Regulation of dendrite formation of Purkinje cells by serotonin through serotonin1A and serotonin2A receptors in culture

Neuroscience Research 48 (2004) 101–109

Regulation of dendrite formation of Purkinje cells by serotonin through serotonin1A and serotonin2A receptors in culture Mayumi Kondoh, Takashi Shiga∗ , Nobuo Okado Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan Received 31 July 2003; accepted 1 October 2003

Abstract Serotonergic fibers and receptors appear in the rat cerebellum during early postnatal development. In the present study, we investigated the actions of serotonin (5-HT) and its receptors in the dendrite formation of Purkinje cells in organotypic cultures of anterior and posterior lobes of the cerebellum at postnatal day 7. In anterior lobes after 4 days in vitro (4DIV), the dendritic areas and branchings of Purkinje cells were increased by the treatment of 2 ␮M 5-HT, but decreased by 20 ␮M 5-HT. In posterior lobes after 4DIV, the dendritic areas of Purkinje cells were increased by 5-HT (2, 20 and 200 ␮M). In contrast, 5-HT treatment decreased dendritic areas of Purkinje cells in both anterior and posterior lobes after 7DIV. Next, we determined the actions of specific 5-HT receptors in mediating the effects of 5-HT by treatment with selective 5-HT receptor agonists. In anterior lobes after 4DIV, dendritic areas of Purkinje cells were increased by a 5-HT1A receptor agonist (8-OH-DPAT), whereas decreased by a 5-HT2A receptor agonist (DOI). The present study suggested that the dendrite formation of Purkinje cells is promoted by 5-HT through 5-HT1A receptors, but inhibited by 5-HT through 5-HT2A receptors. © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. Keywords: Rat; Monoamine; Development; Cerebellum; Slice culture

1. Introduction Serotonin (5-hydroxytryptoamine (5-HT)) is a biogenic amine which is widely distributed in the central nervous system. In addition to its actions in the neurotransmission, studies show that 5-HT regulates various aspects of neural development. Thus, 5-HT regulates cell division (Lauder and Krebs, 1978), neuronal survival (Ahlemeyer et al., 1999), neurite outgrowth (Haydon et al., 1984; Lieske et al., 1999; Lotto et al., 1999), and differentiation of glutamatergic neurons (Lavdas et al., 1997). Furthermore, we and others have shown that 5-HT plays an important role in synaptogenesis (Chubakov et al., 1986a,b, 1993; Okado et al., 1993; Chen et al., 1994; Niitsu et al., 1995; Mazer et al., 1997; Yan et al., 1997a,b; Wilson et al., 1998). The roles of 5-HT in neural development are consistent with on-

Abbreviations: BDNF, brain-derived neurotrophic factor; DIV, day in vitro; DOI, 2,5-dimethoxy-4-iodoamphetamine; 5-HT, serotonin; 8-OHDPAT, 8-hydroxy-DPAT-hydrobromide ∗ Corresponding author. Tel.: +81-29-853-6961; fax: +81-29-853-6960. E-mail address: [email protected] (T. Shiga).

togenetic studies which show early differentiation of 5-HT neurons in raphe nuclei followed by widespread projections in the central nervous system (Levitt and More, 1978; Goto and Sano, 1984; Aitken and Tork, 1988). However, molecular mechanisms underlying the serotonergic regulation of neural development are not clear. In the rat cerebellum, 5-HT neurons project to the inner granule cell layer by postnatal day 6 (P6) with a few 5-HT fibers penetrating the Purkinje cell layer (Lidov and Molliver, 1982). By P10, 5-HT fibers extend into the molecular layer through the Purkinje cell layer. Several 5-HT receptors develop from the early postnatal days. The 5-HT1A receptor is detected on Purkinje cells in the first postnatal week (Daval et al., 1987; Matthiessen et al., 1992, 1993; Miquel et al., 1994). Its expression decreases subsequently and it is not detected in the adult cerebellum. On the other hand, the 5-HT2A receptor is found in the Purkinje cells shortly after birth and its expression is maintained in adulthood (Maeshima et al., 1998; Maeshima, personal communication). During the first three postnatal weeks, cerebellar cortical formation is largely determined, including arborization of Purkinje cell dendrites and migration of external granule cells. Thus, 5-HT

0168-0102/$ – see front matter © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2003.10.001

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fibers and 5-HT1A and 5-HT2A receptors appear during the active stages of cerebellar development, but functional significance of 5-HT and its receptors remains to be examined. In the present study, we examined roles of 5-HT in the dendritic development of Purkinje cells in slice cultures. We showed that dendrite formation of Purkinje cells is stimulated by 5-HT through 5-HT1A receptors, but inhibited by 5-HT through 5-HT2A receptors. These results support the hypothesis that the two receptors have antagonistic actions (Azmitia, 2001).

2.2. Treatment of 5-HT and 5-HT receptor agonists

2. Materials and methods

To investigate roles of 5-HT and 5-HT receptors on dendrite formation of Purkinje cells, we cultured cerebellar slices for 4 or 7 days in the presence of various concentrations (2, 20, 200 ␮M) of 5-HT (Sigma) and 5-HT receptor agonists. Because 5-HT1A and 5-HT2A receptors are expressed on Purkinje cells during early postnatal development, we used 5-HT1A receptor (8-hydroxy-dpathydrobromide, 8-OH-DPAT; Sigma; 200 nM, 2, 20 ␮M) and 5-HT2A receptor (2,5-dimethoxy-4-iodoamphetamine, DOI; Sigma; 10, 100 nM, 1, 10 ␮M) agonists. 5-HT and the receptor agonists were dissolved in DMEM before use.

2.1. Slice culture of the cerebellum

2.3. Immunohistochemical labeling of Purkinje cells

Wistar rats at P7 were used for slice cultures. We cultured P7 parasagittal cerebellar slices, because active elongation and arborization of Purkinje cell dendrites occur between P7 and P21 in vivo (Altman, 1972). As Purkinje cells extend their dendrites two dimensionally in a parasagittal plane (Altman, 1972), we made parasagittal slices, which enables accurate measurements of arborization and areas of the Purkinje cell dendrites. In addition, since in vivo administration of p-chlorophenylalanine (p-CPA), a 5-HT synthesis inhibitor, reduces not only 5-HT but also catecholamines (Reader and Gauthier, 1984), it may be difficult to examine specific roles of 5-HT by in vivo analyses. Therefore, we used organotypic culture by adding 5-HT in the culture medium. After decapitation under deep anesthesia, the head was sterilized in 70% ethanol for a few seconds. The cerebellum was dissected in ice-cold Hank’s balanced salt solution (HBSS; GIBCO BRL), and the pia mater was carefully removed. Sagittal slices were cut at 300 ␮m thickness using a Mcllwain tissue chopper, and were divided into anterior and posterior lobes. The slices were placed on a culture insert for six-well culture dish (Becton Dickinson) which was pre-coated with rat tail collagen. The culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (GIBCO BRL), supplemented with progesterone (20 nM; Sigma), hydrocortisone (20 nM; Sigma), sodium selenite (20 nM; Sigma), putrescine (100 ␮M; Sigma), insulin (5 ␮g/ml; Wako, Osaka, Japan), transferrin (100 ␮g/ml; Sigma), penicillin/streptomycin (1%; Sigma), and glucose (3%; Wako). In addition, we added 20% fetal bovine serum (Sigma) for the first two days of the culture. The level of the medium was adjusted slightly below the surface of the explants so that they could receive a sufficient supply of both the medium and the mixed gas. Half of the medium was exchanged every 2 days. Cultures were maintained at 37 ◦ C in an atmosphere of humidified 95% air and 5% CO2 for 4 or 7 days. All the procedures on live animals were approved by our institution’s Animal Care Committee.

After the culture, slices were immunostained by anticalbindin D-28K antibody (Sigma) which specifically labels Purkinje cells in the cerebellum (Metzger and Kapfhammer, 2000; Sakamoto et al., 2001). Cerebellar slices were fixed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 2 days at 4 ◦ C. After incubation with a blocking solution containing 2% normal horse serum (Sigma) and 0.1% Triton X-100 in PB for 2 h at room temperature, the slices were incubated with a monoclonal antibody against calbindin D-28K (1:3000 dilution) overnight at 4 ◦ C. They were then incubated with a biotinylated secondary anti-mouse IgG antibody (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature, followed by the peroxidase-conjugated avidin–biotin complex (1:100 dilution, Vector Laboratories) for 2 h at room temperature. They were then reacted with diaminobenzidine (DAB) using the ImmunoPure Metal Enhanced DAB Substrate Kit (Pierce, Rockford, IL, USA). After dehydration and clearance by xylene, the slices were mounted on slides by Eukitt (O. Kindler, Freiburg, Germany). 2.4. Analysis of the morphology of Purkinje cells 2.4.1. Analysis of areas of somata and dendrites Slides with immunostained slices were randomly coded so that an investigator cannot know the experimental conditions. Five–ten immuno-labeled Purkinje cells in each slice were traced at magnification of 1000× with a camera lucida (Olympus). These traces were converted to digital files using a scanner (CanoScan D2400U; Canon), and the areas of whole cells and somata were measured by NIH Image software package. To measure the somatic area of Purkinje cells, the border between somata and proximal dendrites was defined as the point at which the spherical shape of the somata was broken by the thin proximal dendrite. To measure dendritic area of Purkinje cells, somatic area was deducted from whole area of each cell.

M. Kondoh et al. / Neuroscience Research 48 (2004) 101–109

2.4.2. Analysis of dendritic length and branchings We analyzed dendritic length and branchings by the branching index and Sholl analysis with a slight modification (Sholl, 1953; Redmond et al., 2002). To calculate the branching index for each Purkinje cell, number of terminal dendritic branches were divided by the number of primary dendrites (Metzger and Kapfhammer, 2000). The primary dendrites and terminal dendritic branches were defined respectively as dendrites arising directly from the somata and terminal dendrites with a length of more than 10 ␮m. For modified Sholl analysis, we measured the number of dendrites which exist within a series of concentric circles from the soma at 20 ␮m interval. 2.5. Statistical analysis Effects of the treatment of 5-HT and 5-HT receptor agonists on the morphology of Purkinje cells were analyzed by Student’s t-test.

3. Results 3.1. Effects of 5-HT on dendritic development of Purkinje cells To elucidate roles of 5-HT on the dendritic development of Purkinje cells, we cultured slices of cerebellar vermis from P7 rats for 4 days or 7 days in the presence of 5-HT (2, 20, 200 ␮M). Dendritic elongation and arborization of Purkinje cells occurred during the culture (Fig. 1). In vivo, Purkinje cells have multiple “somatic processes” emanating directly from the soma during the first postnatal week. Subsequently they lose these processes and establish a single primary dendrite by P15 (Altman, 1972). In contrast, many Purkinje cells had multiple primary dendrites in slice cultures (Fig. 1, see also Metzger and Kapfhammer, 2000; Sakamoto et al., 2001).

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In anterior lobes at 4DIV, both somatic and dendritic areas of Purkinje cells were significantly increased by 30–50% after the treatment with 2 ␮M 5-HT, but decreased slightly by 20 ␮M 5-HT (Fig. 2A). In accordance with the changes in dendritic areas, the number of terminal dendritic branches and the branching index were increased by 2 ␮M 5-HT, whereas decreased by 20 ␮M 5-HT (Fig. 2B). The number of primary dendrites was decreased by both 2 and 20 ␮M 5-HT. In contrast, the treatment with 200 ␮M 5-HT yielded no changes in the somatic and dendritic areas and the branching index (Fig. 2A and B). The 5-HT regulation of dendritic branchings was also confirmed by the modified Sholl analysis which showed that the number of dendritic branches was increased in the wide regions (20–120 ␮m from the soma) by 2 ␮M 5-HT, whereas decreased in the proximal segments (0–40 ␮m by 20 ␮M 5-HT and 0–20 ␮m by 200 ␮M 5-HT; Table 1). Therefore, in the short-term culture of anterior lobes, the low concentration (2 ␮M) of 5-HT promotes the dendritic growth of Purkinje cells, whereas the higher concentration (20 ␮M) of 5-HT inhibits it. In posterior lobes at 4DIV, dendritic areas of Purkinje cells were increased by treatment of all concentrations (2, 20 and 200 ␮M) of 5-HT and somatic areas were increased by 2 ␮M 5-HT (Fig. 2C). The number of terminal branches and the branching index were increased by 2 and 200 ␮M 5-HT, and the number of primary dendrites was increased by 20 ␮M 5-HT. The modified Sholl analysis also indicated the facilitatory effects of 5-HT on dendritic arborization, showing that the number of branches was increased in distal segments (40–100 ␮m from the soma by 2 ␮M 5-HT and 40–80 ␮m by 20 and 200 ␮M 5-HT; Table 1). Therefore, 5-HT promotes dendritic arborization of Purkinje cells in the 4-day culture of posterior lobes. At 7 DIV, 5-HT inhibited the dendritic growth of Purkinje cells in both anterior and posterior lobes (Fig. 3A and C). The number of terminal branches was also reduced by 200 ␮M 5-HT in anterior lobes and by 20 ␮M 5-HT in posterior lobes (Fig. 3B and D). In anterior lobes, the modified Sholl analysis showed the reduction in the number

Fig. 1. Photomicrographs of Purkinje cells in cerebellar slices of P7 rat cultured for 4 days. Immunostained by anti-calbindin D-28K antibody. (A)–(D) show Purkinje cells in anterior lobes and (E)–(H) posterior lobes. Cultures were treated with vehicle (A) and (E), 2 ␮M 5-HT (B) and (F), 20 ␮M 5-HT (C) and (G), 200 ␮M 5-HT (D) and (H). Scale bars: 50 ␮m.

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Fig. 2. Effects of 5-HT on the development of Purkinje cells in anterior lobes (A) and (B) and posterior lobes (C) and (D) cultured for 4 days. (A) and (C) show areas of the somata and the dendrites of Purkinje cells in control and 5-HT treated groups. (B) and (D) show the number of primary dendrites and terminal dendrites and the branching index. Bars represent the mean ± SEM. Data were calculated from 70 to 150 Purkinje cells in each group. (∗ ) P < 0.05, (∗∗ ) P < 0.01, (∗∗∗ ) P < 0.001.

of dendritic branches by 20 ␮M 5-HT (0–60 ␮m from the soma) and 200 ␮M 5-HT (20–40 ␮m from the soma; Table 2). In posterior lobes, the modified Sholl analysis showed that the number of dendritic branches was decreased in 40–120 ␮m from the soma by 20 ␮M and 200 ␮M 5-HT. Therefore, 5-HT inhibits dendritic arborization of Purkinje cells in the 7-day culture of both anterior and posterior lobes. These results suggest that 5-HT has various effects on dendritic development of Purkinje cells, depending on cere-

bellar regions (anterior versus posterior lobes), 5-HT concentration and culture duration. 3.2. Differential effects of 5-HT receptor agonists on the dendritic development of Purkinje cells Next, we tried to determine roles of 5-HT receptors in 5-HT-induced changes in Purkinje cell dendrites. Since the 5-HT1A and 5-HT2A receptors are expressed on Purkinje cells in the neonatal cerebellar cortex, we focussed on the

Table 1 Effects of 5-HT on the number of dendritic branches of Purkinje cells in anterior and posterior lobes for 4DIV Lobes

Treatment

Distance from soma (␮m) 0–20

20–40

40–60

60–80

80–100

100–120

Anterior

Control 2 ␮M 20 ␮M 200 ␮M

7.31 6.65 6.01 6.52

± ± ± ±

0.28 0.35 0.36∗∗ 0.27∗

10.20 11.46 7.96 9.40

± ± ± ±

0.42 0.46∗ 0.46∗∗∗ 0.42

3.35 7.73 3.40 4.09

± ± ± ±

0.35 0.46∗∗∗ 0.41 0.44

0.41 1.81 0.68 0.69

± ± ± ±

0.13 0.31∗∗∗ 0.16 0.20

0.010 0.33 0.015 0.065

± ± ± ±

0.01 0.04∗∗ 0.015 0.37

0 0.82 ± 0.05∗ 0 0

Posterior

Control 2 ␮M 20 ␮M 200 ␮M

6.55 6.39 7.07 5.35

± ± ± ±

0.24 0.36 0.42 0.27∗∗

9.75 10.49 10.85 9.84

± ± ± ±

0.39 0.48 0.69 0.42

4.46 6.05 5.97 6.01

± ± ± ±

0.43 0.52∗∗ 0.69∗ 0.53∗

0.82 1.83 1.67 2.00

± ± ± ±

0.14 0.30∗∗∗ 0.32∗∗ 0.35∗∗∗

0.06 0.40 0.18 0.25

± ± ± ±

0.04 0.13∗∗ 0.08 0.12

0.01 0.06 0.03 0.09

∗

P < 0.05 (as compared with the control group). P < 0.01 (as compared with the control group). ∗∗∗ P < 0.001 (as compared with the control group). ∗∗

± ± ± ±

0.01 0.04 0.02 0.09

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Fig. 3. Effects of 5-HT on the development of Purkinje cells in anterior lobes (A) and (B) and posterior lobes (C) and (D) cultured for 7 days. (A) and (C) show areas of the somata and the dendrites of Purkinje cells in control and 5-HT treated groups. (B) and (D) show the number of primary and terminal dendrites and the branching index. Bars represent the mean ± SEM. Data were calculated from 60 to 130 Purkinje cells in each group. (∗ ) P < 0.05, (∗∗ ) P < 0.01, (∗∗∗ ) P < 0.001.

effects of these receptors on the development of Purkinje cell dendrites using selective receptor agonists. We cultured slices of P7 anterior lobes for 4 days in the presence of various concentrations of a 5-HT1A receptor agonist (8-OH-DPAT; 200 nM, 2, 20 ␮M) or a 5-HT2A receptor agonist (DOI; 10, 100 nM, 1, 10 ␮M). The 8-OH-DPAT treatment promoted the dendritic growth in dendritic areas, number of terminal dendrites and the branching index (Fig. 4A and B). Further examination of the dendrite morphology using the modified Sholl analysis indicated that 8-OH-DPAT treatment increased the number

of dendritic branches (Table 3). In contrast to the dendritic growth, 8-OH-DPAT had a little effect on the somatic area with a slight reduction by the treatment of 2 ␮M (Fig. 4A). Therefore, it was suggested that 5-HT promotes dendritic branch formation of Purkinje cells through the 5-HT1A receptor. In contrast, the DOI treatment resulted in the reduction of dendritic areas and the number of dendritic terminal branches (1 and 10 ␮M DOI) as well as somatic areas (10 ␮M DOI) (Fig. 5A and B). The modified Sholl analysis showed that the number of dendritic branches 20–60 ␮m

Table 2 Effects of 5-HT on the number of dendritic branches of Purkinje cells in anterior and posterior lobes for 7DIV Lobes

Treatment

Distance from soma (␮m) 0–20

20–40

40–60

60–80

80–100

100–120

120–140

Anterior

Control 2 ␮M 20 ␮M 200 ␮M

4.34 4.88 3.61 4.57

± ± ± ±

0.21 0.36 0.29∗ 0.25

10.63 10.94 8.26 9.32

± ± ± ±

0.50 0.67 0.57∗∗ 0.39∗

10.28 10.76 8.11 8.85

± ± ± ±

0.58 0.91 0.61∗ 0.50

4.63 5.84 3.61 3.52

± ± ± ±

0.49 0.87 0.56 0.45

1.11 1.54 0.63 0.70

± ± ± ±

0.27 0.47 0.23 0.21

0.16 0.3 0.065 0.067

± ± ± ±

0.11 0.13 0.065 0.031

0.009 ± 0.009 0 0.016 ± 0.016 0

Posterior

Control 2 ␮M 20 ␮M 200 ␮M

5.37 5.04 5.07 4.98

± ± ± ±

0.25 0.31 0.33 0.31

12.11 12.49 10.86 10.79

± ± ± ±

0.49 0.64 0.55 0.58

11.75 13.11 9.28 9.35

± ± ± ±

0.61 0.66 0.74∗ 0.59∗∗

6.07 6.51 3.09 3.76

± ± ± ±

0.62 0.64 0.53∗∗ 0.54∗∗

2.04 1.37 0.85 0.80

± ± ± ±

0.40 0.30 0.24∗ 0.25∗

0.71 0.22 0.063 0.16

± ± ± ±

0.21 0.10∗ 0.05∗∗∗ 0.064∗

0.086 ± 0.06 0 0.017 ± 0.02 0

∗

P < 0.05 (as compared with the control group). P < 0.01 (as compared with the control group). ∗∗∗ P < 0.001 (as compared with the control group). ∗∗

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Table 3 Effects of 5-HT 1A receptor agonists on the number of dendritic branches of Purkinje cells in anterior lobes for 4DIV Treatment

Distance from soma (␮m) 0–20

20–40

40–60

60–80

80–100

100–120

Control 200 nM

7.00 ± 0.19 7.07 ± 0.291

12.40 ± 0.25 14.19 ± 0.45∗∗∗

7.51 ± 0.30 10.05 ± 0.48∗∗∗

1.71 ± 0.17 3.14 ± 0.35∗∗∗

0.16 ± 0.04 0.43 ± 0.13∗

0.015 ± 0.01 0.017 ± 0.01

2 ␮M 20 ␮M

7.51 ± 0.39 6.08 ± 0.31∗

14.63 ± 0.58∗∗∗ 13.59 ± 0.52∗

10.01 ± 0.70∗∗∗ 11.54 ± 0.60∗∗∗

2.95 ± 0.46∗∗ 4.06 ± 0.45∗∗∗

0.45 ± 0.15∗ 0.67 ± 0.18∗∗∗

0.083 ± 0.05∗ 0.18 ± 0.095∗∗

∗

P < 0.05 (as compared with the control group). P < 0.01 (as compared with the control group). ∗∗∗ P < 0.001 (as compared with the control group). ∗∗

Table 4 Effects of 5-HT 2A receptor agonists on the number of dendritic branches of Purkinje cells in anterior lobes for 4DIV Treatment

Distance from soma (␮m) 0–20

20–40

40–60

60–80

80–100

100–120

Control 10 nM 100 nM

7.07 ± 0.23 7.69 ± 0.35 7.47 ± 0.34

14.38 ± 0.36 15.15 ± 0.42 15.41 ± 0.57

9.85 ± 0.38 9.76 ± 0.59 11.15 ± 0.69

2.77 ± 0.26 3.45 ± 0.50 3.21 ± 0.46

0.38 ± 0.09 0.44 ± 0.11 0.47 ± 0.16

0.020 ± 0.012 0.043 ± 0.025 0.012 ± 0.012

1 ␮M 10 ␮M

6.62 ± 0.27 6.74 ± 0.28

12.90 ± 0.37∗∗ 11.52 ± 0.38∗∗∗

2.47 ± 0.29 1.56 ± 0.20∗∗∗

0.34 ± 0.095 0.2 ± 0.07

0.040 ± 0.033 0.015 ± 0.011

8.56 ± 0.41∗ 7.23 ± 0.41∗∗∗

∗

P < 0.05 (as compared with the control group). P < 0.01 (as compared with the control group). ∗∗∗ P < 0.001 (as compared with the control group). ∗∗

Fig. 4. Effects of 5-HT1A agonist (8-OH-DPAT) on the development of Purkinje cells in anterior lobes cultured for 4 days. Part (A) shows areas of the somata and the dendrites of Purkinje cells in control and 8-OH-DPAT treated groups. Part (B) shows the number of primary dendrites and terminal dendrites and the branching index. Bars represent the mean ± SEM. Data were calculated from 80 to 120 Purkinje cells in each group. (∗∗ ) P < 0.01, (∗∗∗ ) P < 0.001.

Fig. 5. Effects of 5-HT2A agonist (DOI) on the development of Purkinje cells in anterior lobes cultured for 4 days. Part (A) shows areas of the somata and the dendrites of Purkinje cells in control and DOI treated groups. Part (B) shows the number of primary and terminal dendrites and the branching index. Bars represent the mean ± SEM. Data were calculated from 100 to 200 Purkinje cells in each group. (∗∗ ) P < 0.01, (∗∗∗ ) P < 0.001.

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from the soma was decreased by 1 ␮M DOI and in 20–80 ␮m by 10 ␮M (Table 4). The number of primary dendrites and branching index were not affected by DOI. It seems unlikely that the effects of DOI are toxic because similar doses (e.g. 3 ␮M) increase excitatory postsynaptic currents in pyramidal cells of cerebral cortical slices (Aghajanian and Marek, 1999). Therefore, these results suggest that 5-HT inhibits dendritic arborization of Purkinje cells through the 5-HT2A receptors.

4. Discussion The present in vitro study using cerebellar explants demonstrated that 5-HT has both stimulatory and inhibitory effects on the dendrite formation of Purkinje cells, depending on cerebellar regions, developmental stages and 5-HT concentrations. We also suggested using selective receptor agonists that these stimulatory and inhibitory effects may be mediated by 5-HT1A receptors and 5-HT2A receptors, respectively. 4.1. Roles of 5-HT in dendritic development of Purkinje cells Purkinje cells are a good model to examine roles of 5-HT systems in the dendritic development, since 5-HT fibers project to Purkinje cells and these cells express 5-HT receptors during the dendritic growth and arborization (see Section 1). We investigated effects of 5-HT on dendrite formation of Purkinje cells by culturing P7 rat cerebellar slices for 4 or 7 days. When anterior lobes were cultured for 4 days, the dendrite formation in dendritic areas and branchings of Purkinje cells were promoted by the treatment of low concentration (2 ␮M) of 5-HT, but inhibited by higher concentration (20 ␮M) of 5-HT. In posterior lobes, 5-HT treatment promoted the dendritic growth in dendritic areas and the number of distal dendrites. In contrast, in the longer culture for 7 days, 5-HT treatment inhibited the dendrite formation of Purkinje cells in both anterior and posterior lobes. We found effects of 5-HT on the dendrite formation of Purkinje cells were dose-related, especially in the 4-day culture of anterior lobes. As the 5-HT concentration in the extracellular spaces of the neonatal rat cerebellum is not known, the present study cannot conclude in vivo roles of 5-HT. There are, however, several in vitro studies in which effects of 5-HT were examined (Chubakov et al., 1986a,b; Wang et al., 1992; Mitoma et al., 1994; Riad et al., 1994; Lavdas et al., 1997). An electrophysiological experiment reported that the maximum excitability of Purkinje cells in adult rat cerebellar slices is elicited by the treatment of 10–50 ␮M 5-HT (Wang et al., 1992). Therefore, the 5-HT concentration used in the present study (2–200 ␮M) seems to be within the physiological range. Dose-related effects of 5-HT

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may be due to differences of 5-HT receptors (see below), and appropriate concentrations of 5-HT may be necessary for the proper development of Purkinje cell dendrites. It is not clear whether the effects of 5-HT on the Purkinje cell dendrites shown in the present study are direct on Purkinje cells or indirect through other cells. It may be possible that effects of 5-HT are mediated directly through 5-HT1A and 5-HT2A receptors which are expressed on Purkinje cells during the early postnatal weeks. However, we cannot exclude the possibility that effects of 5-HT are mediated by other cells. Astrocytes may participate in the trophic roles of 5-HT, since it is well established that the activation of 5-HT1A receptors on these glial cells induces secretion of S-100␤, a growth-promoting factor (Whitaker-Azmitia et al., 1990; Whitaker-Azmitia and Azmitia, 1994; Azmitia et al., 1995; Eriksen et al., 2002). In addition, analyses using perturbation experiments and mutant animals suggest that afferent inputs from granule cells may regulate dendritic development of Purkinje cells (Baptista et al., 1994). Culture of a single population of Purkinje cells versus co-culture of Purkinje cells with astrocytes or granule cells may be needed to elucidate the actions of 5-HT on these cells. 4.2. Stimulatory and inhibitory effects by 5-HT on dendritic development of Purkinje cells may be mediated through different 5-HT receptors The present study showed that 5-HT had differential effects on the dendritic development of Purkinje cells depending on cerebellar regions, 5-HT concentrations and culture duration. It is possible that different subtypes of 5-HT receptors may contribute to these differential effects. Our functional analyses using selective agonists demonstrated that 5-HT1A receptors stimulate dendritic growth of Purkinje cells, whereas 5-HT2A receptors inhibit it. Antagonistic roles of 5-HT1A and 5-HT2A receptors are also shown in neuronal proliferation and differentiation (for review, see Azmitia, 2001). Both in vitro and in vivo studies using 5-HT1A receptor agonists or antagonists have shown that 5-HT1A receptors have stimulatory effects on elongation and arborization of dendrites and synapse formation in cerebral cortex, hippocampus, septum and hypothalamus (Riad et al., 1994; Azmitia et al., 1995; Nishi et al., 1996; Wilson et al., 1998; Faber and Haring, 1999), although an in vitro study showed that 5-HT1A receptor agonist decreases neurite length and arborization in fetal rat neocortex (Sikich et al., 1990). The present study showed that 5-HT1A receptor agonist increases the dendritic area and branchings of Purkinje cells in the cerebellum. In addition, the present study indicated that 5-HT2A receptor agonist has opposing actions in the dendritic development of Purkinje cells. It has been reported that 5-HT2A receptors activate protein kinase C (for review, Peroutka, 1993, 1994; Raymond et al., 2001) which

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in turn inhibits dendritic growth of Purkinje cells (Metzger and Kapfhammer, 2000). It is, therefore, possible that 5-HT inhibits dendritic development through 5-HT2A receptor by activating protein kinase C. As discussed above, S-100␤ produced by astrocytes may be involved in the trophic effects through 5-HT1A receptors. In addition, neurotrophins may also participate in the trophic effects. Brain-derived neurotrophic factor (BDNF) plays an important role in the differentiation of Purkinje cells (Hirai and Launey, 2000; Mertz et al., 2000; Carter et al., 2002) and dendritic spine formation of Purkinje cells (Shimada et al., 1998). It has been reported that BDNF and its TrkB receptor are detected in Purkinje cells (Maisonpierre et al., 1990; Das et al., 2001; Light et al., 2002). Although not clear in the cerebellum, 5-HT regulates the expression of BDNF mRNA in neocortex and hippocampus (Vaidya et al., 1997; Zetterstrom et al., 1999). Collectively, it is suggested that 5-HT may stimulate the dendritic development of Purkinje cells through BDNF. Our preliminary experiments using 5-HT1A agonists in combination with anti-BDNF antibody suggest that 5-HT1A receptor promotes dendritic growth of Purkinje cells through BDNF (unpublished observation). The differential effects of 5-HT on dendritic development of Purkinje cells, depending on cerebellar regions, 5-HT concentrations and culture duration may be explained by 5-HT receptors. The 5-HT1A and 5-HT2A receptors have several different properties including the expression and ligand affinity (Azmitia, 2001). First, 5-HT1A receptors have higher affinity for 5-HT than 5-HT2A receptors (Peroutka, 1994). Second, the amount of the 5-HT1A receptor expression is higher in posterior lobes than in anterior lobes (Daval et al., 1987; Matthiessen et al., 1992, 1993; Miquel et al., 1994). Finally, although both receptors are detected as early as the first postnatal week, the expression of 5-HT1A receptors is subsequently down-regulated, whereas the expression of 5-HT2A receptors is maintained until adulthood (Daval et al., 1987; Maeshima et al., 1998; Matthiessen et al., 1992, 1993; Miquel et al., 1994). These properties of 5-HT receptors may explain the differential effects of 5-HT on the development of Purkinje cell dendrites. For example, the stimulatory effects of 5-HT in the 4 day cultured posterior lobes may be mediated by 5-HT1A receptors which are highly expressed in this region during early postnatal days. In contrast, the inhibitory effects of 5-HT in 7 day-culture may be mediated by 5-HT2A receptors which remain to be highly expressed, whereas the 5-HT1A receptor expression is down-regulated during the postnatal periods. The dose-related effects of 5-HT on the dendritic development of Purkinje cells in the 4 day cultured anterior lobes may be due to that low concentration of 5-HT predominantly activates 5-HT1A receptors and higher 5-HT concentration activates 5-HT2A receptors as well as 5-HT1A receptors. To clarify contributions of these receptors, the amount of expression of these receptors in cultured cerebellum needs to be investigated.

Acknowledgements We thank Dr. E.C. Azmitia (New York Univ.) for critically reading the manuscript. We also thank Dr. N. Yamamoto (Osaka University) for helpful suggestions of the culture method. This work was supported by a Grant for Special Research Project on Dynamics Brain Function and Amenity for the Mind, University of Tsukuba to T.S. and N.O. We also thank Dr. Hiromitsu Minato for his financial support.

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