Activity-dependent regulation of calcium-binding proteins in the developing rat olfactory bulbкод для вставкиСкачать
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: email@example.com 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 detection of calbindin and parvalbumin. Regardless of the primacy of the change in calcium levels versus the altered calcium-binding protein expression, the data suggest that the activity-dependent down-regulation of parvalbumin and calbindin is associated with a modification in intracellular free calcium. The hypothesis that odor deprivation alters calcium regulation is attractive because a change in calcium levels could modify diverse cellular processes and account for the widespread consequences of naris closure. Calciummediated signaling cascades can activate immediate early genes (Sheng et al., 1990), affect protein expression (Ghosh and Greenberg, 1995), and alter neuronal survival (Choi, 1988; Collins et al., 1991). 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