borror, 1983; Brunjes, 1994). The
remainder of the paper will discuss separately calbindin,
calretinin, and parvalbumin immunoreactivities during
bulb development in normal and naris-occluded rats, and
then general implications will be addressed.
Calbindin
Previous studies suggest that calbindin-immunoreactivity is present in the bulb by P1 (Enderlin et al., 1987;
Bastianelli and Pochet, 1995), although our observations
suggest only rudimentary staining even at P10. This
discrepancy could be either due to procedural differences
or to the fact that many antisera against calbindin crossreact with calretinin (Résibois and Rogers, 1992), which is
precociously expressed in the bulb. Nonetheless, most cells
expressing calbindin are likely born in the first postnatal
week, as x-ray irradiation during this time reduces calbindin-immunoreactivity to 10–30% of control levels (Kosaka
et al., 1992). Our results also support previous findings
that most calbindin-immunoreactive profiles are periglomerular cells, although occasional immunoreactive cells
are present in sub-glomerular layers (Jande et al., 1981;
Baimbridge and Miller, 1982; Halász et al., 1985; Briñón et
al., 1992; Résibois and Rogers, 1992; Kosaka et al., 1995).
Rarely, large-sized calbindin-immunoreactive cells are present in central portions of the subependymal zone (Celio,
1990; Briñón et al., 1992), although their function remains
unclear. These cells may, for example, function in the
guidance of migrating cells, or, as their flattened morphology suggests, they may be migrating cells already express-
ing the protein. In general, labeled profiles increase in
number and staining intensity from P10 until P60.
Unilateral naris closure profoundly affects several patterns of calbindin-immunoreactivity, suggesting that the
protein’s expression is under activity-dependent regulation. Experimental bulbs exhibit both less intensely stained
and smaller somata and almost a complete absence of
stained processes when compared to contralateral controls
(Fig. 4). These differences are unlikely to be an artifact as
both left and right bulbs were processed simultaneously.
Furthermore, large reductions in the number of calbindinimmunostaining profiles were observed in the glomerular
layer (Fig. 1c,d). The reductions in profile density must be
interpreted with caution as soma shrinkage could contribute to lower counts. However, the reductions in profile
density were consistent in animals occluded from P1–P20,
P1–P30, and P30–P60, while the reduction in soma size
was slight and only significant in animals occluded from
P30–P60.
The observed decrease in somata size may be due to
several factors. First, it might be due to general cell
shrinkage, something that occurs in many sensory modalities (Fukuda and Hsiao, 1984; Pasic and Rubel, 1989),
including olfaction (Meisami and Safari, 1981; Meisami
and Nousinfar, 1986), after functional restriction. Second,
large calbindin-immunoreactive cells may lose phenotypic
expression of calbindin, while smaller cells continue to
express the protein, mirroring the experience-induced
changes in tyrosine hydroxylase expression described below. Finally, the shift might be caused by selective cell
death in the bulb (Frazier and Brunjes, 1988; FrazierCierpial and Brunjes, 1989; Najbauer and Leon, 1995).
Perhaps the largest calbindin-immunoreactive cells die
due to deficits in trophic support, as changes in growth
factors affect calbindin expression (Iacopino et al., 1992).
Unlike many changes seen after naris closure, the
reduction in calbindin-immunoreactivity is not age dependent: similar changes are seen in animals deprived from
P1–P30 or P30–P60. As such, the change is similar to the
well-documented reductions in bulb tyrosine hydroxylaseimmunostaining following occlusion (e.g., Kosaka et al.,
1987a; Cho et al., 1996). Indeed, as both calbindin-and
tyrosine hydroxylase-immunopositive cell populations are
found in periglomerular regions, we tested whether the
cell populations overlap. However, our results, using three
double-labeling techniques, supported previous evidence
indicating that the two markers identify entirely unique
populations of cells (Halász et al., 1985; Rogers, 1992;
Kosaka et al., 1995). Interestingly, although levels of
tyrosine hydroxylase and calbindin staining are similar in
normal bulbs (Kosaka et al., 1995, present study), tyrosine
hydroxylase-containing cells are more affected by naris
occlusion (Fig. 3).
Calretinin
Of the three calcium-binding proteins, calretininimmunoreactivity appears earlier in all regions of the
brain, including the bulb, where it is present at birth
(Andressen et al., 1993; Bastianelli and Pochet, 1995). Our
results support previous finding that calretinin-positive
olfactory nerve bundles are common and often fill entire
glomeruli, suggesting the convergence of receptor axons
with strong calretinin expression (Rogers and Résibois,
1992; Bastianelli and Pochet, 1994). Furthermore, our
results support studies demonstrating that calretinin-
Fig. 8. Photomicrographs depicting parvalbumin-immunoreactivity in coronal sections from the developing rat bulb. A: P10, densely
labeled processes are observed in the external plexiform layer (EPL)
and extending into glomerular neuropil (GLM), although immunopositive somatic profiles are rarely present. Some fibers course through
the mitral cell layer (MCL) into the granule cell layer (GCL). B: P20,
immunoreactive profiles are apparent in the EPL. C: P30, darkly
stained profiles and dendritic processes are present throughout the
EPL. The white arrowhead depicts a labeled cell in the GLM. D: P60,
there is a decrease in the number of immunoreactive profiles in the
EPL. Black arrows indicate the MCL. Scale bar 5 100 µm.
22
B.D. PHILPOT ET AL.
acid-containing short-axon cells that make reciprocal synapses with mitral/tufted cells (Kosaka et al., 1987b; Toida
et al., 1994, 1996). Parvalbumin-immunoreactivity increases dramatically during the second and third postnatal weeks (Fig. 8, Kosaka et al., 1994b). The present data
demonstrate that naris closure profoundly reduces the
density of parvalbumin-immunoreactive profiles in rats
occluded from P1–P20, P1–P30, and P30–P60. While a
slight reduction in the average area of parvalbuminimmunoreactive profiles in experimental bulbs might have
contributed to this change, the decrease in profile densities
was so dramatic that it is almost certainly the result of
reduced parvalbumin expression. The present study is the
first direct evidence that short-axon cells in the external
plexiform layer are affected by odor deprivation, a cell
class previously thought to be unaffected (Croul-Ottman
and Brunjes, 1988). Furthermore, parvalbumin expression
is modified even by late-onset functional deprivation. In
the visual system, however, parvalbumin-immunostaining
decreases in visual cortex with early-onset, but not lateonset, deprivation (Cellerino et al., 1992). Thus, activitydependent regulation of parvalbumin in the olfactory bulb
does not coincide with a sensitive period of development.
General implications
Fig. 9. A: Mean percent difference (6 S.E.M.) in parvalbuminimmunoreactive profiles/mm2 between right and left bulbs in narisoccluded (nosx) and sham-operated (sham) rats. In experimental bulbs
from animals occluded from P1–P20, P1–P30, and P30–P60, there is a
large reduction in the number of parvalbumin-immunoreactive profiles. In sham-operated animals, there are similar numbers of immunoreactive profiles between right and left bulbs. B: Mean number of
profiles/mm2 (6 S.E.M.) in experimental and control bulbs from
naris-occluded rats. In control bulbs, there is a non-significant developmental decrease in the density of immunoreactive profiles from P20
until P60. In experimental bulbs from rats occluded from P1–P20,
P1–P30, and P30–P60, the reduction in the number of labeled profiles
is evident (P,0.001, main effect by ANOVA).
immunoreactive cells are prominent in the granule cell
and glomerular layers and relatively sparse in the external plexiform layer. Most mitral cells are lightly immunoreactive (Jacobowitz and Winsky, 1991; Résibois and Rogers, 1992; Bastianelli and Pochet, 1995; Wouterlood and
Härtig, 1995). While unilateral naris closure dramatically
reduces the expression of calbindin-immunoreactivity cells
in the glomerular layer, the manipulation has no effect on
profile number, intensity, or size of calretinin-immunoreactive cells in either the glomerular or granule cell layers.
Notably, calretinin-immunopositive periglomerular cells
are entirely distinct from the populations of tyrosine
hydroxylase and calbindin-immunostaining cells (Résibois
and Rogers, 1992; Kosaka et al., 1995)
Parvalbumin
Parvalbumin-immunoreactive cells in the external plexiform layer have been identified as gamma-aminobutyric
These data demonstrate that calcium-binding proteins
are differentially affected by unilateral naris closure.
There is a precedent for activity-dependent regulation of
calcium-binding proteins. For example, peripheral nervous system denervation alters the number and intensity
of cells immunoreactive for calcium-binding proteins (Olive and Ferrer, 1994; Sánchez-Vives et al., 1995), cochlear
ablation leads to decreased calretinin mRNA expression in
the ventral cochlear nucleus (Winsky and Jacobowitz,
1995), and monocular deprivation results in reduced parvalbumin and calbindin labeling in striate cortex (Cellerino
et al., 1992; Blümcke et al., 1994). Similarly, parvalbumin
and calbindin expression is affected by depolarizations
induced in cultured cells (Pfyffer et al., 1987). Collectively,
these studies illustrate that reduced afferent activity may
be a sufficient cue to regulate the immunohistochemical
expression of calcium-binding proteins.
There are numerous possible explanations for the finding that calbindin and parvalbumin immunoreactivities
decrease with naris closure while calretinin is unaffected.
First, naris closure may differentially alter the temporal
and spatial patterns of activity in these different subpopulations of neurons; little is known about the electrophysiological consequences of naris closure on subpopulations of bulb neurons. A second speculation is that
the properties of the calcium-binding proteins themselves
may confer a certain degree of cellular stability, and
perhaps the calretinin-containing cells are particularly
resilient to activity-dependent modifications following naris closure. Cells that contain calcium-binding proteins are
often remarkably resistant to neurodegeneration (Hof et
al., 1991; Hof and Morrison, 1991; Hof et al., 1993) and
perhaps have varying resilience to shifts in chemical
phenotype. Finally, intrinsic cellular properties of calbindin-and parvalbumin-immunoreactive cells that are as yet
unidentified may cause these cell types to be more susceptible to experience-dependent modifications, as compared
to calretinin-immunoreactive cells.
A change in neurochemical phenotype, rather than a
process of active cell death, probably accounts for the
CALCIUM-BINDING PROTEINS IN DEVELOPING OLFACTORY BULB
23
Fig. 10. Parvalbumin-immunoreactivity in control (left) and experimental (right) bulbs from a P30 rat after 30 days of naris closure. Left:
Dark somatic labeling and extensive process labeling is apparent in
the control bulb. Right: The number of immunopositive cells as well as
the staining intensity is dramatically reduced in experimental bulbs.
Note that there is less dendritic labeling in the experimental bulb.
Scale bar 5 100 µm. ONL, olfactory nerve layer; GLM, glomerular
layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL,
granule cell layer. Black arrow denotes MCL.
observed reductions in immunoreactive profile number.
For example, the decreases occur even after late-onset
naris closure, a procedure that results in few changes in
bulb volume, and therefore presumably does not affect cell
number. The reduced expression of these proteins is reminiscent of the alterations in tyrosine hydroxylase-immunoreactivity that follow either naris closure or deafferentation (Nadi et al., 1981; Kosaka et al., 1987a; Baker, 1990;
Stone et al., 1990, 1991; Cho et al., 1996). While tyrosine
hydroxylase levels decrease following these manipulations, other dopaminergic precursors remain unchanged,
suggesting a shift in cellular phenotype rather than cell
death (Baker et al., 1984).
The reductions in calbindin and parvalbumin immunoreactivities may be a result of either a decrease in their
calcium-binding status or attenuated expression of the
proteins. Recent evidence suggests that calretinin, parvalbumin, and calbindin are recognized preferentially in high
calcium concentrations (Winsky and Kuźnicki, 1996), and
the altered spatial and temporal patterns of neural activity seen after naris occlusion are likely to affect intracellu-
lar calcium levels (see Introduction, Philpot et al., 1997).
Thus, the reductions in calbindin and parvalbumin staining may be a result of a decrease in the calcium load within
these neuronal sub-populations and/or in a decline of
actual protein levels.
A loss of calbindin and parvalbumin expression is almost
certainly commensurate with a change in calcium regulation. Two plausible, and not mutually exclusive, possibilities exist. First, and most obvious, is that a decrease in
these calcium-binding proteins disrupts cellular calcium
buffering. Recent research has demonstrated, for example,
that calbindin-lacking mice have altered calcium signaling
(Airaksinen et al., 1997). Alternatively, the reductions in
calbindin and parvalbumin labeling may be preceded by
an activity-dependent change, either directly in the levels
of intracellular free-calcium due to reduced and asynchronous activity during naris closure, or indirectly through
rapidly-affected mechanisms of intracellular calcium regulation. Previous research has demonstrated that calcium
levels participate in calbindin regulation (Clemens et al.,
1989), suggesting that altered intracellular calcium levels
24
B.D. PHILPOT ET AL.
thermore, as discussed above, a change in intracellular
calcium levels after naris closure may precede the loss of
calcium-binding proteins and could account for the large
reductions in bulb volume.
In sum, these data demonstrate that there is a substantial postnatal increase in the expression of calbindin,
parvalbumin, and calretinin in the bulb, and that these
proteins are present in distinct sub-populations of cells.
Furthermore, olfactory nerve activity regulates, in part,
the expression of calbindin and parvalbumin. The changes
in calcium-binding proteins following odor deprivation
may reflect a general modification in calcium regulation in
the bulb that is associated with the diverse consequences
of this manipulation.
LITERATURE CITED
Fig. 11. Mean area (6 S.E.M.) of parvalbumin-immunoreactive
profiles in control and experimental bulbs from rats receiving unilateral naris closure on P1–P20, P1–P30, and P30–P60. In experimental
bulbs, there is a significant reduction in the area of immunopositive
profiles following unilateral naris closure (P,0.01, main effect by
ANOVA).
following naris closure could affect the expression of
calcium-binding proteins. Furthermore, as discussed above,
a change in calcium load can actually decrease antibody
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calcium-binding protein expression, the data suggest that
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and calbindin is associated with a modification in intracellular free calcium.
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