α2a adrenoceptors regulate phosphorylation of microtubule-associated protein-2 in cultured cortical neurons

Neuroscience 123 (2004) 405– 418

␣2A ADRENOCEPTORS REGULATE PHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN-2 IN CULTURED CORTICAL NEURONS Z.-M. SONG,a* O. ABOU-ZEIDa AND Y.-Y. FANGb1

1995; Berger-Sweeney and Holmann, 1997). Among different subfamilies of adrenoceptors (ARs), ␣2-ARs have been located in developing cortex. The three subtypes of ␣2-ARs, i.e. ␣2A, ␣2B and ␣2C, are encoded by three genes and have different pharmacological profiles and tissue distribution (Civantos Calzada and Alexandre de Artinano, 2001). In situ hybridization with mRNA probes indicates that ␣2A-ARs are expressed in the CNS of embryonic mice (Wang et al., 1996; Wang and Limbird, 1997). In contrast, they did not detect the expression of ␣2B- and ␣2C-AR mRNAs in the fetal brain. The involvement of ␣-ARs, in brain development has been suggested from studies on several animal species. Chemical lesion of the adrenergic afferents at birth modifies the numbers of synapses formed in the visual cortex of rats (Blue and Parnavelas, 1982). Noradrenaline pretreatment of amphibian embryos affects neuronal differentiation in subsequent cell cultures, which is blocked by ␣-AR antagonists (Rowe et al., 1993). Furthermore, the neurotrophic role of ␣2-ARs in synaptogenesis during gestation has been documented in rats (Soto-Moyano et al., 1994). However, the specific roles of ␣2-ARs on dendritic growth, a hallmark of neuronal maturation, are not known. ␣2-ARs belong to the Gi/o protein coupled receptors and exert their regulatory activity predominantly by attenuating cAMP and cAMP-dependent protein kinase A (PKA) signals (Bylund, 1988; Kobilka, 1992; Chabre et al., 1994; Koch et al., 1994; Uchida-Oka and Sugimoto, 2001). Evidence is accumulating that cAMP is a key regulator for the growth of neurons (Song and Poo, 1999). Loss of adenylate cyclase I activity in mutant mice disrupts patterning of mouse somatosensory cortex (Abdel-Majid et al., 1998). Growth of cultured spinal neurons shows attractive turning in a gradient of a membrane permeable cAMP analogues (Lohof et al., 1992). The effect of cAMP can be mediated by PKA, whose substrates include cytoskeleton proteins such as microtubule-associated protein 2 (MAP2; Koszka et al., 1991). MAP2 has a major role in promoting microtubule assembly and maintaining dendritic structures (Maccioni and Cambiazo, 1995; Sanchez et al., 2000). The activity of MAP2 is regulated predominantly via phosphorylation at multiple sites by PKA, PKC, and other protein kinases (Sanchez et al., 2000) and via dephosphorylation by various protein phosphatases (Yamamoto et al., 1988; Hiraga et al., 1993; Gong et al., 2000). Alteration in the phosphorylation state of MAP2 in neurons affects the number, length and branching of the dendrites (Wiche et al., 1991; Johnson and Jope, 1992; Audesirk et al., 1997). We hypothesize that at least one of the mechanisms respon-

a

Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Building 54, Mills Road, Canberra, ACT 0200, Australia b Department of Neurology, University of Maryland, Baltimore, MD 21201, USA

Abstract—Adrenoceptors have been suggested to mediate neuronal development. This study revealed the expression of ␣2A adrenoceptors in the cortical plate of fetal mouse cerebral wall. The effects of ␣2A adrenoceptor on dendrite growth were investigated in primary neuronal cultures. Application of ␣2 adrenoceptor agonists, BHT 933 or UK 14304 for 24 or 72 h resulted in a 1.5–2-fold increase in dendrite lengths. This effect was blocked by ␣2 adrenergic antagonists, RX 821002 or yohimbine, as well as a ␣2A selective antagonist, BRL 44408, but not by ␣2B/␣2C selective antagonists ARC 239, imiloxan and rauwolscine. Guanfacine, a ␣2A selective agonists, also significantly increased the dendrite lengths in culture. These results suggest that the morphological effect is wholly attributable to ␣2A adrenoceptor activation. We further tested the hypothesis that ␣2A adrenoceptors act through altering the phosphorylation state of microtubuleassociated protein 2. The results showed that the phosphorylation of microtubule-associated protein 2 was significantly reduced on both serine and threonine residues by over 40% after 2 h of application of guanfacine and was maintained at this low level for a prolonged time up to 96 h. These findings suggest that ␣2A adrenoceptors regulate the phosphorylation of microtubule-associated protein 2, which in turn mediates dendrite growth of cortical neurons. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: adrenergic receptor, cortex, cell culture, mouse, neuronal development, fetal.

Mounting evidence suggests that developing fetal brain contains high concentration of catecholamines, including adrenaline and noradrenaline. These substances regulate cell differentiation in the developing brain long before they are needed for synaptic transmission in mature brains (Lipton and Kater, 1989; Lauder, 1993; Lidow and Wang, 1

Present address: Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia. *Corresponding author. Tel: ⫹61-2-6125-4963; fax: ⫹61-2-61252687. E-mail address: [email protected] (Z.-M. Song). Abbreviations: AR, adrenoceptor; DAB, diaminobenzidine; DAPI, 4⬘,6-diamedino-2-phenylindole; DARPP-32, dopamine- and cAMPregulated phosphoprotein; E, embryonic day; HRP, horseradish peroxidase; MAP2, microtubule-associated protein 2; PBS, phosphate-buffered saline; PKA, protein kinase A; PP1, protein phosphatase 1; PP2B, Ca2⫹/calmondulin-dependent protein phosphatase.

0306-4522/04$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.09.018

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sible for the ␣2-ARs control of dendritic growth in cortical neurons involves the regulation of the phosphorylation state of MAP2. In this study, we aimed (1) to study the expression profile of ␣2-ARs in different laminae of the fetal mouse cortex, (2) to determine the effects of ␣2-ARs on dendrite growth in cortical cell cultures, and (3) to investigate whether the morphological effects correlate with changes in MAP2 phosphorylation.

EXPERIMENTAL PROCEDURES Immunohistochemistry The localization of ␣2-ARs was investigated in the cerebral wall of the Balb/c mouse at embryonic day 15 (E15) and E18. The pregnant mouse was determined by the presence of a vaginal plug that represents E0. Fetal mice were obtained at E15 or E18 from pregnant mouse anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused intracardinally for 30 s with phosphate-buffered saline (PBS; 0.15 M NaCl in 0.01 M sodium phosphate, pH 7.3), followed by 3 min perfusion with 4% paraformaldehyde in PBS. The fetal brains were dissected out, post-fixed overnight at 4 °C and cryoprotected in 20% sucrose in PBS overnight. Tissues were frozen in ⫺30 °C isopentane and sectioned on a cryostat. Coronal sections of 10 ␮m in thickness were made through the entire brain, mounted on gelatin-coated slides and air-dried. Tissue sections were treated with 0.5% H2O2 in methanol for 10 min, rinsed in PBS and incubated for 1 h in a blocking solution containing 5% normal goat serum in PBS. The sections from frontal, parietal and occipital regions were sampled and incubated overnight at 4 °C in polyclonal rabbit anti-␣2A-AR at 1:100 (Affinity Bioreagents Inc., Golden, CO, USA) After rinses in PBS, tissues labeled with antibody against ␣2A-AR were incubated for 2 h at room temperature in biotinylated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA) at 1:100, followed by incubation with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and a nickel-intensified diaminobenzidine (DAB) reaction. Following rinses in PBS, tissues were dehydrated and mounted in Permount. Images were captured with a CCD camera attached to an Olympus BH2 microscope. Some anti-␣2A-AR labeled tissues were also revealed with Cy3-conjugated goat anti-rabbit IgGs (Jackson; at 1:200), and analyzed with confocal microscopy. In order to test the specificity of the anti ␣ 2A-AR antibody, controls were set up by preabsorption of the antibody for 2 h with 10 ␮g/ml of the synthetic peptide (RIYQIAKRRTRVPPSRRG) used to produce the antibody. To test the presence of ␣2C-AR in the fetal cerebral wall, tissue sections were incubated in polyclonal guinea-pig anti␣2C-AR at 1:200 (Neuromics Inc., Minneapolis, MN, USA) for overnight, followed by Cy3-conjugated goat anti-guinea-pig IgG (Jackson) at 1:200. After rinses, tissues were mounted in buffered glycerol, and viewed with confocal microscopy. The positive control for anti-␣2C-AR was established by staining brain sections containing caudate putamen (Dossin et al., 2000) from adult mouse. Adult mice were anesthetized, perfusion-fixed and their brains were cryostat-sectioned in a way similar to the fetal tissues described above. Negative control experiments were carried out by omitting primary antibodies. To compare the expression patterns of ␣2A-AR in the mouse and the rat, fetal Wistar rats at E18 were obtained from anesthetized dam. The fetal rats were perfused and processed with the same procedure as described for fetal mice. The protocols for animal care and use were approved by the Animal Experimentation Ethics Committee of the Australian National University. All efforts were made to minimize the number of animals used and their suffering.

Confocal laser scanning microscopy Cy3-labeled tissues were viewed on a laser scanning confocal imaging system (Leica TCS 4D), equipped with a krypton/argon mixed gas laser (Leica Lasertechnik GmbH, Heidelberg, Germany). Cy3 was imaged with excitation at 568 nm and emission at 590 nm. The specimens were scanned at 1 ␮m steps throughout the thickness of the tissues, with a 16⫻ or 40⫻ oil immersion lenses. The images were collected and processed with software (Leica TCS 4D). Single images were cropped to form plates using Adobe Photoshop (v7.0; Adobe System Inc., CA, USA) without modification. Digital images were printed out on a photographic printer (Pictrography 4000; Fuji Photo Film USA, Elmsford, NY, USA).

Primary cultures of cortical neurons Primary cultures of cortical neurons were prepared as described previously by Vaccarino et al. (1995) and Song et al. (2002) with some modifications. Briefly, the telencephalon tissue was dissected from Balb/c mouse fetuses at E15 in ice-cold sterile Ca2⫹and Mg2⫹-free Hanks’ balanced salt solution (Invitrogen, Mount Waverley, Vic, Australia). The tissue was triturated into a suspension of single cells with a fire-polished Pasteur pipette and resuspended in DMEM/F12 medium supplemented with 10% (v/v) fetal bovine serum, 25 mM HEPES, 2.5 mM glutamine, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin (Invitrogen). The dispersed cells were plated on 100-mm-diameter culture dishes (Corning, Lindfield, NSW, Australia) or glass coverslips precoated with poly-Lornithine and laminin (Sigma-Aldrich, Sydney, Australia) at a density of 107 cells/dish. Culture dishes and coverslips in 24-well culture plates were placed in a water-jacketed incubator (Forma Scientific, Marietta, OH, USA) in 5% CO2/95% air at 37 °C. Three hours later, the medium was replaced with a serum-free Neurobasal medium with B27 supplement (Invitrogen).

Drug exposure After 1 day in culture, half of the medium was refreshed and ␣2-AR agonists were added. For analysis of the dose-response effects, 10⫺10–10⫺5 M of the ␣2-AR agonists, BHT 933 or UK 14304 (Sigma-Aldrich), which activate all three ␣2-AR subtypes (␣2A, ␣2B and ␣2C), were added for 24 or 72 h. To identify ␣2A-AR specific response, an ␣2A-AR selective agonist, guanfacine (10⫺6M; Tocris Cookson, Bristol, UK) was used. The receptor specificity of the agonists was tested by the addition of ␣2-AR antagonists, RX 821002 or yohimbine (10⫺6M, Tocris Cookson), and subtype specific antagonists including ␣2A-selective BRL 44408 (10⫺6M), ␣2B-selective imiloxan (10⫺6M) and ␣2B/␣2C-selective ARC 239 (10⫺6M) and ␣2C-selective rauwolscine (10⫺6M; all from Tocris Cookson). The subtype selectivity and the optimal concentration of the antagonists were based on previous publications on BRL 44408 (Beeley et al., 1995; Hopwood and Stamford, 2001; Cleary et al., 2002); imiloxan (Michel et al., 1990; Bohmann et al., 1993; Cleary et al., 2002), ARC 239 (Bylund et al., 1988; Fuder and Selbach, 1993; Parsley et al., 1999) and rauwolscine (Fuder and Selbach, 1993; Hieble et al., 1995; Scheibner et al., 2001). The controls consisted of cortical cells cultured for comparable periods of time either without any drugs or with antagonists alone. For analysis of the time course of the effects of agonists on MAP2 phosphorylation, 5-day-old cultures were incubated in 10⫺6 M of guanfacine for periods ranging from 5 min to 4 day. All drugs were dissolved initially in distilled water and final dilutions were made in culture medium.

Immunocytochemistry Cultured neurons were labeled with dendrite-specific MAP2 antibody that did not label axons (Hayashi et al. 2002). Cultured neurons

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Fig. 1. Immunohistochemical localization of ␣2A AR in the developing brain of the mouse at E15. Coronal section through frontal cerebral cortex was labeled with rabbit anti-␣2A-AR antibody and visualized by the chromogen formed from diaminobenzidine reaction (A). The boxed area in (A) is shown at higher magnification in (B). The labeled cells are present only in the cortical plate (CP) and marginal zone of the developing cerebral cortex (B) and the outer part of the ganglionic eminence (GE). No labeling is detectable in the intermediate zone (IMZ) and the ventricular and subventricular zones (VZ & SVZ). V, lateral ventricle. Scale bar⫽1 mm (A) and 200 ␮m (B).

grown on coverslips were fixed for 5 min in 4% formaldehyde in PBS, pH 7.6, and cleared in 0.1% Triton X-100 in PBS for 30 min. The cultures were then preincubated for 1 h in 5% normal goat serum (Sigma). The primary antibodies used were rabbit anti-␣2A-AR at 1:100 (Affinity Bioreagents) and mouse monoclonal anti-MAP2, which label only the neuron-specific high molecular weight a and b isoforms of MAP2 (1:1000 dilution; Sigma). Cultures were incubated with the primary antibodies for 18 –24 h at 4 °C and rinsed in PBS. For ␣2A-AR labeling, cultures were incubated in Cy3-conjugated goat anti-rabbit IgGs (Jackson) for 1 h at room temperature and then counterstained for 3 min with 300 nM 4⬘,6-diamedino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). For MAP2 labeling, cultures were incubated for 1 h in biotinylated goat anti-mouse IgGs (1:1000 dilution; Jackson), followed by Vectastain Elite ABC kit (Vector Laboratories) and fast DAB tablets (Sigma). The specificity of anti-MAP2 has been demonstrated in our previous study (Song et al., 2002).

Analysis of the proportion of ␣2A-AR expressing neurons in cultures Since our immunohistochemistry results showed the lack of expression of ␣2A-ARs in proliferative cells in the ventricular and subventricular zones (see Fig. 1), it was important to determine the proportion of ␣2A-AR-expressing neurons in culture. As demonstrated previously, over 97% of the total number of cells present in these cultures was neurons (Song et al., 2002); we therefore had essentially pure neuronal cultures under the current experimental conditions. Cultures were analyzed by comparing the number of neurons labeled by anti-␣2A-AR antibody with the total number of cells in the same cultures labeled by a nuclei counterstain DAPI as detailed above. Images of the stained cultures were digitized with a CCD camera (IR-1000E, DAGE-MTI, Michigan City, IN, USA) mounted on a Zeiss microscope. Cells were counted from captured images of five randomly selected

0.02 mm2 fields from each of three independent 1-day- or 4-dayold cultures to determine the number of ␣2A immunolabeled cell bodies and DAPI labeled nuclei.

Morphological measurement of MAP2-labeled dendrites For every experimental condition examined, images of MAP2 immunolabeled neurons in three randomly selected 0.02 mm2 fields from each of three independent cultures were captured with a Coolpix 995 digital camera (Nikon, Tokyo, Japan) mounted on a Ziess microscope. All neurons, except for those that were not included in their entirety within a given image, were subject to measurement on NIH image program. The lengths of individual dendrite were measured from their bases at the cell body to the distal ends by tracing the full length with free-hand mouse movement. The lengths measured in pixels were converted into micrometers by measuring a micrometer image at the same magnification. A total of 60 cells that conformed to this criterion were measured for each experimental condition. All measurements were made by an experimenter blind to the experimental condition of the cultures. The data were expressed as mean⫾S.E.M. and the significance of the differences was evaluated by one-way analysis of variance followed by a post-hoc Dunnett’s test. Statistical significance was set at P⬍0.05.

Analysis of MAP2 phosphorylation The phosphorylation of MAP2 on serine, threonine and tyrosine residues was examined using a nonradioisotopic method as previously described (Song et al., 2002). After 5 days in primary cultures with various combinations of drugs, culture medium was aspirated from Petri dishes and 1 ml of ice-cold lysis buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 50 mM sodium fluoride, 40 mM glycerophosphate, 0.5 mM sodium orthovanadate, 1 mM 4-(2-aminoeth-

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yl)-benzenesulfonyl fluoride, 20 ␮g/ml aprotinin, 2 ␮g/ml leupeptin, 2 ␮g/ml pepstatin A, 10 mM molybdic acid; all from Sigma) was added to each dish for 10 min. The cells were scraped off, collected and agitated on ice for 30 min. The lysates were clarified by centrifugation at 16,000⫻g for 30 min at 4 °C in a refrigerated centrifuge (Eppendorf 5415R, Hamburg, Germany), and the supernatants were processed for MAP2 purification with an Immunocatcher protein immunoprecipitation kit (CytoSignal, Irvine, CA, USA). MAP2 was immunoprecipitated with monoclonal MAP2 antibody specific for the a and b isoforms of MAP2 (Sigma) at a concentration of 20 ␮g/1000 ␮g of the total sample protein (determined using Bradford Protein Assay Kit; Sigma). The resultant immunoprecipitates were mixed 1:1 with the 2⫻ Laemmli Sample Buffer (Sigma) and boiled for 5 min. The proteins in the boiled mixtures (50 ␮l per well) were resolved by SDS-PAGE for 2.5 h at 100 V using Ready-Made 4 –15% Gradient SDS Gel in a Ready Gel Cell containing Tris/Glycine/SDS Running Buffer (Bio-Rad, Regents Park, NSW, Australia). The resolved proteins were transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences, Castle Hill, NSW, Australia) for 2 h at 100 V using a the same Bio-Rad Gel Cell containing Bio-Rad Tris/Glycine Buffer with 20% methanol (v/v) and 0.1% SDS (w/v; Sigma) to ensure a complete transfer of high molecular weight MAP2 (Song et al., 2002). The completeness of transfer was verified by post-staining of gels with Gelcode Blue Stain Reagent (Pierce, Rockford, IL, USA). The membranes were preincubated for 2 h with a Membrane Blocking Solution (Zymed Laboratories, San Francisco, CA, USA). To reveal the phosphorylation states of serine, threonine, and tyrosine residues, blots were incubated with one of the three antibodies from Zymed: rabbit anti-phosphoserine (4 ␮g), antiphosphothreonine (4 ␮g) or rabbit anti-phosphotyrosine (2 ␮g) diluted in 2 ml Membrane Blocking Solution for 3 h at room temperature. Membranes were rinsed five times in TBS containing 0.1% (v/v) Tween 20 (Sigma) and then incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson; 1:4000). After five washes in TBS, bands were visualized using SuperSignal Chemiluminescence kit (Pierce) and exposed to X-Omat AR Film (Kodak). In some later experiments, the chemiluminescence was directly digitized into a computer using a Luminescent Image Analyzer LAS-1000 (Fuji Photo Film Ltd.). The lack of cross-reactivity between antibodies to different phospho-amino acids has been demonstrated with pre-absorption test in our previous study (Song et al., 2002). To detect MAP2 in the same blots, the anti-phospho-amino acid antibodies were stripped from the membranes by washing in a buffer containing 62.5 mM Tris–HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol at 65 °C for 30 min. Membranes were rinsed in PBS, blocked in PBS/ containing 0.1% (v/v) Tween 20 and 5% non-fat milk for 1 h, and incubated for 2 h at room temperature with monoclonal anti-MAP2 antibodies (1:1000 dilution; the same clone used for immunocytochemistry and immunoprecipitation). This was followed by 1 h incubation at room temperature with HRP-conjugated goat anti-mouse IgG (Jackson Laboratories; 1:4000 dilution) and visualization by chemiluminescence as described above. These optical densities of bands were within the linear range of the films as described in Song et al. (2002). The films were digitized using a Luminescent Image Analyzer LAS-1000, and the density of the bands was determined by the software ImageGauge (Fuji Photo Film). Values were quantitatively represented in arbitrary units relative to the density of each band. Samples from all the experimental cell cultures were always processed together with their controls, and each experiment was repeated five times. The relative levels of serine, threonine or tyrosine-phosphorylated MAP2 were obtained by normalizing the anti-phosphoserine, anti-phosphothreonine or anti-phosphotyrosine immunoblot against the corresponding MAP2 immunoblot from the same membrane. Subsequently, the arbitrary values of

bands from drug-treated samples were normalized against untreated controls on the same film. The statistical analysis of the time course of ␣2-AR-specific drugs in cell cultures was performed with one-way ANOVAs followed by Tukey’s post hoc comparisons between individual groups. The differences were considered significant at P⬍0.05.

RESULTS Localization of ␣2-ARs in the fetal cerebral cortex of the mouse and the rat Immunohistochemical labeling with anti-␣2A-AR antibody revealed intensely stained cell bodies in the cortical plate and marginal zone of the developing cortex on E15 (Fig. 1) and E18 (Fig. 2A, B). No immunolabeled cells were found in other layers including the ventricular and subventricular zones and the intermediate zone (Fig. 1; Fig. 2A, B). This pattern of distribution of labeled cells was true throughout the cerebral cortex from frontal to occipital areas of E15 and E18 mice. With the dramatic increase in the thickness of the cortical plate from E15 to E18, much more cells were labeled in the cortical plate of E18 brain (Fig. 2A). The fluorescence labeling appeared to be cytoplasmic (Fig. 2B), confirming our DAB-stained sections (Fig. 1B). The exclusive expression of ␣2A-AR in post-migration cells suggests that ␣2A-ARs may be involved in the differentiation of the neurons instead of the proliferation of neuronal progenitors or the migration of immature neurons. Furthermore, intense labeled cells were located in the outer layer of the ganglionic eminence, the future basal ganglia (Fig. 1). Previous studies using in situ hybridization with ␣2A-AR mRNA probes showed that ␣2A-ARs were expressed in dividing cells, migrating neurons and post-migrating neurons in the cerebral cortex of developing rats (Winzer-Serhan et al., 1997a; Wang et al., 1996). This is different from the mouse as described above. To determine whether this was due to species difference or the sensitivity of the techniques (in situ hybridization vs. immunohistochemistry), we studied the distribution of this receptor in fetal rats with immunohistochemistry. In E18 rat cortex, the ␣2A-AR expressing cells were located exclusively in the cortical plate but not in the intermediate and ventricular zones (Fig. 2C, D). Again, the labeling appeared to be cytoplasmic (Fig. 2D). The results from the mouse and the rat indicate that the ␣2A-AR proteins are only expressed in the differentiated cells in the developing cerebral cortex. The apparent discrepancy of the expression pattern revealed in this immunohistochemical study and previous in situ hybridization results may reflect the levels of expression of the receptor at protein level and at mRNA level. In control sections, which were incubated in an anti-␣2A-AR antibody preabsorbed with the antigenic peptide prior to staining procedures, no staining was observed (Fig. 2E) Anti-␣2C-ARs antibody did not yield any labeling in the cerebral cortex of E15 and E18 mice (Fig. 2G). To check whether this was due to the failure of our immunohistochemical detection, we stained brain sections from adult mouse in parallel. We could detect a large number of

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Fig. 2. Characterization of ␣2A-AR and ␣2C-AR in the developing cortex of the mouse and the rat. Fluorescence images were captured with a confocal microscope. Immunoreactivity for ␣2A-AR was revealed in the cortical plate of the E18 mouse (A) and E18 rat (C). A portion of the cortical plate in (A) and (C) was shown at higher magnification in (B) and (D), respectively. No ␣2A-AR immunoreactivity was revealed in the intermediate zone (IMZ) and the ventricular zone (VZ) in either the mouse (A) or the rat (B). Staining was blocked by preabsorption of the anti ␣2A-AR with the antigenic peptide (E, mouse). ␣2C-AR was not located in the E18 mouse cortex (G), although the same antibody stained neurons in the caudate putamen (CPu) of the adult mouse (F). ec, external capsule. Scale bar⫽100 ␮m (A, C, E–G); 50 ␮m (B, D).

␣2C-AR expressing cells in the caudate putamen of adult mouse (Fig. 2F) as previously described (Dossin et al., 2000). Expression of ␣2A-ARs in all cultured cortical neurons As has been characterized previously (Song et al., 2002), the cortical cell cultures under this experimental condition were essentially pure neuronal cultures. However, since we plated all the cells from the developing cortex, which was a mixture of undifferentiated cells from the ventricular and subventricular zones and differentiating cells from the cortical plate, the proportion of ␣2A-AR expressing neurons had to be determined in cortical cell cultures. Cultured cells were labeled with antibodies against ␣2A-ARs and counter-stained with a nucleus marker DAPI. Cultures were analyzed on 1-day, the time-point when most drug treatments were started. Cells were counted from five randomly selected 0.02 mm2 fields from each of three independent cultures. Of these, a total

of 325 cells were counted, and a complete co-localization of ␣2A-AR immunolabeled cell bodies and DAPI labeled nuclei was revealed. No DAPI labeled nuclei was unaccounted for by ␣2A-ARs (data not shown). This indicates that all the plated cells, including those originated from proliferation zones, started to differentiate and express ␣2A-ARs within 1 day in culture, and therefore, the application of ␣2-AR active drugs would have effects on all cultured neurons. Cell counting on 4-day-old cultures (348 cells in total from three experiments) also demonstrated a complete co-localization of ␣2A-AR containing cell bodies and DAPI labeled nuclei (data not shown). These experiments showed that ␣2A-ARs were expressed in neurons throughout the culture period. Activation of ␣2A-ARs increases the length of dendrites in culture The effect of ␣2-AR activation on the growth of neuronal processes in primary cultures is shown in Figs. 3 and 4.

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Fig. 3. Effects of ␣2-AR agonists (BHT 933 and UK 14304) at different concentrations on dendritic length in primary neuronal culture. Cortical cells from E15 mice were cultured in drug-free medium for the first day, followed by drug application for the second day. ** Denotes significant difference (P⬍0.001, one way ANOVA) compared with the drug-free control.

BHT 933 or UK 14304, both ␣2-AR selective agonists, was added 24 h after the cells were plated and remained for additional 24 or 72 h. Cultured neurons were stained with MAP2 antibody that specifically labeled cell bodies and dendrites but not axons (Reinoso et al., 1996; Hayashi et al., 2002). In control cultures without any drugs neurons grew slender dendrites after 2 days in culture (Fig. 4A). Concentration-response curves of the effects of the ␣2-AR agonists, BHT 933 or UK 14304, were constructed in cultures in the presence of concentrations of 10⫺10, 10⫺9, 10⫺8, 10⫺7 and 10⫺6 M of both drugs. At concentrations at 10⫺6 and 10⫺7 M of either agonist, these drugs caused a significant increase (P⬍0.001) in the dendrite length as compared with the drug-free controls (Fig. 3; Fig. 4B, C). The effects of both agonists were not significantly different at the same molar concentration (Table 1). At all agonist concentrations examined, the increase in dendrite growth was abolished by ␣2-AR antagonists, 10⫺6M RX 821002 or 10⫺6M yohimbine (Table 1). The growth of dendrites was not significantly altered in cultures applied with antagonists alone (Fig. 4D). Since both our immunohistochemical studies and previous results with in situ hybridization (Wang et al., 1996) showed that ␣2A-AR was likely to be the only subtype of ␣2-AR expressed in the fetal mouse cerebral cortex plus that all the cultured cells expressed ␣2A-AR, it was important to determine whether ␣2A-AR alone could promote dendritic growth. We co-incubated ␣2-AR agonists, BHT 933 or UK 14303, with subtype selective antagonists including BRL 44408 (␣2A-selective), ARC 239 (␣2B/␣2Cselective), imiloxan (␣2B-selective) and rauwolscine (␣2C selective). After 48 or 72 h of drug application, cells were processed for MAP2 immunocytochemistry. As demon-

strated in Fig. 4B, C and Table 1, the lengths of the dendrites were significantly increased in BHT 933- and UK 14304-treated groups. This effect was completely abolished by ␣2A-selective antagonist BRL 44408 (Fig. 4H, I; Table 1), indicating an effect through ␣2A receptor subtype in these neurons. However, the effects of BHT 933 and UK 14304 were not blocked by any of the ␣2B or ␣2C selective antagonist: ARC 239, imiloxan or rauwolscine. The antagonists alone did not significantly change the length of dendrites (Table 1). These results demonstrated that the effect of BHT 933 and UK 14304 was wholly attributable to ␣2A-AR activation. The effect of ␣2A-AR on dendritic growth was further verified by using selective agonist guanfacine. Treatment with guanfacine at 10⫺6 M caused significant increase in dendrite length as compared with controls (Fig. 5B; Table 2) in both 2d and 4d cultures. The effect was blocked by BRL 44408 (Fig. 5C; Table 2). These results demonstrate that ␣2A-AR activation promotes the growth of dendrites in vitro. ␣2A-ARs regulation of MAP2 phosphorylation on serine and threonine residues in neuronal cultures Western blot of neuron-specific high molecular weight MAP2 immunoprecipitated from cultured cortical neurons revealed a single band of approximately 280 kDa (Figs. 6A, 7A), which was likely the b isoform of this protein (Song et al., 2002). This MAP2 always displayed phosphorylation of serine, threonine and tyrosine residues at all time-points of cultures examined (Figs. 6, 7). After cerebral cortical cells were cultured for 1 day when virtually all neurons in the culture expressed ␣2AAR, we exposed cultures for additional 4 days with the ␣2A-AR selective agonists, guanfacine at 10⫺6 M, a concentration that significant increased length of dendrites as showed above. Guanfacine resulted in a significant decrease in MAP2 phosphorylation on both serine (Fig. 6A, B) and threonine residues (Fig. 6C, D). The effects of guanfacine were abolished by the addition of 10⫺6 M of the ␣2A-AR selective antagonist, BRL 44408 (Fig. 6), whereas this antagonist alone did not affect MAP2 phosphorylation (Fig. 6). In contrast, the MAP2 phosphorylation on tyrosine residues was unchanged after guanfacine treatment for the same period (Fig. 6E, F). The time course of MAP2 phosphorylation in response to ␣2-AR stimulation was examined in 5-day-old neuronal cultures incubated with 10⫺6 M of guanfacine for periods from 5 min to 4 day, to determine both the short- and long-term effects of the agonist. Age-matched drug-free cultures served as controls. Incubation with guanfacine resulted in a statistically significant decrease in the phosphorylation of MAP2 on both serine (Fig. 7A, B) and threonine (Fig. 7C, D) residues in a time dependent manner. The phosphorylation started to decrease after 5 min agonist exposure and progressively decreased after 30 min, 2 h and 24 h (Fig. 7). The phosphorylation maintained at a low level after 4 day of drug incubation (Fig. 7). Once again, MAP2 phosphorylation on tyrosine residues was not

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Fig. 4. Photomicrographs showing the effects of ␣2-AR activation on dendrite growth in primary neuronal cultures. Cortical cells from E15 mice were maintained for 1 day in drug-free medium and then grown for an additional 1 day in the presence of culture medium alone (A), 10⫺6 M BHT 933, a ␣2-AR agonist (B), 10⫺6 M UK 14304, a ␣2-AR agonist (C), 10⫺6 M RX 821002, a ␣2-AR antagonist (D), BHT 933 in combination with RX821002 (E), UK 14304 and RX 821002 (F), 10⫺6 M BRL 44408, a ␣2A-AR selective antagonist (G), BHT in combination with BRL (H) and UK in combination with BRL (I). The soma and dendrites of neurons were visualized by immunolabeling of the heavy molecular weight neuron-specific MAP2. Note that BHT 933 and UK 14304 increased the lengths of MAP2-positive dendrites and this effect was completely abolished by both ␣2-AR antagonists and ␣2A-AR selective antagonist. Scale bar⫽100 ␮m.

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Table 1. Effects of ␣2-AR on the lengths of the longest dendrites in cultured cortical neuronsa Drugs

Dendrite length (48 h)

Control BHT 933 UK 14304 BHT 933⫹RX 821002 BHT 933⫹yohimbine UK 14304⫹RX 821002 UK 14304⫹yohimbine RX 821002 Yohimbine BHT 933⫹BRL 44408 UK 14304⫹BRL 44408 BRL 44408 BHT 933⫹ARC 239 UK 14304⫹ARC 239 ARC 239 BHT 933⫹imiloxan UK 14304⫹imiloxan Imiloxan BHT 933⫹rauwolscine UK 14304⫹rauwolscine Rauwolscine

39.00⫾2.31 66.32⫾3.41 74.91⫾2.41 39.95⫾3.03 39.24⫾2.53 37.53⫾2.19 35.97⫾2.48 35.25⫾2.08 34.66⫾1.63 46.99⫾1.81 39.58⫾1.71 42.08⫾1.97 67.57⫾2.41 66.87⫾2.29 46.15⫾1.87 57.30⫾3.14 58.17⫾3.79 35.31⫾2.14 63.55⫾4.10 56.18⫾3.01 34.52⫾1.89

P value

⬍0.001 ⬍0.001 NS NS NS NS NS NS NS NS NS ⬍0.001 ⬍0.001 NS ⬍0.001 ⬍0.001 NS ⬍0.001 ⬍0.001 NS

Dendrite length (96 h) 61.49⫾2.76 97.09⫾3.59 91.03⫾3.44 -b 60.38⫾2.44 63.23⫾2.53 67.90⫾2.75 93.08⫾3.64 88.31⫾4.18 61.51⫾2.97 81.34⫾4.39 90.29⫾5.14 49.24⫾2.81 90.71⫾5.16 93.03⫾5.52 46.55⫾1.97

P value

⬍0.001 ⬍0.001 NS NS NS ⬍0.001 ⬍0.001 NS ⬍0.001 ⬍0.001 NS ⬍0.001 ⬍0.001 NS

a The lengths of the longest MAP2-positive dendrites (␮m, mean⫾S.E.M.) of cultured cerebral cortical cells from E15 mouse fetuses. The neurons were cultured for a total of 48 or 96 h with the first 24 h drug-free and an additional 24 or 72 h in the presence of ␣2-AR selective agonists and/or ␣2-AR subtype selective antagonists. A total of 60 cells from three experiments were measured for each experimental condition. The concentrations of all drugs were 10⫺6 M. The data were statistically analyzed with one-way ANOVA. NS, non-significant differences. b -, indicates no data.

altered at any time point of guanfacine application (data not shown).

DISCUSSION This study has confirmed that ␣2A-AR is expressed in the fetal cerebral cortex of the mouse on E15. We further

reveal that the expression of ␣2A-AR is highly localized at the cortical plate, where differentiating cells are situated. In contrast, progenitor cells in the ventricular and subventricular zones do not express ␣2A-AR. This indicates that ␣2A-AR is exclusively expressed in differentiated cells and hence may regulate neuronal matura-

Fig. 5. Examples of the effects of ␣2A-AR activation on dendrite growth in primary neuronal culture. Cortical cells from E15 mice were maintained for 1 day in drug-free medium and then grown for an additional 1 day in the presence of culture medium alone (A), 10⫺6 M guanfacine, a ␣2A-AR agonist (B) and guanfacine in combination with BRL 44408 (C). The soma and dendrites of neurons were immunolabeled with an antibody against the dendrite specific MAP2. Note that guanfacine increased the lengths of MAP2-positive dendrites and this effect was completely prevented by ␣2A-AR antagonist. Scale bar⫽100 ␮m.

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Table 2. Effects of ␣2A-AR on the lengths of the longest dendrites in cultured cortical neuronsa Drugs

Dendrite length (48 h)

Control Guanfacine Guanfacine⫹BRL 44408 BRL 44408

30.63⫾2.12 57.35⫾3.11 39.34⫾2.57 32.85⫾1.80

P value

⬍0.001 NS NS

Dendrite length (96 h) 46.43⫾2.04 93.43⫾8.49 47.63⫾2.52 45.52⫾3.04

P value

⬍0.001 NS NS

a The lengths of the longest MAP2-positive dendrites (␮m, mean⫾S.E.M.) of cultured cerebral cortical cells from E15 mouse fetuses. The neurons were cultured for a total of 48 or 96 h with the first 24 h drug-free and an additional 24 or 72 h in the presence of ␣2A-AR selective agonist, guanfacine, and/or ␣2A-AR selective antagonist, BRL 44408. A total of 60 cells from three experiments were measured for each experimental condition. The concentrations of all drugs were 10⫺6 M. The data were statistically analyzed with one-way ANOVA. NS, non-significant differences.

tion. This is further evidenced by the fact that virtually all differentiated neurons in cell culture express ␣2A-AR. In these neuronal cultures, we demonstrated that activation of ␣2A-AR significantly promotes the dendrite growth by up to two-fold. To further illustrate the mechanism of the morphological effect, we studied the state of phosphorylation of MAP2, which has a direct bearing on the neuronal growth. The results show that ␣2A-AR activation significantly reduces MAP2 phosphorylation on both serine and threonine residues but not on tyrosine residues. Furthermore, the hypo-phosphorylation of MAP2 occurs 2 h after ␣2A-AR activation and maintains at low levels after 4-day treatment with ␣2A-AR agonists. The MAP2 hypo-phosphorylation correlates well with the increase in dendrite growth in culture, as documented in previous studies (Johnson and Jope, 1992; Audesirk et al., 1997; Sanchez et al., 2000). Expression profile of ␣2-ARs in embryonic cerebral cortex of mammals It has been shown that ␣2-ARs are highly expressed in the developing cerebral cortex of various mammals, including the monkey (Lidow and Rakic, 1995; Lidow and Wang, 1995; Wang and Lidow, 1997) the rat (Winzer-Serhan et al., 1997a) and the mouse (Wang and Limbird, 1997). In this study, we found that ␣2A-AR was only expressed in post-migrating cells in the cortical plate, but not in dividing cells in the ventricular and subventricular zones in the fetal mouse cortex. Our immunohistochemical staining of the developing rat at E18 revealed specific labeling of the cortical plate, whereas no labeling was revealed in the intermediate zones and proliferating zones. This is essentially the same as the developing mouse cortex as shown in this study. It appears likely that ␣2A-ARs do not mediate cellular mechanisms related to the multiplication of progenitor cells and the migration of neurons during the corticogenesis of the fetal mouse and rat brain. In contrast, receptor binding autoradiography (Wang and Lidow, 1997) and in situ hybridization with mRNA probes for each subtype of ␣2-AR (Winzer-Serhan et al., 1997a; Wang et al., 1996) show that the ␣2A-ARs are widely expressed in dividing cells, migrating neurons and post-migrating neurons in the cerebral cortex of developing rats and monkey. The discrepancy in the expression profiles in ␣2A-ARs between our immunohistochemical study and

previous in situ hybridization in the rat may reflect the inherent difference of the two techniques. It is known that in situ hybridization technique based on the detection of mRNA, which may have markedly different levels of expression from the protein that they encode (Chesselet 1998). The mismatch between the level of receptor mRNA and the protein had been reported previously in the mouse striatum, where only a small proportion of GluR1 mRNA expressing neurons were detectable with an antibody against the GluR1 protein (Noblett and Ariano, 1998). Although the lack of protein labeling was suggested to result from a transportation of the protein to other parts of a matured neuron, this leaves an answered question why dividing and migrating cortical cells lack staining in our study. We chose to use immunohistochemistry because it not only locates the receptor protein, which reflects the expression of functional receptors, but also offers the advantage of excellent cellular resolution. The two other subtypes of ␣2-AR, ␣2B and ␣2C, are not expressed in fetal cerebral cortex of the mouse and rat (Wang et al., 1996; Wang and Limbird, 1997; Nicholas et al., 1993; Winzer-Serhan et al., 1997b; WinzerSerhan and Leslie, 1997). The immunohistochemical results from this study confirmed the lack of expression of ␣2C-ARs in the cerebral cortex of the fetal mouse, as demonstrated in a previous in situ hybridization study (Wang et al., 1996). Origins of noradrenaline and adrenaline in developing cerebral cortex The cerebral cortical cells are exposed to noradrenaline and adrenaline during fetal and postnatal development. Noradrenaline in the developing cerebral cortex has two sources. (1) It is released from adrenergic axons that originate mainly from the locus coeruleus. These axons enter the developing cerebral cortex as early as E13 in the mouse, immediately after cortical plate emerges (Schlumpf et al., 1980; Caviness and Korde, 1981). The density of innervation increases with development, paralleled by an increase in noradrenaline concentration in the cortex until about 1 month old (Elias et al., 1982). (2) It comes from the fetal adrenal medulla toward the end of gestation. Similarly, developing cerebral cortex contains high levels of adrenaline comparable to those in the adult cerebral cortex (Masudi and Gilmore, 1983). Adrenaline is diffused

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Fig. 6. Effects of ␣2A-AR agonist on the level of MAP2 phosphorylation on serine (A, B) and threonine (C, D) residues in cultures of E15 mouse cortical neurons. After 1 day of drug-free culture, neurons were incubated for additional 4 day with 10⫺6 M of the ␣2A-AR agonist, guanfacine. The top blot shows the immunoreactive bands against phosphoserine (A) or phosphothreonine (C) after immunoprecipitation of cultures cells with anti-MAP2 antibodies. The bottom blot shows immunolabeling with anti-MAP2 after stripping and reprobing the same membrane previously probed with anti-phosphoserine or phosphothreonine antibody. Comparison of the levels of serine (B) or threonine (D) phosphorylation normalized to corresponding MAP2 bands. The relative phosphoserine or phosphothreonine levels in drug-treated cultures are expressed as a percentage of the drug-free culture. Data are the means⫾S.E.M. from five experiments. Asterisks represent significant differences from control cultures (* P⬍0.05; one way ANOVA, Tukey’s post-test). Note that the ␣2A-AR agonist decreases the levels of MAP2 phosphorylation on serine (A, B) or threonine (C, D) residues and that this effect is blocked by the ␣2A-AR antagonist.

through undeveloped blood– brain barrier from the fetal plasma, which is rich in adrenaline originated from fetal

adrenal medulla and from mothers through the placenta (Ben-Jonathan and Maxson, 1978).

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Fig. 7. The time course of the level of MAP2 phosphorylation on serine and threonine residues during ␣2A-AR agonist application in cultures of E15 mouse cortical neurons. During 5 days in culture the cells were exposed to ␣2A-AR agonist, guanfacine from 5 min to 4 days before the termination of cultures. A, C, The top blot shows the immunoreactive bands against phosphoserine (A) or phosphothreonine (C) after immunoprecipitation of cultures cells with anti-MAP2 antibodies. The bottom blot shows immunolabeling with anti-MAP2 after stripping and reprobing the same membrane previously probed with anti-phosphoserine or anti-phosphothreonine antibody. The last two lanes (control and 4 day guanfacine treatment) in both A and C were blotted separately from the first five lanes. B, D, Summary data of the levels of serine (B) or threonine (D) phosphorylation normalized to corresponding MAP2 bands. The relative phosphoserine (B) or phosphothreonine (D) levels in drug-treated cultures are expressed as a percentage of the drug-free culture. Asterisks represent significant differences from control cultures (* P⬍0.05; ** P⬍0.01; one way ANOVA, Tukey’s post-test).

␣2-ARs have a neurotrophic role in vitro and in vivo We have shown in this study that activation of ␣2A-ARs promotes the dendrite growth of cultured neurons, suggesting that ␣2A-ARs have a neurotrophic role. This effect may be associated with the well-documented neuroprotective roles in the CNS under various experimental conditions. In cultures of the retinal ganglion cells or cortical neurons incubated with high concentration of glutamate to induce cell death, activation of ␣2-ARs with clonidine or UK 14034 significantly increases the neuronal survival (Baptiste et al., 2002) and decreases the numbers of pycnotic neurons (Laudenbach et al., 2002). These effects are blocked by ␣2-AR antagonists, yohimbine or rauwolscine, suggesting that the activation of post-synaptic ␣2-AR protects these neurons from glutamate-induced excitoxicity. Furthermore, topically applied clonidine or UK 14034 to the rat retina protects retinal structures and increases survival of retinal ganglion cells during retinal ischemia (Lafuente et al., 2001; Chao and Osborne, 2001). This effect can be so potent that a single systemic treatment of rat with UK14304 completely prevents the death of retinal ganglion cells in ischemia-injured retina or mechanically injured optic nerve (Yoles et al., 1999; Vidal-Sanz et al., 2001). Cerebral cortical neurons are also protected by ␣2-AR activation in vivo. For example, administration of ␣2-AR agonists prevents cortical damages caused by ischemia (Jolkkonen et al., 1999) or by glutamate agonists (Laudenbach et al.,

2002). The neuroprotective effects of ␣2-ARs have also been observed during fetal development. Clonidine treatment during gestation prevents functional deficits induced by prenatal malnutrition in the rat visual cortex (SotoMoyano et al., 1994). All these data are consistent with our view of a direct neurotrophic effect of ␣2-ARs, although the in vivo experiments on whole animals (Soto-Moyano et al., 1994; Baptiste et al., 2002) could also be explained by pre-synaptic mechanisms, in addition to a post-synaptic effect of ␣2-ARs on these protected neurons. Correlation of dendrite growth and reduction of phosphorylation of MAP2 mediated by ␣2-ARs We demonstrate in this study that the increase in dendrite growth is well correlated with a reduction in the levels of MAP2 phosphorylation on both serine and threonine residues. Activation of ␣2A-AR with agonists causes up to 60% reduction of MAP2 phosphorylation after 2 h to 96 h of drug treatment. It is well known that ␣2-ARs, as a Gi/o protein-coupled receptor, regulate cellular functions predominantly through its negative coupling to adenylate cyclase and the attenuation of cAMP production (Bylund, 1988). Subsequently, the activity of cAMP-dependent PKA signal is reduced. PKA is directly or indirectly involved in the phosphorylation of MAP2 (Vallee, 1980; Sanchez et al., 2000; Song et al., 2002). Reduction in PKA activity will result in the production of hypo-phosphorylated MAP2.

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Therefore, at least one of the mechanisms for ␣2-ARs to influence the neuronal growth is through its regulation of the phosphorylation of MAP2 via PKA pathway. Furthermore, activation of ␣2ARs on neurons may trigger mechanisms that de-phosphorylate MAP2. For example, ␣2ARs can act through the G protein system to open L-type voltage-gated Ca2⫹ channels (Ichihara et al., 1993; De Luca et al., 2002), which are known to exist in neurons (Bean, 1989; Lo´pez et al., 2001; Mize et al., 2002), leading to the influx of extracellular Ca2⫹. ␣2-ARs activation may also indirectly increase the release of Ca2⫹ from internal stores (Keularts et al., 2000). The resultant increase in cytoplasmic Ca2⫹ activates the Ca2⫹/calmondulin-dependent protein phosphatase (PP2B), calcineurin (Aperia et al., 1992), which is able to de-phosphorylate MAP2 at serine and threonine residues (Goto et al., 1985; Alexa et al., 2002). Calcineurin may also trigger MAP2 dephosphorylation through the dis-inhibition of protein phosphatase 1 (PP1), resulting from the calcineurin-mediated dephosphorylation of a selective PP1 inhibitor, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein; Sanchez et al., 2000). There is evidence that PP1, PP2A and PP2B, as phosphoseryl/phosphothreonyl protein phosphatases, dephosphorylated MAP2 at specific sites phosphorylated by PKA (Alexa et al., 2002; Sanchez et al., 1996; 2000). It is likely that both the production of hypo-phosphorylated MAP2 through down-regulated PKA pathway and the dephosphorylation of MAP2 by various PPs contribute to the overall reduction of MAP2 phosphorylation. It is well known that the microtubule stabilizing activity of MAP2 is inversely proportional to the extent of its phosphorylation (Tucker, 1990; Hely et al., 2001); the decrease in MAP2 phosphorylation during ␣2-ARs activation correlates well with the increase of dendrite growth in culture. Our finding that the MAP2 phosphorylation at tyrosine residues was not significantly changed after treatment with ␣2-AR agonists is consistent with the current view that only phosphoseryl/ phosphothreonyl PPs are involved in the dynamic regulation of the phosphorylation of MAP2 (Gong et al., 2000). Acknowledgements—This study was supported by a Young Investigator Award of National Alliance for Research on Schizophrenia and Depression (Z.-M.S.). We are very grateful to Prof. Stephen Redman of the Australian National University and Prof. Michael Lidow of the University of Maryland for their support to this work and for their helpful discussions of the manuscript.

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(Accepted 19 September 2003)