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Microtubule-associated protein MAP2 expression in olfactory bulb in schizophrenia
Psychiatry Research 128 (2004) 1 – 7 www.elsevier.com/locate/psychres
Microtubule-associated protein MAP2 expression in olfactory bulb in schizophrenia Lise Rioux *, Delta Ruscheinsky, Steven Edward Arnold Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Received 13 October 2003; received in revised form 26 March 2004; accepted 22 May 2004
Abstract Previous studies have described alterations in presynaptic and postsynaptic elements in various parts of the CNS in schizophrenia, which may, at least in part, be due to abnormalities in neurodevelopmental processes. The olfactory bulb (OB) is a unique CNS area for examining synaptic development and plasticity in schizophrenia because it undergoes continuous reinnervation throughout life. Moreover, olfactory deficits and reduced OB volume have been observed in schizophrenia. We investigated the expression in the OB of the microtubule-associated protein MAP2, which has been shown to be abnormally expressed in the hippocampal region in schizophrenia. In both developing and mature neurons, MAP2 is an important structural component of dendrites and participates in the modification of synaptic organization. We used immunocytochemistry with phosphoepitope-specific and phosphorylation-state-independent antibodies to examine MAP2 expression in the glomerular layer of the OB in elderly subjects with chronic schizophrenia and controls. Phosphorylation-independent MAP2 expression was significantly reduced in schizophrenia, while phosphorylated MAP2 expression did not differ between groups. These results are consistent with faulty OB innervation in schizophrenia. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: AP18; Glomerulus; Dendrite; Plasticity
1. Introduction Psychophysical studies have documented deficits in odor detection, identification, and memory in schizophrenia (Moberg et al., 1999). Clinical neurobiological studies have shown that the olfactory bulb (OB) is smaller and that olfactory evoked response potentials
* 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).
are abnormal in schizophrenia (Moberg et al., 1999; Turetsky et al., 2000). Additionally, postmortem studies have described cellular and molecular abnormalities in the olfactory epithelium (Arnold et al., 2001). Given the mounting evidence for abnormal neurodevelopment in schizophrenia, the OB provides a valuable window into the cellular and molecular processes of axon guidance, synaptogenesis and synaptic plasticity that may go awry in schizophrenia (Arnold and Rioux, 2001). Its value as a model system lies on the fact that the OB undergoes continuous reinnervation throughout life as olfactory receptor neurons (ORNs) in the neuroepithelium of the nose turn over and send new axons to synapse with the glomeruli of
0165-1781/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.psychres.2004.05.022
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L. Rioux et al. / Psychiatry Research 128 (2004) 1–7
the OB (Farbman, 1992). The development and appearance of glomeruli depend on influences imparted by ingrowing axon terminals from the olfactory epithelium (Philpot et al., 1997). Within the glomeruli, the ORN axons make excitatory synapses with the dendrites from mitral, tufted, and periglomerular neurons of the OB (Kratskin, 1997) (Fig. 1). Additionally, the intraglomerular circuit consists of the mitral and tufted cell reciprocal dendrodendritic synapses with the periglomerular cells. Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein important for the genesis and maintenance of dendrites (Matus, 1988). In both developing and mature neurons, MAP2 participates in the modification of synaptic and dendritic
morphology. MAP2 expression is dynamic, with decreases of MAP2 mRNA and protein reported in neurons following axonal injury (Svensson and Aldskogius, 1992a,b) and increases in MAP2 immunoreactivity observed with remodeling of dendrites after denervation (Ca´ceres et al., 1988). MAP2 has multiple phosphorylation sites recognized by a variety of kinases and phosphatases (Walaas and Nairn, 1989). MAP2 phosphorylation affects microtubule stability and growth, as well as MAP2’s binding to neurofilaments and microfilaments (Hirokawa et al., 1988; Audesirk et al., 1997). Neural activity in hippocampus modifies dendritic organization, in part, by regulating MAP2 phosphorylation (Quinlan and Halpain, 1996). Experience-dependent
Fig. 1. Diagram of the olfactory bulb. The olfactory bulb (OB) receives input from the receptor neurons in the olfactory epithelium (OE) and projects to the olfactory cortex (CTX). The olfactory bulb is composed of six layers (from inside out): AON, accessory olfactory nucleus; GCL, granule cell layer; ML, mitral cell layer; EPL, external plexiform layer, GLM, glomerular layer; ON, olfactory nerve layer. The main neural elements of the olfactory bulb include: GC, granule cell; MC, mitral cell; PG, periglomerular cell; TC, tufted cell. Abbreviations: olfactory tract (OT); cribriform plate (CP).
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modifications in MAP2 phosphorylation also have been observed in the developing OB (Philpot et al., 1997). While still controversial, MAP2 expression has been reported to be altered in the subiculum (Rosoklija et al., 2000; Arnold et al., 1991), entorhinal cortex (Arnold et al., 1991;), hippocampus (Cotter et al., 1997, 2000) and prefrontal cortex (Jones et al., 2002) in schizophrenia, but no changes have been observed in the cerebellum (Mukaetova-Ladinska et al., 2002). Its expression has not been investigated in the olfactory system. The present study used immunocytochemistry to examine changes in MAP2 expression and phosphorylation in glomeruli of the OB in schizophrenia.
2. Material and methods Olfactory bulbs were obtained at autopsy from 11 subjects with schizophrenia and seven nonpsychiatric control subjects comparable for age, sex and postmortem interval (PMI) (Table 1). Schizophrenic subjects had been elderly participants in a prospective clinicopathological studies program and diagnosed according to DSM-III-R/DSM-IV criteria based on medical history, interviews with caregivers and direct clinical examination (Arnold et al., 1995). All schizophrenia subjects had required chronic hospitalization because of severe symptomatology including cognitive and functional impairment. Patients were excluded from the study if they had additional psychiatric or neurologic disorders predating or subsequent to the onset of psychiatric symptoms. Written informed consent for antemortem evaluation and autopsy was obtained from
Table 1 Clinical and demographic data on human subjects Schizophrenic Mean S.D. Age (years) 78.5 7.4 PMI (h) 12.2 6.3 Age of onset (years) 26.9 5.1 Duration (years) 52.7 7.7 CPZ1MO 175 351
Control Range
Mean S.D. Range
67 – 87 6.5 – 30 16 – 33 39 – 60 0 – 800
73.9 11.8 na na na
10.7 6.0 na na na
60 – 91 4.0 – 21 na na na
CPZ1MO = antipsychotic dosage 1 month before death; S.D. = standard deviation; na = not available.
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next of kin. Controls were seven non-neurologic subjects obtained through the University of Pennsylvania’s Center for Neurodegenerative Disease Research. While none of them had undergone antemortem assessments, a review of their clinical histories found no evidence of psychiatric or neurological illness. At the time of death, subjects were excluded because of anoxic injury or extensive injury due to trauma, stroke, neoplasm, infection or toxins. Gross and microscopic diagnostic neuropathological examinations were conducted in all cases and were normal. Olfactory bulbs were dissected at autopsy, fixed in formalin (3.7% formaldehyde in 0.1 M Tris, 0.9 g/ l NaCl (TBS)) or ethanol (70% ethyl alcohol, 150 mM NaCl) for 24 h, paraffin-embedded and cut into 20-Am-thick sections. Mounted sections were labeled with a phosphorylation-independent mouse monoclonal antibody to MAP2, clone C, and a mouse monoclonal phosphospecific antibody, AP18. Both well-characterized antibodies specifically recognized all isoforms of MAP2. AP18 recognized MAP2 containing a phosphorylated Serine 136 residue (Riederer and Innocenti, 1992; Riederer et al., 1995). Sections were deparaffinized, rehydrated and then incubated for 30 min in 1% H2O2. After two washes in TBS, sections were incubated in TBS containing 4% normal horse serum (NHS) for 60 min at room temperature. The sections were then incubated overnight at 4 jC in TBS containing 4% NHS and AP18 (neat) or clone C (neat), rinsed three times with TBS and incubated in biotinylated secondary antibody in TBS with 4% NHS (1:100, BioGenex) for 1 h at room temperature. After three rinses with TBS, they were incubated 1 h in streptavidin –horseradish peroxidase (1:100, BioGenex) 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, and coded for blind analysis by one observer (D.R.). A section incubated without primary antibody was processed in parallel and served as a negative control. All cases were included in a single, precisely timed run. One section per case was used in this study. To determine the optical density (OD) of the DAB reaction product in the glomeruli of OBs, we used
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Brain 3.0 (Nissanov and McEachron, 1991) 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. To correct for unevenness in background illumination and non-square pixels, shading correction and aspect ratio correction were also performed on each captured image. A slide-mounted OD step tablet was imaged and a calibration curve generated. OD of negative control slides served as background OD. For each OB, 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. Between-group differences for glomerular values, age and PMI were assessed with the Mann-Whitney U-test with an alpha level of 0.05 used to determine significance. Gender differences between schizophrenia and control groups were assessed with the v2 test. We assessed effects of potentially confounding factors, including age, PMI and antipsychotic medication exposure, on glomerular MAP2 ODs with Spearman Rank correlation analysis.
mm2 (AP-18) and 0.012 (S.D. 0.04) mm2 (clone C) for schizophrenia subjects. OD values were reduced in schizophrenia subjects compared with controls for phosphorylation-independent (PI) MAP2 using the MAP2 antibody clone C (Mann – Whitney, U = 15, n = 18, P = 0.03) while the difference for phosphorylated (P) MAP2 using AP18 was less in schizophrenia, but did not reach significance (Mann –Whitney, U = 22, n = 18, P = 0.14) (Fig. 2). There were no between-group differences for age (Mann – Whitney, U = 28, n = 18, P = 0.34), sex (v2 = 0.51, n = 18, P = 0.47) or PMI (Mann –Whitney, U = 37, n = 18, P = 0.89). Within the control group, there was no correlation between glomerular PI MAP2 OD and age (Z = 1.6, P = 0.12) or PMI (Z = 1.5, P = 0.14). There was no correlation either between glomerular P MAP2 OD and age (Z = 0.5, P = 0.60) or PMI (Z = 0.5, P = 0.64) in this group. Within the schizophrenia group, there was no correlation between glomerular PI MAP2 OD and age (Z = 0.05, P = 0.96), PMI (Z = 1.8, P = 0.07) or antipsychotic medication dose (Z = 0.14, P = 0.89). Similarly, there was no correlation between glomerular P MAP2 OD and age (Z = 0.2, P = 0.52), PMI (Z = 1.9, P = 0.06), or antipsychotic medication dose (Z = 0.14, P = 0.89) in this same group.
3. Results MAP2-immunoreactive dendrites were abundant in the glomeruli of both control and schizophrenia subjects. No significant difference between groups were observed in the number of glomeruli per section identified with AP18 (Mann – Whitney, U = 31, n = 18, P = 0.70) or clone C (Mann – Whitney, U = 30.5, n = 18, P = 0.66). The mean number of glomeruli per section was 25.86 (S.D. 20.78) glomeruli (AP-18) and 12.86 (S.D. 8.19) glomeruli (clone C) for controls and 22.82 (S.D. 18.87) glomeruli (AP-18) and 10.91 (7.94) glomeruli (clone C) for schizophrenia subjects. There was also no difference in the mean cross-sectional areas of glomeruli labeled with AP18 (Mann – Whitney, U = 35, n = 18, P = 0.75) or clone C (Mann – Whitney, U = 38, n = 18, P = 0.96). The mean area of the glomeruli was 0.012 (S.D. 0.005) mm2 (AP-18) and 0.014 (S.D. 0.006) mm2 (clone C) for controls and 0.017 (S.D. 0.015)
Fig. 2. Scatterplot of OD values for phosphorylated MAP2 (AP18) and total MAP2 (clone C) immunoreactivity from control (Co) and schizophrenia (Sz) subjects. Bars represent mean OD for each group. *indicates P < 0.05 when compared with control.
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4. Discussion The present study relies on optical density measurement to evaluate the relative concentration of immunolabeled MAP2 in postmortem OB. It shows that schizophrenia is associated with a change in MAP2 expression in the OB using an antibody that recognizes MAP2 in a phosphorylation-independent state. Our results also indicate a trend toward a reduction of phosphorylated MAP2. Optical densitometry allows rapid, objective and automatic evaluation of immunohistochemical label intensity. The use of this method for quantitative analysis requires that the various steps in processing and analysis be standardized, from the histological treatments to image analysis. Even then, concerns exist that this method of quantification may not reflect the amount of a specific protein in the brain sections. This immunohistochemical procedure involving DAB as the chromogen leads to the formation of antigen, primary antibody, secondary biotinylated antibody, avidin-biotin-peroxidase, and DAB in which each step of the processing can react nonlinearly. This nonlinearity is thought to be the source of significant variation in the evaluation of the relative concentration of an immunolabeled protein across tissue sections. However, a recent study has shown that the measure of OD of an immunolabeled protein using DAB as a chromogen is in fact proportional to its concentration in a tissue section as long as the range of OD measured in the tissue falls within the linear range of the OD calibration curve (Rieux et al., 2002). Is the observed reduction in MAP2 expression in the OB related to the disease process? With increasing postmortem interval, MAP dendritic staining has been shown to diminish and be replaced by neuronal staining (Schwad et al., 1994). However, since both experimental groups had similar postmortem intervals and there was no significant correlation between MAP2 levels and PMIs for either the control or schizophrenia groups, a reduction in MAP2 expression is likely to represent a disease-related change. The observed MAP2 reduction could result from reduction of dendritic arborization in the glomeruli or from a decrease in the amount of MAP2 per glomerular dendrite. One consequence of the ongoing normal
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neurogenesis of ORNs in the olfactory epithelium is that the OB is being continually reinnervated (Farbman, 1992). The OB neurons and dendrites that synapse with the ingrowing ORN axons within the glomeruli must, therefore, remain in a state of high plasticity. MAP2 expression has complex functions and its expression precedes the morphological appearance of mature, functional dendrites (Moore et al., 1998). A lower MAP2 expression in schizophrenia may reflect reduced capacity for dendrogenesis by any or all of the OB neurons extending their dendrites into the glomeruli and connecting to ORN axons. In a preliminary study, we screened a number of other dendritic and spine markers in OB glomeruli, including neurofilaments, Homer 1b and PSD 95, but found no altered expression of these proteins in schizophrenia subjects (Rioux et al., 2002). This indicates that the density of dendrites in OB glomeruli of schizophrenia subjects is similar to control values and that the MAP2 deficit represents a molecule or pathway specific abnormality. The formation of abnormal synapses between the ORNs and its OB targets could affect the expression of MAP2 per dendrite. This would be compatible with the dysregulation of olfactory receptor neuron lineage that we have observed in the olfactory epithelium of schizophrenia subjects (Arnold and Rioux, 2001), which suggests the persistence of ORNs in an immature state that may be incapable of establishing mature synaptic connections (Verhaagen et al., 1989). A reduction in the activity of ORNs could also affect the differentiation of dendrites in the neurons of the OB (Matsutani and Yamamoto, 2000) and reduce the expression of MAP2 in the glomeruli. However, a reduction of ORN activity may more likely affect the phosphorylation of MAP2 as previously shown (Philpot et al., 1997). Finally, the output neurons of the OB, the mitral and tufted cells, synapse on neurons in the entorhinal cortex, an area that also exhibits abnormalities in schizophrenia (Arnold, 2000). Change in glomerular MAP2 expression may therefore result from the failure of the OB neurons to receive adequate trophic support from their entorhinal targets (Shipley and Ennis, 1996). Postmortem interval has been shown to produce MAP2 dephosphorylation, making it difficult to attri-
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bute the findings of altered postmortem phosphorylated MAP2 levels to in vivo phosphorylation levels (Schwab et al., 1994). Since both groups had similar postmortem intervals and there was no significant correlation between PMAP2 levels and PMIs for either the control or the schizophrenia group, a change in MAP2 phosphorylation could have represented a disease state. While the reduction in glomerular phosphorylated MAP2 did not reach statistical significance, this study does not completely exclude a change in the phosphorylation state of MAP2. AP18 only recognizes MAP2 phosphorylated at Ser136, and it is possible that additional phosphorylation state changes occur at other residues (Walaas and Nairn, 1989). Moreover, due to the small sample size, it is possible that a modest change in the level of phosphorylated MAP2 could not be detected. A final possibility we cannot exclude is an increase in phosphorylation due to chronic neuroleptic treatment (Lidow et al., 2001). In conclusion, the decrease in MAP2 expression in the OB extends previous findings in the hippocampal region, another highly plastic brain area (Arnold et al., 1991; Rosoklija et al., 2000). We speculate that at least one pathophysiological process underlying schizophrenia is a disturbance in the ability to successfully establish and remodel functional synapses. If so, then these molecular – structural abnormalities would be most evident in areas of high plasticity. Dynamic areas of the CNS such as the OB are important windows into these processes.
Acknowledgements MAP2 antibodies were gifts from Dr. Beat M. Riederer. We want to thank Williams Bug and Dr. Jonathan Nissanov for their comments on the manuscript. We are grateful to Louise Bertrand for her technical advice. This work was supported by NIH grants MH00978, MH43880 and MH57401. We express our gratitude to the patients, families, and caregivers that participated in this work and our appreciation to the residents and staff of the University of Pennsylvania Schizophrenia Center, Department of Psychiatry, and Department of Pathology and Laboratory Medicine.
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L. Rioux et al. / Psychiatry Research 128 (2004) 1–7 Matsutani, S., Yamamoto, N., 2000. Differentiation of mitral cell dendrites in the developing main olfactory bulbs of normal and naris-occluded rats. Journal of Comparative Neurology 418, 402 – 410. Matus, A., 1988. Microtubule-associated proteins: their potential role in determining neuronal morphology. Annual Review of Neuroscience 11, 29 – 44. Moberg, P.J., Agrin, R., Gur, R.E., Gur, R.C., Turetsky, B.I., Doty, R.L., 1999. Olfactory dysfunction in schizophrenia: a qualitative and quantitative review. Neuropsychopharmacology 21, 325 – 340. Moore, J.K., Guan, Y.L., Shi, S.R., 1998. MAP2 expression in developing dendrites of human brainstem auditory neurons. Journal of Chemical Neuroanatomy 16 (1), 1 – 15. Mukaetova-Ladinska, E.B., Hurt, J., Honer, W.G., Harrington, C.R., Wischik, C.M., 2002. Loss of synaptic but not cytoskeletal proteins in the cerebellum of chronic schizophrenics. Neuroscience Letters 317, 161 – 165. Nissanov, J., McEachron, D.L., 1991. Advances in image processing for autoradiography. Journal of Chemistry & Neuroanatomy 4, 329 – 342. Philpot, B.D., Lim, J.H., Halpain, S., 1997. Experience-dependent modifications in MAP2 phosphorylation in rat olfactory bulb. Journal of Neuroscience 24, 9596 – 9604. Quinlan, E.M., Halpain, S., 1996. Postsynaptic mechanisms for bidirectional control of MAP2 phosphorylation by glutamate receptors. Neuron 16, 357 – 368. Riederer, B.M., Innocenti, G.M., 1992. MAP2 isoforms in developing cat cerebral cortex and corpus callosum. European Journal of Neuroscience 4, 1376 – 1386. Riederer, B.M., Draberova, E., Viklicky, V., Draber, P., 1995. Changes of MAP2 phosphorylation during brain development. Journal of Histochemistry and Cytochemistry 43, 1269 – 1284. Rieux, C., Carney, R., Lupi, D., Dkhissi-Benyahya, O., Jansen, K., Chounlamountri, N., Foster, R.G., Cooper, H.M., 2002. Analysis of immunohistochemical label of Fos protein in the supra-
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chiasmatic nucleus: comparison of different methods of quantification. Journal of Biological Rhythms 17 (2), 121 – 136. Rioux, L., Kazi, H.A., Arnold, S.E., 2002. Abstracts-Society for Neuroscience 32 Program no. 706.2. Rosoklija, G., Toomayan, G., Ellis, S.P., Keilp, J.G., Mann, J.J., Latov, N., Hays, A.P., Dwork, A.J., 2000. Structural abnormalities of subicular dendrites in subjects with schizophrenia and mood disorders: preliminary findings. Archives of General Psychiatry 57 (4), 349 – 356. Schwab, C., Bondada, V., Sparks, D.L., Cahan, L.D., Geddes, J.W., 1994. Postmortem changes in the levels and localization of microtubule-associated proteins (tau, MAP2 and MAP1B) in the rat and human hippocampus. Hippocampus 4 (2), 210 – 225. Shipley, M.T., Ennis, M., 1996. Functional organization of olfactory system. Journal of Neurobiology 30 (1), 123 – 176. Svensson, M., Aldskogius, H., 1992a. The effect of axon injury on microtubule-associated protein MAP2, 3 and 5 in the hypoglossal nucleus of the adult rat. Journal of Neurocytology 21, 222 – 231. Svensson, M., Aldskogius, H., 1992b. The effect of axon injury on microtubule-associated protein MAP2 mRNA in the hypoglossal nucleus of the adult rat. Brain Research 581, 319 – 322. Turetsky, B.I., Moberg, P.J., Yousem, D.M., Doty, R.L., Arnold, S.E., Gur, R.E., 2000. Reduced olfactory bulb volume in patients with schizophrenia. American Journal of Psychiatry 157, 828 – 830. Verhaagen, J., Ostreicher, A.B., Gispen, W.H., Margolis, F.L., 1989. The expression of the growth associated protein B50/GAP43 in the olfactory system of neonatal and adult rats. Journal of Neuroscience 9, 683 – 691. Walaas, S.J., Nairn, A.C., 1989. Multisite phosphorylation of microtubule-associated protein 2 (MAP2) in rat brain: peptide mapping distinguishes between cyclic AMP-, calcium/ calmodulin-, and calcium/phospholipid-regulated phosphorylation mechanisms. Journal of Molecular Neuroscience 1, 117 – 127.
Microtubule-associated protein MAP2 expression in olfactory bulb in schizophrenia Lise Rioux *, Delta Ruscheinsky, Steven Edward Arnold Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Received 13 October 2003; received in revised form 26 March 2004; accepted 22 May 2004
Abstract Previous studies have described alterations in presynaptic and postsynaptic elements in various parts of the CNS in schizophrenia, which may, at least in part, be due to abnormalities in neurodevelopmental processes. The olfactory bulb (OB) is a unique CNS area for examining synaptic development and plasticity in schizophrenia because it undergoes continuous reinnervation throughout life. Moreover, olfactory deficits and reduced OB volume have been observed in schizophrenia. We investigated the expression in the OB of the microtubule-associated protein MAP2, which has been shown to be abnormally expressed in the hippocampal region in schizophrenia. In both developing and mature neurons, MAP2 is an important structural component of dendrites and participates in the modification of synaptic organization. We used immunocytochemistry with phosphoepitope-specific and phosphorylation-state-independent antibodies to examine MAP2 expression in the glomerular layer of the OB in elderly subjects with chronic schizophrenia and controls. Phosphorylation-independent MAP2 expression was significantly reduced in schizophrenia, while phosphorylated MAP2 expression did not differ between groups. These results are consistent with faulty OB innervation in schizophrenia. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: AP18; Glomerulus; Dendrite; Plasticity
1. Introduction Psychophysical studies have documented deficits in odor detection, identification, and memory in schizophrenia (Moberg et al., 1999). Clinical neurobiological studies have shown that the olfactory bulb (OB) is smaller and that olfactory evoked response potentials
* 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).
are abnormal in schizophrenia (Moberg et al., 1999; Turetsky et al., 2000). Additionally, postmortem studies have described cellular and molecular abnormalities in the olfactory epithelium (Arnold et al., 2001). Given the mounting evidence for abnormal neurodevelopment in schizophrenia, the OB provides a valuable window into the cellular and molecular processes of axon guidance, synaptogenesis and synaptic plasticity that may go awry in schizophrenia (Arnold and Rioux, 2001). Its value as a model system lies on the fact that the OB undergoes continuous reinnervation throughout life as olfactory receptor neurons (ORNs) in the neuroepithelium of the nose turn over and send new axons to synapse with the glomeruli of
0165-1781/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.psychres.2004.05.022
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L. Rioux et al. / Psychiatry Research 128 (2004) 1–7
the OB (Farbman, 1992). The development and appearance of glomeruli depend on influences imparted by ingrowing axon terminals from the olfactory epithelium (Philpot et al., 1997). Within the glomeruli, the ORN axons make excitatory synapses with the dendrites from mitral, tufted, and periglomerular neurons of the OB (Kratskin, 1997) (Fig. 1). Additionally, the intraglomerular circuit consists of the mitral and tufted cell reciprocal dendrodendritic synapses with the periglomerular cells. Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein important for the genesis and maintenance of dendrites (Matus, 1988). In both developing and mature neurons, MAP2 participates in the modification of synaptic and dendritic
morphology. MAP2 expression is dynamic, with decreases of MAP2 mRNA and protein reported in neurons following axonal injury (Svensson and Aldskogius, 1992a,b) and increases in MAP2 immunoreactivity observed with remodeling of dendrites after denervation (Ca´ceres et al., 1988). MAP2 has multiple phosphorylation sites recognized by a variety of kinases and phosphatases (Walaas and Nairn, 1989). MAP2 phosphorylation affects microtubule stability and growth, as well as MAP2’s binding to neurofilaments and microfilaments (Hirokawa et al., 1988; Audesirk et al., 1997). Neural activity in hippocampus modifies dendritic organization, in part, by regulating MAP2 phosphorylation (Quinlan and Halpain, 1996). Experience-dependent
Fig. 1. Diagram of the olfactory bulb. The olfactory bulb (OB) receives input from the receptor neurons in the olfactory epithelium (OE) and projects to the olfactory cortex (CTX). The olfactory bulb is composed of six layers (from inside out): AON, accessory olfactory nucleus; GCL, granule cell layer; ML, mitral cell layer; EPL, external plexiform layer, GLM, glomerular layer; ON, olfactory nerve layer. The main neural elements of the olfactory bulb include: GC, granule cell; MC, mitral cell; PG, periglomerular cell; TC, tufted cell. Abbreviations: olfactory tract (OT); cribriform plate (CP).
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modifications in MAP2 phosphorylation also have been observed in the developing OB (Philpot et al., 1997). While still controversial, MAP2 expression has been reported to be altered in the subiculum (Rosoklija et al., 2000; Arnold et al., 1991), entorhinal cortex (Arnold et al., 1991;), hippocampus (Cotter et al., 1997, 2000) and prefrontal cortex (Jones et al., 2002) in schizophrenia, but no changes have been observed in the cerebellum (Mukaetova-Ladinska et al., 2002). Its expression has not been investigated in the olfactory system. The present study used immunocytochemistry to examine changes in MAP2 expression and phosphorylation in glomeruli of the OB in schizophrenia.
2. Material and methods Olfactory bulbs were obtained at autopsy from 11 subjects with schizophrenia and seven nonpsychiatric control subjects comparable for age, sex and postmortem interval (PMI) (Table 1). Schizophrenic subjects had been elderly participants in a prospective clinicopathological studies program and diagnosed according to DSM-III-R/DSM-IV criteria based on medical history, interviews with caregivers and direct clinical examination (Arnold et al., 1995). All schizophrenia subjects had required chronic hospitalization because of severe symptomatology including cognitive and functional impairment. Patients were excluded from the study if they had additional psychiatric or neurologic disorders predating or subsequent to the onset of psychiatric symptoms. Written informed consent for antemortem evaluation and autopsy was obtained from
Table 1 Clinical and demographic data on human subjects Schizophrenic Mean S.D. Age (years) 78.5 7.4 PMI (h) 12.2 6.3 Age of onset (years) 26.9 5.1 Duration (years) 52.7 7.7 CPZ1MO 175 351
Control Range
Mean S.D. Range
67 – 87 6.5 – 30 16 – 33 39 – 60 0 – 800
73.9 11.8 na na na
10.7 6.0 na na na
60 – 91 4.0 – 21 na na na
CPZ1MO = antipsychotic dosage 1 month before death; S.D. = standard deviation; na = not available.
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next of kin. Controls were seven non-neurologic subjects obtained through the University of Pennsylvania’s Center for Neurodegenerative Disease Research. While none of them had undergone antemortem assessments, a review of their clinical histories found no evidence of psychiatric or neurological illness. At the time of death, subjects were excluded because of anoxic injury or extensive injury due to trauma, stroke, neoplasm, infection or toxins. Gross and microscopic diagnostic neuropathological examinations were conducted in all cases and were normal. Olfactory bulbs were dissected at autopsy, fixed in formalin (3.7% formaldehyde in 0.1 M Tris, 0.9 g/ l NaCl (TBS)) or ethanol (70% ethyl alcohol, 150 mM NaCl) for 24 h, paraffin-embedded and cut into 20-Am-thick sections. Mounted sections were labeled with a phosphorylation-independent mouse monoclonal antibody to MAP2, clone C, and a mouse monoclonal phosphospecific antibody, AP18. Both well-characterized antibodies specifically recognized all isoforms of MAP2. AP18 recognized MAP2 containing a phosphorylated Serine 136 residue (Riederer and Innocenti, 1992; Riederer et al., 1995). Sections were deparaffinized, rehydrated and then incubated for 30 min in 1% H2O2. After two washes in TBS, sections were incubated in TBS containing 4% normal horse serum (NHS) for 60 min at room temperature. The sections were then incubated overnight at 4 jC in TBS containing 4% NHS and AP18 (neat) or clone C (neat), rinsed three times with TBS and incubated in biotinylated secondary antibody in TBS with 4% NHS (1:100, BioGenex) for 1 h at room temperature. After three rinses with TBS, they were incubated 1 h in streptavidin –horseradish peroxidase (1:100, BioGenex) 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, and coded for blind analysis by one observer (D.R.). A section incubated without primary antibody was processed in parallel and served as a negative control. All cases were included in a single, precisely timed run. One section per case was used in this study. To determine the optical density (OD) of the DAB reaction product in the glomeruli of OBs, we used
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Brain 3.0 (Nissanov and McEachron, 1991) 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. To correct for unevenness in background illumination and non-square pixels, shading correction and aspect ratio correction were also performed on each captured image. A slide-mounted OD step tablet was imaged and a calibration curve generated. OD of negative control slides served as background OD. For each OB, 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. Between-group differences for glomerular values, age and PMI were assessed with the Mann-Whitney U-test with an alpha level of 0.05 used to determine significance. Gender differences between schizophrenia and control groups were assessed with the v2 test. We assessed effects of potentially confounding factors, including age, PMI and antipsychotic medication exposure, on glomerular MAP2 ODs with Spearman Rank correlation analysis.
mm2 (AP-18) and 0.012 (S.D. 0.04) mm2 (clone C) for schizophrenia subjects. OD values were reduced in schizophrenia subjects compared with controls for phosphorylation-independent (PI) MAP2 using the MAP2 antibody clone C (Mann – Whitney, U = 15, n = 18, P = 0.03) while the difference for phosphorylated (P) MAP2 using AP18 was less in schizophrenia, but did not reach significance (Mann –Whitney, U = 22, n = 18, P = 0.14) (Fig. 2). There were no between-group differences for age (Mann – Whitney, U = 28, n = 18, P = 0.34), sex (v2 = 0.51, n = 18, P = 0.47) or PMI (Mann –Whitney, U = 37, n = 18, P = 0.89). Within the control group, there was no correlation between glomerular PI MAP2 OD and age (Z = 1.6, P = 0.12) or PMI (Z = 1.5, P = 0.14). There was no correlation either between glomerular P MAP2 OD and age (Z = 0.5, P = 0.60) or PMI (Z = 0.5, P = 0.64) in this group. Within the schizophrenia group, there was no correlation between glomerular PI MAP2 OD and age (Z = 0.05, P = 0.96), PMI (Z = 1.8, P = 0.07) or antipsychotic medication dose (Z = 0.14, P = 0.89). Similarly, there was no correlation between glomerular P MAP2 OD and age (Z = 0.2, P = 0.52), PMI (Z = 1.9, P = 0.06), or antipsychotic medication dose (Z = 0.14, P = 0.89) in this same group.
3. Results MAP2-immunoreactive dendrites were abundant in the glomeruli of both control and schizophrenia subjects. No significant difference between groups were observed in the number of glomeruli per section identified with AP18 (Mann – Whitney, U = 31, n = 18, P = 0.70) or clone C (Mann – Whitney, U = 30.5, n = 18, P = 0.66). The mean number of glomeruli per section was 25.86 (S.D. 20.78) glomeruli (AP-18) and 12.86 (S.D. 8.19) glomeruli (clone C) for controls and 22.82 (S.D. 18.87) glomeruli (AP-18) and 10.91 (7.94) glomeruli (clone C) for schizophrenia subjects. There was also no difference in the mean cross-sectional areas of glomeruli labeled with AP18 (Mann – Whitney, U = 35, n = 18, P = 0.75) or clone C (Mann – Whitney, U = 38, n = 18, P = 0.96). The mean area of the glomeruli was 0.012 (S.D. 0.005) mm2 (AP-18) and 0.014 (S.D. 0.006) mm2 (clone C) for controls and 0.017 (S.D. 0.015)
Fig. 2. Scatterplot of OD values for phosphorylated MAP2 (AP18) and total MAP2 (clone C) immunoreactivity from control (Co) and schizophrenia (Sz) subjects. Bars represent mean OD for each group. *indicates P < 0.05 when compared with control.
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4. Discussion The present study relies on optical density measurement to evaluate the relative concentration of immunolabeled MAP2 in postmortem OB. It shows that schizophrenia is associated with a change in MAP2 expression in the OB using an antibody that recognizes MAP2 in a phosphorylation-independent state. Our results also indicate a trend toward a reduction of phosphorylated MAP2. Optical densitometry allows rapid, objective and automatic evaluation of immunohistochemical label intensity. The use of this method for quantitative analysis requires that the various steps in processing and analysis be standardized, from the histological treatments to image analysis. Even then, concerns exist that this method of quantification may not reflect the amount of a specific protein in the brain sections. This immunohistochemical procedure involving DAB as the chromogen leads to the formation of antigen, primary antibody, secondary biotinylated antibody, avidin-biotin-peroxidase, and DAB in which each step of the processing can react nonlinearly. This nonlinearity is thought to be the source of significant variation in the evaluation of the relative concentration of an immunolabeled protein across tissue sections. However, a recent study has shown that the measure of OD of an immunolabeled protein using DAB as a chromogen is in fact proportional to its concentration in a tissue section as long as the range of OD measured in the tissue falls within the linear range of the OD calibration curve (Rieux et al., 2002). Is the observed reduction in MAP2 expression in the OB related to the disease process? With increasing postmortem interval, MAP dendritic staining has been shown to diminish and be replaced by neuronal staining (Schwad et al., 1994). However, since both experimental groups had similar postmortem intervals and there was no significant correlation between MAP2 levels and PMIs for either the control or schizophrenia groups, a reduction in MAP2 expression is likely to represent a disease-related change. The observed MAP2 reduction could result from reduction of dendritic arborization in the glomeruli or from a decrease in the amount of MAP2 per glomerular dendrite. One consequence of the ongoing normal
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neurogenesis of ORNs in the olfactory epithelium is that the OB is being continually reinnervated (Farbman, 1992). The OB neurons and dendrites that synapse with the ingrowing ORN axons within the glomeruli must, therefore, remain in a state of high plasticity. MAP2 expression has complex functions and its expression precedes the morphological appearance of mature, functional dendrites (Moore et al., 1998). A lower MAP2 expression in schizophrenia may reflect reduced capacity for dendrogenesis by any or all of the OB neurons extending their dendrites into the glomeruli and connecting to ORN axons. In a preliminary study, we screened a number of other dendritic and spine markers in OB glomeruli, including neurofilaments, Homer 1b and PSD 95, but found no altered expression of these proteins in schizophrenia subjects (Rioux et al., 2002). This indicates that the density of dendrites in OB glomeruli of schizophrenia subjects is similar to control values and that the MAP2 deficit represents a molecule or pathway specific abnormality. The formation of abnormal synapses between the ORNs and its OB targets could affect the expression of MAP2 per dendrite. This would be compatible with the dysregulation of olfactory receptor neuron lineage that we have observed in the olfactory epithelium of schizophrenia subjects (Arnold and Rioux, 2001), which suggests the persistence of ORNs in an immature state that may be incapable of establishing mature synaptic connections (Verhaagen et al., 1989). A reduction in the activity of ORNs could also affect the differentiation of dendrites in the neurons of the OB (Matsutani and Yamamoto, 2000) and reduce the expression of MAP2 in the glomeruli. However, a reduction of ORN activity may more likely affect the phosphorylation of MAP2 as previously shown (Philpot et al., 1997). Finally, the output neurons of the OB, the mitral and tufted cells, synapse on neurons in the entorhinal cortex, an area that also exhibits abnormalities in schizophrenia (Arnold, 2000). Change in glomerular MAP2 expression may therefore result from the failure of the OB neurons to receive adequate trophic support from their entorhinal targets (Shipley and Ennis, 1996). Postmortem interval has been shown to produce MAP2 dephosphorylation, making it difficult to attri-
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bute the findings of altered postmortem phosphorylated MAP2 levels to in vivo phosphorylation levels (Schwab et al., 1994). Since both groups had similar postmortem intervals and there was no significant correlation between PMAP2 levels and PMIs for either the control or the schizophrenia group, a change in MAP2 phosphorylation could have represented a disease state. While the reduction in glomerular phosphorylated MAP2 did not reach statistical significance, this study does not completely exclude a change in the phosphorylation state of MAP2. AP18 only recognizes MAP2 phosphorylated at Ser136, and it is possible that additional phosphorylation state changes occur at other residues (Walaas and Nairn, 1989). Moreover, due to the small sample size, it is possible that a modest change in the level of phosphorylated MAP2 could not be detected. A final possibility we cannot exclude is an increase in phosphorylation due to chronic neuroleptic treatment (Lidow et al., 2001). In conclusion, the decrease in MAP2 expression in the OB extends previous findings in the hippocampal region, another highly plastic brain area (Arnold et al., 1991; Rosoklija et al., 2000). We speculate that at least one pathophysiological process underlying schizophrenia is a disturbance in the ability to successfully establish and remodel functional synapses. If so, then these molecular – structural abnormalities would be most evident in areas of high plasticity. Dynamic areas of the CNS such as the OB are important windows into these processes.
Acknowledgements MAP2 antibodies were gifts from Dr. Beat M. Riederer. We want to thank Williams Bug and Dr. Jonathan Nissanov for their comments on the manuscript. We are grateful to Louise Bertrand for her technical advice. This work was supported by NIH grants MH00978, MH43880 and MH57401. We express our gratitude to the patients, families, and caregivers that participated in this work and our appreciation to the residents and staff of the University of Pennsylvania Schizophrenia Center, Department of Psychiatry, and Department of Pathology and Laboratory Medicine.
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