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Characterization of olfactory bulb glomeruli in schizophrenia
Schizophrenia Research 77 (2005) 229 – 239 www.elsevier.com/locate/schres
Characterization of olfactory bulb glomeruli in schizophrenia Lise Riouxa,*, Edward Isaac Gelbera, Leila Paranda, Hala Altaf Kazia, Joannie Yeha, Rebecca Winteringa, Warren Bilkerb, Steven Edward Arnolda a Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
b
Received 11 January 2005; received in revised form 15 April 2005; accepted 19 April 2005 Available online 8 June 2005
Abstract Olfactory deficits, observed in schizophrenia, may be associated with a disruption of synaptic transmission in the olfactory system. Using immunohistochemistry and optical densitometry, we assessed the integrity of the synaptic connection between olfactory receptor neurons and olfactory bulb target neurons in schizophrenia by comparing the level of eight proteins, expressed in the olfactory bulb glomeruli, among schizophrenia and control subjects. In schizophrenia, no change was observed in the levels of OMP, GAP43 and NCAM, proteins expressed by olfactory receptor neurons, suggesting an intact innervation of the olfactory bulb by these neurons. This was supported by the absence of change in calbindin level, which has been shown to decrease after the destruction of the olfactory epithelium. The level of synaptophysin, a pre-synaptic protein, was also unchanged. These findings suggested that axons of olfactory receptor neurons establish synapses with their olfactory bulb targets in schizophrenia. The absence of change in the level of poorly phosphorylated neurofilament of moderate and high molecular weight (NFM/HP) suggested no lack of dendritic innervation despite a previously seen reduction of glomerular MAP2 level in schizophrenia subjects. This and above findings were consistent with the absence of change in the level of htubulin III, a protein expressed by neurons of both olfactory epithelium and bulb. Finally, we noted no significant decrease in trkB level, a neurotrophin receptor involved in the olfactory epithelium maintenance. This study showed no evidence of major structural alteration of the synapse between the olfactory epithelium and bulb in schizophrenia. D 2005 Elsevier B.V. All rights reserved. Keywords: Olfaction; Plasticity; Neurotrophin; Synaptogenesis
1. Introduction * Corresponding author. Present address: Laboratory for BioImaging and Anatomical Informatics, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA, 19129-1096, USA. Tel.: +1 215 991 8410; fax: +1 215 843 9367. E-mail address: [email protected] (L. Rioux). 0920-9964/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2005.04.022
Findings from various avenues of research have highlighted the importance of the olfactory system in understanding schizophrenia (Arnold and Rioux, 2001; Moberg and Turetsky, 2003). While not a cardinal feature, olfactory abnormalities are nonethe-
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less likely mediated by the same neurobiological mechanisms underlying the signature symptoms of schizophrenia. Because both neurogenesis and activity-dependent plasticity occur throughout life in the olfactory system (Shipley and Ennis, 1996), it is particularly suited for studying schizophrenia, a disease believed to be of developmental nature (Weinberger, 1995). Deficits in odor identification, detection threshold sensitivity, discrimination and memory have been reported in schizophrenia (Brewer et al., 1996; Hurwitz et al., 1988; Malaspina et al., 1994; Moberg et al., 1997). Efforts to uncover the pathoetiology of these deficits have not yet determined if they are caused by abnormalities in the olfactory epithelium, olfactory bulb, or central olfactory regions but some evidence points to a disruption of the synaptic connection between the olfactory epithelium and the olfactory bulb (Arnold et al., 2001; Rioux et al., 2004). The olfactory epithelium, located at the top of the nasal passages, continuously generates new olfactory receptor neurons that send their axons through the cribriform plate to reinnervate the glomeruli of the olfactory bulb. There, these axons form new glutamatergic synapses with dendrites of target neurons that include mitral and tufted cells that project directly to limbic cortex and, periglomerular neurons that modulate neurotransmission within the glomeruli. Additionally, the intraglomerular circuit consists of the mitral and tufted cell reciprocal dendrodendritic synapses with the periglomerular cells (Shipley and Ennis, 1996). Deafferentation and naris obstruction experiments have demonstrated that input from the olfactory epithelium is important for the normal expression of transmitters, enzymes and trophic factors in its target olfactory bulb neurons (Shipley and Ennis, 1996). Reciprocally, bulbectomy studies have shown the presence of the olfactory bulb to be necessary for the survival of olfactory receptor neurons (Chuah and Farbman, 1983). MRI volumetry has revealed that the olfactory bulb of subjects with schizophrenia is 23% smaller than healthy control (Turetsky et al., 2000). This could be the result of a reduction in olfactory receptor neurons activity or inputs, required for the survival of bulbar interneurons (Henegar and Maruniak, 1991; Mandairon et al., 2003). A significant increase in the proportion of immature olfactory receptors but no
corresponding increase in mature olfactory receptor neurons has also been found in the olfactory epithelium of subjects with schizophrenia (Arnold et al., 2001). Similar alterations in olfactory receptor neuron lineage have been observed in rodents after experimental interruption of the synaptic connection between the olfactory epithelium and its olfactory bulb target (Verhaagen et al., 1989). This increase in olfactory receptor neuron turnover could be due to intrinsic factors controlling the proliferation, differentiation and apoptosis in the olfactory epithelium. It could also results from a failure in access or response to growth factors produced by the olfactory bulb. The latter possibility would be consistent with the reduction in glomerular MAP2 level observed in schizophrenia (Rioux et al., 2004), indicating dendritic abnormalities in olfactory receptor neurons targets. In the present study, we tested the hypothesis that the integrity of the connection between the olfactory epithelium and olfactory bulb is altered in schizophrenia. We measured, using immunohistochemistry, the level of eight proteins expressed in olfactory bulb glomeruli. h-Tubulin III was used as a general marker for neuronal processes forming the glomeruli (Be´dard and Parent, 2004; Roskams et al., 1998). OMP, GAP43 and NCAM immunoreactivity was used to evaluate olfactory receptor neurons innervation of the glomeruli (Hoogland et al., 2003; Smith et al., 1991). These proteins are expressed by adult human olfactory receptor neurons (Arnold et al., 2001; Smutzer et al., 1998). To further examine the integrity of the olfactory receptor neurons innervation of the olfactory bulb, we also looked at the expression of calbindin, a protein strongly expressed in the glomeruli (Ohm et al., 1991), which is decreased by the olfactory epithelium destruction (Philpot et al., 1997). Poorly phosphorylated NFM/HP protein, largely confined to somatodendritic domain of neurons (Arnold et al., 1991), was used to assess the integrity of the dendritic innervation of the glomeruli (Chien et al., 1998). To assess the presence of synapses, we used synaptophysin, a pre-synaptic protein (Wiedenmann and Franke, 1985), strongly expressed in the glomeruli (Karowski et al., 1999). Finally, we measured the level of trkB, the receptor for BDNF (Klein et al., 1991), which plays an important role in the maintenance of the olfactory epithelium (Roskams et al., 1996; Simpson et al., 2002). A reduction
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in trkB level would be consistent with our findings of increased olfactory receptor neurons turnover in schizophrenia (Arnold et al., 2001).
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infection or toxins. Gross and microscopic diagnostic neuropathological examinations were conducted in all cases and were normal. 2.2. Tissue processing and immunocytochemistry
2. Material and methods 2.1. Patients The olfactory bulbs were removed at autopsy from 14 prospectively assessed elderly subjects with schizophrenia and 17 non-psychiatric control subjects comparable for age, postmortem interval (PMI) and gender (Table 1). Subjects with schizophrenia had been elderly participants in a prospective clinicopathological studies program (Arnold et al., 1995) and, diagnosed according to DSM-III-R/DSM-IV criteria based on medical history, interview with caregivers and clinical examination of the patient. To eliminate any hormonal confounds, all female subjects were postmenopausal (Kopala et al., 1995). All patients had required chronic hospitalization because of severe schizophrenia-related symptomatology including cognitive and functional impairment. Written informed consent for antemortem evaluation and autopsy in the event of death were obtained from next of kin according to approved institutional review board protocols. Brain tissues from non-neuropsychiatric elderly controls were obtained through the University of Pennsylvania’s Center for Neurodegenerative Disease Research. While none of these control subjects had undergone antemortem assessments, a review of their clinical histories found no evidence of any neurological or psychiatric illness. At the time of death, subjects were excluded because of anoxic injury or extensive injury due to trauma, stroke, neoplasm,
The olfactory bulbs were fixed in formalin (3.7% formaldehyde in 0.1 M Tris, 0.9 g/l NaCl) or ethanol (70% ethyl alcohol, 150 mM NaCl) for 24 h, paraffin embedded, cut into 10-Am-thick sections and mounted on poly-l-lysine coated slides. Immunohistochemistry experiments were conducted according to a previously described procedure (Arai et al., 1992), using mostly commercial antibodies, to assess the expression of h-tubulin III (SDL-3D10/Sigma, 1:500), OMP (Goat#255/Margolis, 1:2000), GAP43 (GAP-7B10/Sigma, 1:1000), NCAM (MOC-1/Trojanowski, 1:250), synaptophysin (SY38/Dako, 1:500), poorly phosphorylated NFM/HP (RMDO20/Lee, neat), trkB (794/Santa Cruz, 1:2000) and calbindin (AB1778/Chemicon, 1:50) in the glomeruli. Specificity of GAP43, synaptophysin, OMP, h-tubulin III, TrkB, NFM/HP, NCAM antibodies has been previously demonstrated by others (Eastwood and Harrison, 2001; Gomez et al., 2000; Lambert et al., 2001; Quartu et al., 2003; Lee et al., 1988; Smutzer et al., 1998). Calbindin antibody specificity was determined by Western blot in rat brain (Kawaguchi and Hirano, 2002). It also produces a specific staining of neuron cell bodies and processes in human anterior cingulated cortex (Cotter et al., 2002) that is consistent with expected distribution of calbindin (DeFelipe, 1997). Briefly, sections were deparaffinized, rehydrated and then incubated for 30 min in 1% H2O2 at room temperature to remove endogenous peroxidase activity. Antigen retrieval techniques
Table 1 Clinical and demographic data on human subjects Schizophrenic (10 females/4 males)
Age (year) PMI (h) Age of onset (year) Duration (year) CPZ1MO
Control (10 females/7 males)
Mean
S.D.
Range
Mean
S.D.
Range
75.1 10.8 25.0 52.4 322.7
15.2 3.2 5.8 8.9 389.5
28–90 6.5–17 16–30 39a–70 0–1350
69.4 13.1 na na na
11.8 6.0 na na na
47–91 4.0–23 na na na
CPZ1MO = antipsychotic dosage 1 month before death; S.D. = standard deviation; na = not available. a Excludes 28 year old subject.
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were used for h-tubulin III (boiling for 30 min in 10 mM trisodium citrate, pH 6) as well as for calbindin (boiling for 10 min in 1 mM EDTA, 0.1 M Tris, pH 8). After two washes in TBS (0.1 M Tris, 0.9 g/ l NaCl), sections were incubated in TBS containing 4% normal (goat or horse) serum (NS) for 60 min at room temperature. The sections were then incubated overnight at 4 8C in TBS containing 4% NS and the primary antibodies at their respective dilution, rinsed three times with TBS and incubated in biotinylated secondary antibody in TBS with 4% NS (1:200, Vector) for 1 h. After being rinsed three times with TBS, they were incubated 1 h in Vectastain ABC reagent (1:100, Vector) at room temperature. After three more rinses, they were incubated with the chromogen diaminobenzidine (0.1% DAB in 0.1 M Tris, 0.01% triton, pH 7.3) and 0.03% H2O2 for 6 min and rinsed twice with water. Sections were then dehydrated, coverslipped with Cytoseal, and coded for analysis blind to case identity. For each antibody, two immunohistochemical assays were run. One section per case was used in each run. For each assay, all cases were processed in a single, precisely timed run. Sections incubated without primary antibody or with an IgG of the same species than the primary antibody were run in parallel and served as negative controls. The concentration of the primary antibodies was based on company’s recommendations and our titration studies. Any tissue sections, which was damaged or unlabeled during the procedures, could not be assessed subsequently for that particular investigation. 2.3. Quantitative analysis To determine the optical density (OD) of the DAB reaction product in the glomeruli of the olfactory bulbs, we used customized NIH Image 1.61 software run on a Macintosh computer attached to a Leitz DMRB microscope (Leica) and Pulnix video camera. At the start of each image capture session, the scope illumination was adjusted to a standard level using a blank slide. A slide mounted OD step tablet was imaged and a calibration curve generated. OD of negative control slides served as background OD. For each olfactory bulb, a field of view was captured and all glomeruli contained in it were delineated. The gray value for each glomerulus was recorded and converted to an OD value. Background OD was
then subtracted from that value. The number of glomeruli per section and the area of each glomerulus were also determined. 2.4. Statistical analysis The normality of the data was assessed using the Shapiro–Wilk test. Between group differences for mean glomerular immunodensities were assessed with a non-parametric one factor repeated measures ANOVA test (Brunner et al., 2001). Between group differences for age, PMI, mean glomerular counts and mean glomerular areas were assessed with Mann– Whitney–Wilcoxon test. Gender differences between schizophrenia and control groups were assessed with the v 2 test. To assess effects of potentially confounding factors, including age, PMI, the effects of antipsychotic medication exposure and other demographic data on OD, we used Spearman Rank correlation analysis on each individual assay. An alpha level of 0.05 was used to determine significance.
3. Results 3.1. Immunolabeling distribution in olfactory bulb OMP Immunoreactivity (Fig. 1A) was strong and present only in axon processes within the olfactory receptor neuron layer and in glomeruli. No cell demonstrated immunostaining in any of the layers of the olfactory bulb. Similar pattern of staining was observed for GAP43 and NCAM (Fig. 1B and C, respectively). In addition, we observed mildly to moderately stained fibers in the granular and internal plexiform layer of several subjects. Poorly phosphorylated NFM/HP immunoreactivity (Fig. 1D) was present in all layers of the olfactory bulb except for the olfactory receptor neuron layer. NFM/HP labeling was very strong in glomeruli and what look like mitral cells, moderately strong in the external plexiform layer but quite faint in neurons and processes within the other layers. Immunohistochemistry for h-tubulin (Fig. 1E) was present in all layers of the olfactory bulb. Strong labeling was expressed within the olfactory receptor neuron layer and in glomeruli. Some juxtaglomerular and granular cells were also strongly labeled. Moderate labeling of processes was observed in the external and internal plexiform layers. Faint labeling was also present in the granular layer. Synaptophysin immunoreactivity (Fig. 1F) was strongly expressed in the glomeruli. Faint labeling of processes was
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Fig. 1. Photomicrographs depicting immunoreactivity for (A) OMP, (B) GAP43, (C) NCAM, (D) NFM/HP, (E) h-tubulin III, (F) synaptophysin, (G) calbindin and (H) trkB in coronal sections of human olfactory bulbs. Arrows indicate (A) glomeruli and (G) periglomerular cells. Window (H) shows mitral cells. Scale bar, 100 Am.
also present in external and internal plexiform layers. No cell demonstrated immunostaining in any of the layers of the olfactory bulb. Calbindin immunoreactivity (Fig. 1G) was observed in all layers of the olfactory bulb. Strong immunohistochem-
istry for calbindin was seen in glomeruli and some juxtaglomerular cells. Some labeled cells were also observed in other layers except for the olfactory receptor neuron layer. Strong immunohistochemistry for trkB (Fig. 1H) was expressed within the olfactory receptor neuron layer and
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in glomeruli. Granular, juxtaglomerular and what look like mitral cells showed moderate labeling. Unless indicated, the morphologic characteristics and distribution of labeled elements were consistent across the subjects studied for each of the antibodies used in this study. 3.2. Quantification of immunoreactivity in glomeruli As shown in Table 2, the glomerular level of each proteins investigated in this study was not different in subjects with schizophrenia when compared to controls. Correlation analysis found no significant relationship between OD values for most proteins and age for both control (rho V 0.52, p z 0.08) and schizophrenia (rho V 0.47, p z 0.15) groups. In the control group, correlations with age were inconclusive for calbindin (rho = 0.17, p = 0.59 and rho = 0.62, p = 0.04) and trkB (rho = 0.14, p = 0.66 and rho = 0.66, p = 0.02). No significant correlation was found between OD values for most proteins and PMI for both control (rho V 0.48, p z 0.16) and schizophrenia (rho V 0.63, p z 0.07) groups. In the control group, correlations with PMI were inconclusive for calbindin (rho = 0.04, p = 0.92 and rho = 0.61, p = 0.03) and NCAM (rho = 0.25, p = 0.49 and rho = 0.65, p = 0.03). In the schizophrenia group, correlations with PMI were inconclusive for RMDO (rho = 0.50, p = 0.10 and rho = 0.73, p = 0.01) and almost reach significance for one tubulin assay (rho = 0.76, p = 0.05 and rho = 0.07, p = 0.82). In conclusion, we have not demonstrated any definitive correlation between age or PMI and the glomerular level of any of the studied proteins. No significant correlation was found either between OD values for most proteins and age of onset (rho V 0.57,
p z 0.17), duration of illness (rho V 0.62, p z 0.10) or antipsychotic dosage (rho V 0.50, p z 0.21) for the schizophrenia group. Correlation between OD and antipsychotic dosage almost reached significance for one of the tubulin assays (rho = 0.66, p = 0.05 and rho = 0.19, p = 0.69). Correlation between OD and age of onset was inconclusive for calbindin (rho = 0.08, p = 0.80 and rho = 0.63, p = 0.04). Again, we have not demonstrated any definitive correlation between these three factors and the glomerular level of any of the studied protein. No significant difference between groups were observed in the number of glomeruli per section (Z V 1.33, p z 0.18) or in the mean area of the glomeruli (Z V 0.83, p z 0.32) labeled with any of the antibodies. There were no group differences in age (Z V 1.78, p z 0.08) for the cases labeled with each of the antibodies except for calbindin (Z = 2.21, p = 0.03) and RMDO20 (Z = 2.07, p = 0.04). There were no group differences in PMI (Z V 1.04, p z 0.30) for the cases labeled with each of the antibodies. The relative frequencies of male and female was the same for both schizophrenia and control group (v 2 V 0.22, p z 0.64).
4. Discussion The present study reports the protein expression patterns of several pre- and post-synaptic markers in the human olfactory bulb. While the distribution of these proteins varies widely, they are all located in the glomeruli.
Table 2 Glomerular level of various proteins expressed in mean optical density (OD) Antigen
Location/function
OD schizophrenia, total n (2 repeats)
OD control, total n (2 repeats)
ANOVA test t (df), p
OMP
0.12)
1.42 (24.68), 0.17
Growth cone/axon guidance
0.08)
0.47 (26.84), 0.64
NCAM
ORNs/adhesion molecule
0.12)
0.83 (20.77), 0.41
TrkB
ORNs and OB cells/BDNF receptor Dendrite/forms neurofilaments
0.43 (S.D. 22 subjects 0.31 (S.D. 23 subjects 0.17 (S.D. 26 subjects 0.28 (S.D. 21 subjects 0.09 (S.D. 24 subjects 0.35 (S.D. 24 subjects 0.07 (S.D. 23 subjects 0.20 (S.D. 20 subjects
1.11 (23.70), 0.28
GAP43
0.48 (S.D. 0.11) 21 subjects 0.27 (S.D. 0.15) 18 subjects 0.18 (S.D. 0.08) 23 subjects 0.26 (S.D. 0.12) 21 subjects 0.06 (S.D. 0.03) 20 subjects 0.39 (S.D. 0.12) 24 subjects 0.06 (S.D. 0.05) 28 subjects 0.23 (S.D. 0.08) 19 subjects
0.16)
Synaptophysin
ORNs marker/neural transmission Synaptic vesicle/exocytosis
0.06)
1.93 (23.02), 0.07
0.10)
1.50 (22.96), 0.15
0.05)
1.44 (23.99), 0.16
0.10)
0.93 (20.78), 0.36
NFM/HP Calbindin h-Tubulin III
PG neurons/Ca2+ binding protein Neurons/forms microtubules
ORN = olfactory receptor neuron, OB = olfactory bulb, PG = periglomerular neuron.
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As previously shown, OMP, GAP43 and NCAM immunoreactivity was only present within the olfactory receptor neuron layer and in glomeruli of human olfactory bulb (Hoogland et al., 2003; Smith et al., 1991). Like in rodent, poorly phosphorylated NFM/ HP labeling was present in all layers of the olfactory bulb except for the olfactory receptor neuron layer (Chien et al., 1998). Consistent with earlier studies in human and rodents, h-tubulin III was present in all layers of the olfactory bulb, strongly labeling glomeruli as well as periglomerular and granular cells (Be´dard and Parent, 2004; Gonzales and Silver, 1994; Roskams et al., 1998). Calbindin immunoreactivity was observed in all layers of the olfactory bulb but was particularly strong in the glomeruli and juxtaglomerular cells, as previously reported (Alonzo et al., 2001; Ohm et al., 1991). While negative controls did not show any nonspecific labeling, no Western blot or pre-adsorption study was done with human brain tissue to exclude the possibility of detecting other proteins sharing a domain homolog to the peptide used to raise the calbindin antibody. As expected from a previous study in rodent, synaptophysin was strongly expressed in the glomeruli and also present in the plexiform layers (Karowski et al., 1999). TrkB immunoreactivity was found in various levels in the glomeruli as well as in the mitral, granular and juxtaglomerular cells of the human olfactory bulb. A similar distribution of trkB has been reported in the rat with the difference that the glomeruli were only lightly stained (Yan et al., 1997). While this suggests that glomerular trkB could be located in dendrites of mitral and periglomerular cells, it could also be located in axon terminals of olfactory receptor neurons. TrkB has also been shown to be expressed by human olfactory receptor neurons (Nibu et al., 1999). In rodents, it has been shown that trkB is expressed by post-mitotic, immature olfactory receptor neurons, first in their cell bodies and dendrites and then primarily in their axons as they mature (Deckner et al., 1993; Holcomb et al., 1995; Roskams et al., 1996). The glomerular level of these proteins was measured to assess the integrity of the connection between the olfactory epithelium and olfactory bulb in schizophrenia. The present study relies on optical density
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(OD) measurement to evaluate the relative concentration of these proteins in postmortem olfactory bulb glomeruli. While optical densitometry allows rapid, objective and automatic evaluation of immunohistochemical label intensity, concerns exist that this method of quantification may not reflect the amount of a specific protein across tissue sections because of the non-linearity of the immunohistochemical procedure. However, several studies have shown that the measure of OD of an immunolabeled protein using DAB as a chromogen is in fact consistently proportional to its concentration in a tissue section as long as the range of OD measured in the tissue fall within the linear range of the OD calibration curve (Jojich and Pourcho, 1996; Nabors et al., 1988; Rieux et al., 2002; Schipper and Tilders, 1983). Given that the various steps in processing and analysis are standardized, from the histological treatments to image analysis, the use of this method for quantitative analysis is reliable. We noted no difference in the immunodensity of OMP, a protein exclusively expressed by mature olfactory receptor neurons, in the glomeruli (Arnold et al., 2001; Nakashima et al., 1985). This finding suggests intact olfactory receptor neuron innervation of the olfactory bulb. While OMP expressing olfactory receptor neurons have been shown to decline following olfactory bulbectomy, OMP level increase in remaining olfactory receptor neurons (Carr et al., 1998). OMP expression in glomeruli disappears after destruction of olfactory epithelium (Ehrlich et al., 1990). However, since OMP immunostaining persists in axon terminals severed from their parent cell body for several days, it may not be the best marker of intact olfactory receptor neuron axons (Slotnick et al., 2001). On the other hand, the concomitant absence of change in the level of GAP43 and NCAM in the glomeruli also suggests no major loss of olfactory receptor neuron innervation or major insults to the olfactory bulb in schizophrenia. This is consistent with a previous study showing no change in the number of OMP cells in the olfactory epithelium in schizophrenia (Arnold et al., 2001). This is also supported by the absence of change in the glomerular level of calbindin, which is decreased by the destruction of the olfactory epithelium (Philpot et al., 1997). We noted no difference either in the immunodensity of synaptophysin, a synaptic vesicle membrane
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component, which is often used as a general marker of the presence of synapses. This and above findings suggest that axons of olfactory receptor neurons establish synapses with their targets in the olfactory bulb glomeruli and do not support the notion of a generalized deficit in pre-synaptic proteins in schizophrenia. Studies of pre-synaptic proteins in cortical areas in schizophrenia are too inconsistent to support that notion either (Halim et al., 2003; Honer et al., 2000; Honer and Young, 2004; Mirnics et al., 2000). However, they do not exclude discrete changes in selected pre-synaptic proteins. No significant changes were found in the expression of poorly phosphorylated NFM/HP, a cytoskeletal element selective for dendrites. As with the presynaptic proteins, our finding suggests no general lack of dendritic innervation in the glomeruli. These findings are consistent with the absence of change in the level of h-tubulin III, a protein expressed by both olfactory receptor neurons and its target neurons in the olfactory bulb (Be´dard and Parent, 2004; Gonzales and Silver, 1994; Roskams et al., 1998). Finally, we observed no significant decrease in trkB expression, the receptor for BDNF, which is an important regulator of growth and survival of mature olfactory receptor neurons. This finding contrasts with other studies suggesting a reduction of neuronal trophic support in schizophrenia (Hashimoto et al., 2005; Takahashi et al., 2000; Weickert et al., 2003). Observed reduction in trkB may be restricted to specific brain areas that do not include the olfactory bulb. However, since the decrease in glomerular trkB reached a trend level of significance, a net decrease in trkB level may have been masked by an increase in packing density resulting from the reduced volume of the olfactory bulb in schizophrenia. Using only two sections might have not been sufficient to detect changes that may also be more pronounced in certain areas of the olfactory bulb. These last two confounds might equally apply to other proteins as well. One other consideration in the interpretation of our results is the potential effect of neuroleptics on the expression of these eight proteins. Studies on the effects of neuroleptics on the brain level of each protein have been negative or inconclusive (Eastwood et al., 1997; Lidow et al., 2001; Linden et al., 2000). Because in our study, neuroleptic dosage was not correlated to the level of any of these proteins, neu-
roleptic exposure may not be the main determinant of their expression. Moreover, no study has noted differences between neuroleptic-naive, neuroleptic withdrawn, and currently medicated schizophrenia patients on a variety of olfactory tests (Geddes et al., 1991; Kohler et al., 2001). Studies searching for molecular correlates of synaptic deficits in schizophrenia have found reduced, increased and no change in the expression of pre- and post-synaptic markers (Honer et al., 2000; Mirnics et al., 2001), inconsistent with a generalized deficit in synaptic proteins. As caution must be heeded because of the small sample size in our study, our findings suggest that axons of olfactory receptor neurons establish synapses with their targets in the olfactory bulb glomeruli. They also indicate no general lack of dendritic innervation of the glomeruli by these targets. Despite the absence of changes in the pre- and post-synaptic elements in glomeruli, it is possible that with other markers we could detect selective abnormalities in the molecular integrity of these structures. MAP2, a microtubule-associated protein that binds and stabilizes microtubules in dendrites, is significantly decreased in the olfactory bulb glomeruli in schizophrenia (Rioux et al., 2004). While these findings suggest that changes in the olfactory epithelium are not associated with major structural changes in the olfactory bulb glomeruli, one must not forget that the volume of olfactory bulb in schizophrenia is decreased when compared to control (Turetsky et al., 2000). Although a stereological approach might be able to detect glomerular changes not otherwise observable, they would not be sufficient to account for the 23% reduction of the olfactory bulb volume reported in schizophrenia. Changes in other layers of the olfactory epithelium have to contribute to the size reduction observed in patients with schizophrenia and, could impact the olfactory epithelium by indirectly altering the synaptic transmission between the olfactory receptor neurons and its targets.
Acknowledgement OMP, RMDO20 and MOC-1 antibodies were gifts from Drs. F.L. Margolis, V.M.-Y. Lee and J.Q. Trojanowski, respectively. We want to acknowledge the
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Characterization of olfactory bulb glomeruli in schizophrenia Lise Riouxa,*, Edward Isaac Gelbera, Leila Paranda, Hala Altaf Kazia, Joannie Yeha, Rebecca Winteringa, Warren Bilkerb, Steven Edward Arnolda a Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
b
Received 11 January 2005; received in revised form 15 April 2005; accepted 19 April 2005 Available online 8 June 2005
Abstract Olfactory deficits, observed in schizophrenia, may be associated with a disruption of synaptic transmission in the olfactory system. Using immunohistochemistry and optical densitometry, we assessed the integrity of the synaptic connection between olfactory receptor neurons and olfactory bulb target neurons in schizophrenia by comparing the level of eight proteins, expressed in the olfactory bulb glomeruli, among schizophrenia and control subjects. In schizophrenia, no change was observed in the levels of OMP, GAP43 and NCAM, proteins expressed by olfactory receptor neurons, suggesting an intact innervation of the olfactory bulb by these neurons. This was supported by the absence of change in calbindin level, which has been shown to decrease after the destruction of the olfactory epithelium. The level of synaptophysin, a pre-synaptic protein, was also unchanged. These findings suggested that axons of olfactory receptor neurons establish synapses with their olfactory bulb targets in schizophrenia. The absence of change in the level of poorly phosphorylated neurofilament of moderate and high molecular weight (NFM/HP) suggested no lack of dendritic innervation despite a previously seen reduction of glomerular MAP2 level in schizophrenia subjects. This and above findings were consistent with the absence of change in the level of htubulin III, a protein expressed by neurons of both olfactory epithelium and bulb. Finally, we noted no significant decrease in trkB level, a neurotrophin receptor involved in the olfactory epithelium maintenance. This study showed no evidence of major structural alteration of the synapse between the olfactory epithelium and bulb in schizophrenia. D 2005 Elsevier B.V. All rights reserved. Keywords: Olfaction; Plasticity; Neurotrophin; Synaptogenesis
1. Introduction * Corresponding author. Present address: Laboratory for BioImaging and Anatomical Informatics, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA, 19129-1096, USA. Tel.: +1 215 991 8410; fax: +1 215 843 9367. E-mail address: [email protected] (L. Rioux). 0920-9964/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2005.04.022
Findings from various avenues of research have highlighted the importance of the olfactory system in understanding schizophrenia (Arnold and Rioux, 2001; Moberg and Turetsky, 2003). While not a cardinal feature, olfactory abnormalities are nonethe-
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less likely mediated by the same neurobiological mechanisms underlying the signature symptoms of schizophrenia. Because both neurogenesis and activity-dependent plasticity occur throughout life in the olfactory system (Shipley and Ennis, 1996), it is particularly suited for studying schizophrenia, a disease believed to be of developmental nature (Weinberger, 1995). Deficits in odor identification, detection threshold sensitivity, discrimination and memory have been reported in schizophrenia (Brewer et al., 1996; Hurwitz et al., 1988; Malaspina et al., 1994; Moberg et al., 1997). Efforts to uncover the pathoetiology of these deficits have not yet determined if they are caused by abnormalities in the olfactory epithelium, olfactory bulb, or central olfactory regions but some evidence points to a disruption of the synaptic connection between the olfactory epithelium and the olfactory bulb (Arnold et al., 2001; Rioux et al., 2004). The olfactory epithelium, located at the top of the nasal passages, continuously generates new olfactory receptor neurons that send their axons through the cribriform plate to reinnervate the glomeruli of the olfactory bulb. There, these axons form new glutamatergic synapses with dendrites of target neurons that include mitral and tufted cells that project directly to limbic cortex and, periglomerular neurons that modulate neurotransmission within the glomeruli. Additionally, the intraglomerular circuit consists of the mitral and tufted cell reciprocal dendrodendritic synapses with the periglomerular cells (Shipley and Ennis, 1996). Deafferentation and naris obstruction experiments have demonstrated that input from the olfactory epithelium is important for the normal expression of transmitters, enzymes and trophic factors in its target olfactory bulb neurons (Shipley and Ennis, 1996). Reciprocally, bulbectomy studies have shown the presence of the olfactory bulb to be necessary for the survival of olfactory receptor neurons (Chuah and Farbman, 1983). MRI volumetry has revealed that the olfactory bulb of subjects with schizophrenia is 23% smaller than healthy control (Turetsky et al., 2000). This could be the result of a reduction in olfactory receptor neurons activity or inputs, required for the survival of bulbar interneurons (Henegar and Maruniak, 1991; Mandairon et al., 2003). A significant increase in the proportion of immature olfactory receptors but no
corresponding increase in mature olfactory receptor neurons has also been found in the olfactory epithelium of subjects with schizophrenia (Arnold et al., 2001). Similar alterations in olfactory receptor neuron lineage have been observed in rodents after experimental interruption of the synaptic connection between the olfactory epithelium and its olfactory bulb target (Verhaagen et al., 1989). This increase in olfactory receptor neuron turnover could be due to intrinsic factors controlling the proliferation, differentiation and apoptosis in the olfactory epithelium. It could also results from a failure in access or response to growth factors produced by the olfactory bulb. The latter possibility would be consistent with the reduction in glomerular MAP2 level observed in schizophrenia (Rioux et al., 2004), indicating dendritic abnormalities in olfactory receptor neurons targets. In the present study, we tested the hypothesis that the integrity of the connection between the olfactory epithelium and olfactory bulb is altered in schizophrenia. We measured, using immunohistochemistry, the level of eight proteins expressed in olfactory bulb glomeruli. h-Tubulin III was used as a general marker for neuronal processes forming the glomeruli (Be´dard and Parent, 2004; Roskams et al., 1998). OMP, GAP43 and NCAM immunoreactivity was used to evaluate olfactory receptor neurons innervation of the glomeruli (Hoogland et al., 2003; Smith et al., 1991). These proteins are expressed by adult human olfactory receptor neurons (Arnold et al., 2001; Smutzer et al., 1998). To further examine the integrity of the olfactory receptor neurons innervation of the olfactory bulb, we also looked at the expression of calbindin, a protein strongly expressed in the glomeruli (Ohm et al., 1991), which is decreased by the olfactory epithelium destruction (Philpot et al., 1997). Poorly phosphorylated NFM/HP protein, largely confined to somatodendritic domain of neurons (Arnold et al., 1991), was used to assess the integrity of the dendritic innervation of the glomeruli (Chien et al., 1998). To assess the presence of synapses, we used synaptophysin, a pre-synaptic protein (Wiedenmann and Franke, 1985), strongly expressed in the glomeruli (Karowski et al., 1999). Finally, we measured the level of trkB, the receptor for BDNF (Klein et al., 1991), which plays an important role in the maintenance of the olfactory epithelium (Roskams et al., 1996; Simpson et al., 2002). A reduction
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in trkB level would be consistent with our findings of increased olfactory receptor neurons turnover in schizophrenia (Arnold et al., 2001).
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infection or toxins. Gross and microscopic diagnostic neuropathological examinations were conducted in all cases and were normal. 2.2. Tissue processing and immunocytochemistry
2. Material and methods 2.1. Patients The olfactory bulbs were removed at autopsy from 14 prospectively assessed elderly subjects with schizophrenia and 17 non-psychiatric control subjects comparable for age, postmortem interval (PMI) and gender (Table 1). Subjects with schizophrenia had been elderly participants in a prospective clinicopathological studies program (Arnold et al., 1995) and, diagnosed according to DSM-III-R/DSM-IV criteria based on medical history, interview with caregivers and clinical examination of the patient. To eliminate any hormonal confounds, all female subjects were postmenopausal (Kopala et al., 1995). All patients had required chronic hospitalization because of severe schizophrenia-related symptomatology including cognitive and functional impairment. Written informed consent for antemortem evaluation and autopsy in the event of death were obtained from next of kin according to approved institutional review board protocols. Brain tissues from non-neuropsychiatric elderly controls were obtained through the University of Pennsylvania’s Center for Neurodegenerative Disease Research. While none of these control subjects had undergone antemortem assessments, a review of their clinical histories found no evidence of any neurological or psychiatric illness. At the time of death, subjects were excluded because of anoxic injury or extensive injury due to trauma, stroke, neoplasm,
The olfactory bulbs were fixed in formalin (3.7% formaldehyde in 0.1 M Tris, 0.9 g/l NaCl) or ethanol (70% ethyl alcohol, 150 mM NaCl) for 24 h, paraffin embedded, cut into 10-Am-thick sections and mounted on poly-l-lysine coated slides. Immunohistochemistry experiments were conducted according to a previously described procedure (Arai et al., 1992), using mostly commercial antibodies, to assess the expression of h-tubulin III (SDL-3D10/Sigma, 1:500), OMP (Goat#255/Margolis, 1:2000), GAP43 (GAP-7B10/Sigma, 1:1000), NCAM (MOC-1/Trojanowski, 1:250), synaptophysin (SY38/Dako, 1:500), poorly phosphorylated NFM/HP (RMDO20/Lee, neat), trkB (794/Santa Cruz, 1:2000) and calbindin (AB1778/Chemicon, 1:50) in the glomeruli. Specificity of GAP43, synaptophysin, OMP, h-tubulin III, TrkB, NFM/HP, NCAM antibodies has been previously demonstrated by others (Eastwood and Harrison, 2001; Gomez et al., 2000; Lambert et al., 2001; Quartu et al., 2003; Lee et al., 1988; Smutzer et al., 1998). Calbindin antibody specificity was determined by Western blot in rat brain (Kawaguchi and Hirano, 2002). It also produces a specific staining of neuron cell bodies and processes in human anterior cingulated cortex (Cotter et al., 2002) that is consistent with expected distribution of calbindin (DeFelipe, 1997). Briefly, sections were deparaffinized, rehydrated and then incubated for 30 min in 1% H2O2 at room temperature to remove endogenous peroxidase activity. Antigen retrieval techniques
Table 1 Clinical and demographic data on human subjects Schizophrenic (10 females/4 males)
Age (year) PMI (h) Age of onset (year) Duration (year) CPZ1MO
Control (10 females/7 males)
Mean
S.D.
Range
Mean
S.D.
Range
75.1 10.8 25.0 52.4 322.7
15.2 3.2 5.8 8.9 389.5
28–90 6.5–17 16–30 39a–70 0–1350
69.4 13.1 na na na
11.8 6.0 na na na
47–91 4.0–23 na na na
CPZ1MO = antipsychotic dosage 1 month before death; S.D. = standard deviation; na = not available. a Excludes 28 year old subject.
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were used for h-tubulin III (boiling for 30 min in 10 mM trisodium citrate, pH 6) as well as for calbindin (boiling for 10 min in 1 mM EDTA, 0.1 M Tris, pH 8). After two washes in TBS (0.1 M Tris, 0.9 g/ l NaCl), sections were incubated in TBS containing 4% normal (goat or horse) serum (NS) for 60 min at room temperature. The sections were then incubated overnight at 4 8C in TBS containing 4% NS and the primary antibodies at their respective dilution, rinsed three times with TBS and incubated in biotinylated secondary antibody in TBS with 4% NS (1:200, Vector) for 1 h. After being rinsed three times with TBS, they were incubated 1 h in Vectastain ABC reagent (1:100, Vector) at room temperature. After three more rinses, they were incubated with the chromogen diaminobenzidine (0.1% DAB in 0.1 M Tris, 0.01% triton, pH 7.3) and 0.03% H2O2 for 6 min and rinsed twice with water. Sections were then dehydrated, coverslipped with Cytoseal, and coded for analysis blind to case identity. For each antibody, two immunohistochemical assays were run. One section per case was used in each run. For each assay, all cases were processed in a single, precisely timed run. Sections incubated without primary antibody or with an IgG of the same species than the primary antibody were run in parallel and served as negative controls. The concentration of the primary antibodies was based on company’s recommendations and our titration studies. Any tissue sections, which was damaged or unlabeled during the procedures, could not be assessed subsequently for that particular investigation. 2.3. Quantitative analysis To determine the optical density (OD) of the DAB reaction product in the glomeruli of the olfactory bulbs, we used customized NIH Image 1.61 software run on a Macintosh computer attached to a Leitz DMRB microscope (Leica) and Pulnix video camera. At the start of each image capture session, the scope illumination was adjusted to a standard level using a blank slide. A slide mounted OD step tablet was imaged and a calibration curve generated. OD of negative control slides served as background OD. For each olfactory bulb, a field of view was captured and all glomeruli contained in it were delineated. The gray value for each glomerulus was recorded and converted to an OD value. Background OD was
then subtracted from that value. The number of glomeruli per section and the area of each glomerulus were also determined. 2.4. Statistical analysis The normality of the data was assessed using the Shapiro–Wilk test. Between group differences for mean glomerular immunodensities were assessed with a non-parametric one factor repeated measures ANOVA test (Brunner et al., 2001). Between group differences for age, PMI, mean glomerular counts and mean glomerular areas were assessed with Mann– Whitney–Wilcoxon test. Gender differences between schizophrenia and control groups were assessed with the v 2 test. To assess effects of potentially confounding factors, including age, PMI, the effects of antipsychotic medication exposure and other demographic data on OD, we used Spearman Rank correlation analysis on each individual assay. An alpha level of 0.05 was used to determine significance.
3. Results 3.1. Immunolabeling distribution in olfactory bulb OMP Immunoreactivity (Fig. 1A) was strong and present only in axon processes within the olfactory receptor neuron layer and in glomeruli. No cell demonstrated immunostaining in any of the layers of the olfactory bulb. Similar pattern of staining was observed for GAP43 and NCAM (Fig. 1B and C, respectively). In addition, we observed mildly to moderately stained fibers in the granular and internal plexiform layer of several subjects. Poorly phosphorylated NFM/HP immunoreactivity (Fig. 1D) was present in all layers of the olfactory bulb except for the olfactory receptor neuron layer. NFM/HP labeling was very strong in glomeruli and what look like mitral cells, moderately strong in the external plexiform layer but quite faint in neurons and processes within the other layers. Immunohistochemistry for h-tubulin (Fig. 1E) was present in all layers of the olfactory bulb. Strong labeling was expressed within the olfactory receptor neuron layer and in glomeruli. Some juxtaglomerular and granular cells were also strongly labeled. Moderate labeling of processes was observed in the external and internal plexiform layers. Faint labeling was also present in the granular layer. Synaptophysin immunoreactivity (Fig. 1F) was strongly expressed in the glomeruli. Faint labeling of processes was
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Fig. 1. Photomicrographs depicting immunoreactivity for (A) OMP, (B) GAP43, (C) NCAM, (D) NFM/HP, (E) h-tubulin III, (F) synaptophysin, (G) calbindin and (H) trkB in coronal sections of human olfactory bulbs. Arrows indicate (A) glomeruli and (G) periglomerular cells. Window (H) shows mitral cells. Scale bar, 100 Am.
also present in external and internal plexiform layers. No cell demonstrated immunostaining in any of the layers of the olfactory bulb. Calbindin immunoreactivity (Fig. 1G) was observed in all layers of the olfactory bulb. Strong immunohistochem-
istry for calbindin was seen in glomeruli and some juxtaglomerular cells. Some labeled cells were also observed in other layers except for the olfactory receptor neuron layer. Strong immunohistochemistry for trkB (Fig. 1H) was expressed within the olfactory receptor neuron layer and
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in glomeruli. Granular, juxtaglomerular and what look like mitral cells showed moderate labeling. Unless indicated, the morphologic characteristics and distribution of labeled elements were consistent across the subjects studied for each of the antibodies used in this study. 3.2. Quantification of immunoreactivity in glomeruli As shown in Table 2, the glomerular level of each proteins investigated in this study was not different in subjects with schizophrenia when compared to controls. Correlation analysis found no significant relationship between OD values for most proteins and age for both control (rho V 0.52, p z 0.08) and schizophrenia (rho V 0.47, p z 0.15) groups. In the control group, correlations with age were inconclusive for calbindin (rho = 0.17, p = 0.59 and rho = 0.62, p = 0.04) and trkB (rho = 0.14, p = 0.66 and rho = 0.66, p = 0.02). No significant correlation was found between OD values for most proteins and PMI for both control (rho V 0.48, p z 0.16) and schizophrenia (rho V 0.63, p z 0.07) groups. In the control group, correlations with PMI were inconclusive for calbindin (rho = 0.04, p = 0.92 and rho = 0.61, p = 0.03) and NCAM (rho = 0.25, p = 0.49 and rho = 0.65, p = 0.03). In the schizophrenia group, correlations with PMI were inconclusive for RMDO (rho = 0.50, p = 0.10 and rho = 0.73, p = 0.01) and almost reach significance for one tubulin assay (rho = 0.76, p = 0.05 and rho = 0.07, p = 0.82). In conclusion, we have not demonstrated any definitive correlation between age or PMI and the glomerular level of any of the studied proteins. No significant correlation was found either between OD values for most proteins and age of onset (rho V 0.57,
p z 0.17), duration of illness (rho V 0.62, p z 0.10) or antipsychotic dosage (rho V 0.50, p z 0.21) for the schizophrenia group. Correlation between OD and antipsychotic dosage almost reached significance for one of the tubulin assays (rho = 0.66, p = 0.05 and rho = 0.19, p = 0.69). Correlation between OD and age of onset was inconclusive for calbindin (rho = 0.08, p = 0.80 and rho = 0.63, p = 0.04). Again, we have not demonstrated any definitive correlation between these three factors and the glomerular level of any of the studied protein. No significant difference between groups were observed in the number of glomeruli per section (Z V 1.33, p z 0.18) or in the mean area of the glomeruli (Z V 0.83, p z 0.32) labeled with any of the antibodies. There were no group differences in age (Z V 1.78, p z 0.08) for the cases labeled with each of the antibodies except for calbindin (Z = 2.21, p = 0.03) and RMDO20 (Z = 2.07, p = 0.04). There were no group differences in PMI (Z V 1.04, p z 0.30) for the cases labeled with each of the antibodies. The relative frequencies of male and female was the same for both schizophrenia and control group (v 2 V 0.22, p z 0.64).
4. Discussion The present study reports the protein expression patterns of several pre- and post-synaptic markers in the human olfactory bulb. While the distribution of these proteins varies widely, they are all located in the glomeruli.
Table 2 Glomerular level of various proteins expressed in mean optical density (OD) Antigen
Location/function
OD schizophrenia, total n (2 repeats)
OD control, total n (2 repeats)
ANOVA test t (df), p
OMP
0.12)
1.42 (24.68), 0.17
Growth cone/axon guidance
0.08)
0.47 (26.84), 0.64
NCAM
ORNs/adhesion molecule
0.12)
0.83 (20.77), 0.41
TrkB
ORNs and OB cells/BDNF receptor Dendrite/forms neurofilaments
0.43 (S.D. 22 subjects 0.31 (S.D. 23 subjects 0.17 (S.D. 26 subjects 0.28 (S.D. 21 subjects 0.09 (S.D. 24 subjects 0.35 (S.D. 24 subjects 0.07 (S.D. 23 subjects 0.20 (S.D. 20 subjects
1.11 (23.70), 0.28
GAP43
0.48 (S.D. 0.11) 21 subjects 0.27 (S.D. 0.15) 18 subjects 0.18 (S.D. 0.08) 23 subjects 0.26 (S.D. 0.12) 21 subjects 0.06 (S.D. 0.03) 20 subjects 0.39 (S.D. 0.12) 24 subjects 0.06 (S.D. 0.05) 28 subjects 0.23 (S.D. 0.08) 19 subjects
0.16)
Synaptophysin
ORNs marker/neural transmission Synaptic vesicle/exocytosis
0.06)
1.93 (23.02), 0.07
0.10)
1.50 (22.96), 0.15
0.05)
1.44 (23.99), 0.16
0.10)
0.93 (20.78), 0.36
NFM/HP Calbindin h-Tubulin III
PG neurons/Ca2+ binding protein Neurons/forms microtubules
ORN = olfactory receptor neuron, OB = olfactory bulb, PG = periglomerular neuron.
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As previously shown, OMP, GAP43 and NCAM immunoreactivity was only present within the olfactory receptor neuron layer and in glomeruli of human olfactory bulb (Hoogland et al., 2003; Smith et al., 1991). Like in rodent, poorly phosphorylated NFM/ HP labeling was present in all layers of the olfactory bulb except for the olfactory receptor neuron layer (Chien et al., 1998). Consistent with earlier studies in human and rodents, h-tubulin III was present in all layers of the olfactory bulb, strongly labeling glomeruli as well as periglomerular and granular cells (Be´dard and Parent, 2004; Gonzales and Silver, 1994; Roskams et al., 1998). Calbindin immunoreactivity was observed in all layers of the olfactory bulb but was particularly strong in the glomeruli and juxtaglomerular cells, as previously reported (Alonzo et al., 2001; Ohm et al., 1991). While negative controls did not show any nonspecific labeling, no Western blot or pre-adsorption study was done with human brain tissue to exclude the possibility of detecting other proteins sharing a domain homolog to the peptide used to raise the calbindin antibody. As expected from a previous study in rodent, synaptophysin was strongly expressed in the glomeruli and also present in the plexiform layers (Karowski et al., 1999). TrkB immunoreactivity was found in various levels in the glomeruli as well as in the mitral, granular and juxtaglomerular cells of the human olfactory bulb. A similar distribution of trkB has been reported in the rat with the difference that the glomeruli were only lightly stained (Yan et al., 1997). While this suggests that glomerular trkB could be located in dendrites of mitral and periglomerular cells, it could also be located in axon terminals of olfactory receptor neurons. TrkB has also been shown to be expressed by human olfactory receptor neurons (Nibu et al., 1999). In rodents, it has been shown that trkB is expressed by post-mitotic, immature olfactory receptor neurons, first in their cell bodies and dendrites and then primarily in their axons as they mature (Deckner et al., 1993; Holcomb et al., 1995; Roskams et al., 1996). The glomerular level of these proteins was measured to assess the integrity of the connection between the olfactory epithelium and olfactory bulb in schizophrenia. The present study relies on optical density
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(OD) measurement to evaluate the relative concentration of these proteins in postmortem olfactory bulb glomeruli. While optical densitometry allows rapid, objective and automatic evaluation of immunohistochemical label intensity, concerns exist that this method of quantification may not reflect the amount of a specific protein across tissue sections because of the non-linearity of the immunohistochemical procedure. However, several studies have shown that the measure of OD of an immunolabeled protein using DAB as a chromogen is in fact consistently proportional to its concentration in a tissue section as long as the range of OD measured in the tissue fall within the linear range of the OD calibration curve (Jojich and Pourcho, 1996; Nabors et al., 1988; Rieux et al., 2002; Schipper and Tilders, 1983). Given that the various steps in processing and analysis are standardized, from the histological treatments to image analysis, the use of this method for quantitative analysis is reliable. We noted no difference in the immunodensity of OMP, a protein exclusively expressed by mature olfactory receptor neurons, in the glomeruli (Arnold et al., 2001; Nakashima et al., 1985). This finding suggests intact olfactory receptor neuron innervation of the olfactory bulb. While OMP expressing olfactory receptor neurons have been shown to decline following olfactory bulbectomy, OMP level increase in remaining olfactory receptor neurons (Carr et al., 1998). OMP expression in glomeruli disappears after destruction of olfactory epithelium (Ehrlich et al., 1990). However, since OMP immunostaining persists in axon terminals severed from their parent cell body for several days, it may not be the best marker of intact olfactory receptor neuron axons (Slotnick et al., 2001). On the other hand, the concomitant absence of change in the level of GAP43 and NCAM in the glomeruli also suggests no major loss of olfactory receptor neuron innervation or major insults to the olfactory bulb in schizophrenia. This is consistent with a previous study showing no change in the number of OMP cells in the olfactory epithelium in schizophrenia (Arnold et al., 2001). This is also supported by the absence of change in the glomerular level of calbindin, which is decreased by the destruction of the olfactory epithelium (Philpot et al., 1997). We noted no difference either in the immunodensity of synaptophysin, a synaptic vesicle membrane
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component, which is often used as a general marker of the presence of synapses. This and above findings suggest that axons of olfactory receptor neurons establish synapses with their targets in the olfactory bulb glomeruli and do not support the notion of a generalized deficit in pre-synaptic proteins in schizophrenia. Studies of pre-synaptic proteins in cortical areas in schizophrenia are too inconsistent to support that notion either (Halim et al., 2003; Honer et al., 2000; Honer and Young, 2004; Mirnics et al., 2000). However, they do not exclude discrete changes in selected pre-synaptic proteins. No significant changes were found in the expression of poorly phosphorylated NFM/HP, a cytoskeletal element selective for dendrites. As with the presynaptic proteins, our finding suggests no general lack of dendritic innervation in the glomeruli. These findings are consistent with the absence of change in the level of h-tubulin III, a protein expressed by both olfactory receptor neurons and its target neurons in the olfactory bulb (Be´dard and Parent, 2004; Gonzales and Silver, 1994; Roskams et al., 1998). Finally, we observed no significant decrease in trkB expression, the receptor for BDNF, which is an important regulator of growth and survival of mature olfactory receptor neurons. This finding contrasts with other studies suggesting a reduction of neuronal trophic support in schizophrenia (Hashimoto et al., 2005; Takahashi et al., 2000; Weickert et al., 2003). Observed reduction in trkB may be restricted to specific brain areas that do not include the olfactory bulb. However, since the decrease in glomerular trkB reached a trend level of significance, a net decrease in trkB level may have been masked by an increase in packing density resulting from the reduced volume of the olfactory bulb in schizophrenia. Using only two sections might have not been sufficient to detect changes that may also be more pronounced in certain areas of the olfactory bulb. These last two confounds might equally apply to other proteins as well. One other consideration in the interpretation of our results is the potential effect of neuroleptics on the expression of these eight proteins. Studies on the effects of neuroleptics on the brain level of each protein have been negative or inconclusive (Eastwood et al., 1997; Lidow et al., 2001; Linden et al., 2000). Because in our study, neuroleptic dosage was not correlated to the level of any of these proteins, neu-
roleptic exposure may not be the main determinant of their expression. Moreover, no study has noted differences between neuroleptic-naive, neuroleptic withdrawn, and currently medicated schizophrenia patients on a variety of olfactory tests (Geddes et al., 1991; Kohler et al., 2001). Studies searching for molecular correlates of synaptic deficits in schizophrenia have found reduced, increased and no change in the expression of pre- and post-synaptic markers (Honer et al., 2000; Mirnics et al., 2001), inconsistent with a generalized deficit in synaptic proteins. As caution must be heeded because of the small sample size in our study, our findings suggest that axons of olfactory receptor neurons establish synapses with their targets in the olfactory bulb glomeruli. They also indicate no general lack of dendritic innervation of the glomeruli by these targets. Despite the absence of changes in the pre- and post-synaptic elements in glomeruli, it is possible that with other markers we could detect selective abnormalities in the molecular integrity of these structures. MAP2, a microtubule-associated protein that binds and stabilizes microtubules in dendrites, is significantly decreased in the olfactory bulb glomeruli in schizophrenia (Rioux et al., 2004). While these findings suggest that changes in the olfactory epithelium are not associated with major structural changes in the olfactory bulb glomeruli, one must not forget that the volume of olfactory bulb in schizophrenia is decreased when compared to control (Turetsky et al., 2000). Although a stereological approach might be able to detect glomerular changes not otherwise observable, they would not be sufficient to account for the 23% reduction of the olfactory bulb volume reported in schizophrenia. Changes in other layers of the olfactory epithelium have to contribute to the size reduction observed in patients with schizophrenia and, could impact the olfactory epithelium by indirectly altering the synaptic transmission between the olfactory receptor neurons and its targets.
Acknowledgement OMP, RMDO20 and MOC-1 antibodies were gifts from Drs. F.L. Margolis, V.M.-Y. Lee and J.Q. Trojanowski, respectively. We want to acknowledge the
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