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Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex
Journal of Chemical Neuroanatomy 30 (2005) 212–220 www.elsevier.com/locate/jchemneu
Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex Ken Takumi, Takuma Mori, Keiko Shimizu, Motoharu Hayashi * Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan Received 5 April 2005; received in revised form 8 August 2005; accepted 12 August 2005 Available online 10 October 2005
Abstract Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells. # 2005 Elsevier B.V. All rights reserved. Keywords: Cerebellar cortex; Primate; BDNF; NT-3; TrkC; ELISA; Immunohistochemistry
1. Introduction The mammalian cerebellar cortex is comprised of six distinct neuronal cell types, Purkinje cells, granule cells, stellate cells, basket cells, Golgi cells and unipolar brush cells. The distributions of those cells and the synaptic connections between them have been thoroughly elucidated (Palay and Chan-Palay, 1974; Ito, 1984). In addition, several developmentally critical events, such as neurogenesis, neuroblast migration and neuronal differentiation occur extensively in postnatal stages in the cerebellum. The cerebellum has therefore been used as a model system to study the role of epigenetic factors in the development of the central nervous system (CNS). Despite the fact that the cerebellum has been considered to control mainly motor systems, recent studies have demonstrated that it may also be involved in higher cognitive
* Corresponding author. Tel.: +81 568 63 0572; fax: +81 568 63 0576. E-mail address: [email protected] (M. Hayashi). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.08.004
functions. For example, cerebellar output through thalamic neurons has been shown to extend to the prefrontal cortex (Middleton and Strick, 1994) which is of importance for planning and working memory. Several functional neuroimaging studies have also demonstrated activation of the cerebellum during various cognitive tasks, such as verbal tasks, passive sensory tasks and visual attention shifting (Raichle et al., 1994; Gao et al., 1996; Allen et al., 1997). Neurotrophins are a family of structurally related proteins, which includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5) and neurotrophin-3 (NT-3). Neurotrophins and their receptors have been reported to be expressed widely in the developing and adult cerebellum of various experimental animals (Maisonpierre et al., 1990; Rocamora et al., 1993; Kawamoto et al., 1996; Lindholm et al., 1997; Friedman et al., 1998; Das et al., 2001; Dieni and Rees, 2002; Ohira and Hayashi, 2003; Ohira et al., 2004). Studies using primary cultures of rat cerebellar neurons have demonstrated promotion of survival, movement and neurite extension by neurotrophins (Segal et al., 1992;
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Lindholm et al., 1993b; Gao et al., 1995; Kubo et al., 1995; Nonomura et al., 1996; Tanaka et al., 2000). Furthermore, genetargeting studies have shown that BDNF (Schwartz et al., 1997) and NT-3 (Bates et al., 1999) are required for normal foliation. Although developmental changes in levels and localizations of neurotrophins have been investigated intensively in the rodent cerebellum (Lindholm et al., 1997), less is known about the primate cerebellum. In view of the above, we considered it worthwhile to investigate developmental patterns in the levels and cellular distributions of neurotrophins in the primate cerebellum. In this study, we examined developmental changes in the protein levels of BDNF, NT-4/5 and NT-3 by enzyme-linked immunosorbent assays (ELISAs) and the distributions of those three neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellar cortex of macaque monkeys. Most previous reports have focused on the localizations of neurotrophins in the Purkinje cells and granule cells, and precise studies on the cerebellar interneurons have not been performed. Therefore, we also analyzed the localizations of neurotrophins in inhibitory interneurons, such as stellate cells, basket cells, Golgi cells and unipolar brush cells, which are of importance in the cerebellar circuitry.
2. Materials and methods 2.1. Experimental animals and tissue preparation Twenty-three crab-eating monkeys (Macaca fascicularis) and six rhesus monkeys (Macaca mulatta) from embryonic day 120 (E120) to 15 years old were the subjects for ELISAs and twelve rhesus monkeys and three crab-eating monkeys from postnatal day 1 (P1d) to 15 years old were used for immunohistochemical analysis (Table 1). The method used to determine the embryonic day of age of fetal monkeys was previously described by Shimizu (Shimizu, 1988). Embryonic monkeys were obtained by Caesarian section under anesthesia with ketamine hydrochloride (10 mg/kg, i.m.) and maintained with halothane (1%)–N2O–oxygen inhalation. For non-survival procedures, all monkeys were pretreated with ketamine hydrochloride (10 mg/kg, i.m.) and deeply anaesthetized with pentobarbital sodium (25 mg/kg, i.v.). For ELISAs, monkeys were killed by bloodletting from Table 1 Monkeys used in this study Age (abbreviation)
ELISAs Embryonic day 120 (E120) Embryonic day 140 (E140) Postnatal day 1 (P1d) Postnatal 2 months (P2m) Postnatal 2 years (P2y) Postnatal 4 years (P4m) Postnatal 7–15 years (AD) Immunohistochemistry Postnatal day 1 (P1d) Postnatal 1 month (P1m) Postnatal 6 months (P6m) Postnatal 5–15 years (AD)
Macaca facicularis
Macaca mulatta
Male
Female
3 3 2 3 1
1 1 2 1
3
1
Male
Female
1 2
1 1
2 1 1
2
2 2 3 2
1
2
213
the carotid artery. The skulls were quickly opened and dissection of cerebellar tissue was performed on ice. The dissection of cerebellum included cerebellar cortex of lobule VI to VIII, but not cerebellar nuclei. The dissected tissues were stored at 80 8C until use. For immunohistochemistry, monkeys were perfused through the heart with 0.15 M NaCl followed by one of the fixation buffers (1) 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4 (PB), (2) 2% PFA and 0.5% glutaraldehyde (GA) in PB, (3) 4% PFA in PB or (4) 4% PFA and 0.1% GA in PB. After perfusion, the cerebellum were immediately removed and immersed in 2% PFA and 5% sucrose in PB at 4 8C for 24 h, followed by successive immersion in 10% sucrose in 0.1 M phosphate-buffered saline, pH 7.4 (PBS), 20% sucrose in PBS and 30% sucrose in PBS containing 0.02% sodium azide. The cerebellum was sagittally cut into blocks 5 mm thick and mounted in Tissue-Tek (Miles, Elkhart, IN, USA) and frozen rapidly in a dry-ice/acetone bath, and stored at 80 8C until dissection. All procedures were carried out in accordance with The Guide for the Care and Use of Laboratory Animals established by the NIH (1985) and The Guide for the Care and Use of Laboratory Primates established by the Primate Research Institute of Kyoto University (2002).
2.2. Antibodies For ELISAs, we used each of three kinds of antibodies as the coating antibody: anti-BDNF mouse monoclonal IgG, R43-01 (Radka et al., 1996), antiNT-4 mouse monoclonal IgG (Promega) and anti-NT-3 mouse monoclonal IgG (Promega). For the primary antibody, we used the following: anti-BDNF chick polyclonal IgY (Promega), anti-NT-4 chick polyclonal IgY (Promega), anti-NT3 chick polyclonal IgY (Promega) and normal chick IgY (Promega). Horseradish peroxidase (HRP) conjugated anti-chick IgY antibody (Promega) was used as secondary antibody. For immunohistochemistry, we used anti-BDNF rabbit polyclonal IgG (1:1600, sc-546, Santa Cruz Biotech., Calif., USA), NT-4/5 (1:3200, sc-545, Santa Cruz Biotech.), NT-3 (1:3200, sc-547, Santa Cruz Biotech.) and TrkC (1:400, sc-117, Santa Cruz Biotech.) as the primary antibody. As the secondary antibody, we used biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA).
2.3. ELISAs ELISA methods we used in this study were based on the procedure described by Mori et al. (2002, 2004). The monkey cerebellar tissue was homogenized in 10 volumes of extraction buffer (1 mM EDTA, 1 M guanidine hydrochloride, 10 mg/ml aprotinin, 0.2 mM benzethonium chloride, 2 mM benzamidine and 1 mM PMSF in 0.1 M phosphate buffer, pH 7.2). The homogenate was centrifuged for 30 min at 8000 g at 4 8C, the supernatants (first extraction) were collected and the sediments were rehomogenized and centrifuged as described above. The supernatant (second extraction) were collected, added to the first extraction and stored at 80 8C until use. Hundred microliters of the supernatant was used to determine the concentrations of neurotrophins. To estimate the recovery of exogenous neurotrophins in our ELISA methods, we added recombinant human (rh) neurotrophins (200 pg/ml of BDNF, 50 pg/ml of NT-3 and 50 pg/ml of NT-4; Peprotech, London, UK) into the cerebellar extract. Ninety six-well plates (Nunc, Roskilde, Denmark) were coated with 100 ml of the coating antibody in coating buffer (50 mM sodium carbonate–bicarbonate buffer, pH 9.6) and incubated overnight at 4 8C. After aspiration of excess antibody and three washes with washing buffer (50 mM Tris–HCl, 0.6 M NaCl and 0.1% Triton X-100), 300 ml of coating buffer containing 1% BSA was added to each well and the plates were incubated at room temperature for 1 h. The standard amounts of rh-neurotrophins were diluted in sample buffer (1 M guanidine hydrochloride and 1% BSA in washing buffer). After aspiration and three washes with washing buffer, 100 ml of standards or duplicated test samples were added per well and wells were incubated overnight at 4 8C. The plates were then washed three times with washing buffer, followed by incubation with 100 ml of the primary antibody in washing buffer overnight at 4 8C. After three washes, 100 ml of a 1:1000 dilution of the secondary antibody in washing buffer were added per well, and the plates were incubated for 6 h at 4 8C. After further washing, the reaction was developed for 15 min with 100 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB; Kirkegaard and Perry Laboratories, Gaithersburg, MD) and
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stopped with 100 ml of 1 M phosphoric acid. The optical density of the plates was read on a multilabel counter (Wallac 1420 ARVOsx; Wallac, Turku, Finland) at 460 nm.
2.4. Immunohistochemistry The immunohistochemical methods used in this study were similar to those described by Hayashi et al. (2000) (Ohira and Hayashi, 2003). The sections were cut at 40 mm thickness with a cryostat (HM500-M, Micron, Walldorf, Germany). Free-floating sections were washed for 30 min in PBS and incubated with PBS containing 0.1% Triton X-100, 1% bovine serum albumin and 4% normal goat serum (PBS-GB) for at least 1 h. The sections were then incubated at 4 8C for 48 h with one of the primary antibodies in PBS-GB. After several washes with PBS, the sections were incubated with the secondary antibody at 4 8C overnight. The immunoreactive sites were visualized by the avidin–biotin complex peroxidase method using the ABC Elite kit (Vector Laboratories). A solution of 0.3% H2O2, 20 mg/ml 3,30 -diamino benzidine tetrahydrochloride and 0.3% nickel ammonium sulfate in 0.05 M Tris–HCl, pH 7.6, was used as the substrate for peroxidase. The sections were mounted on slide glasses that had been precoated with VECTABOND (Vector Laboratories), and allowed to dry overnight. Adjacent sections were stained with cresyl violet (Merck, Darmstadt, Germany). They were then dehydrated in ethanol, cleared in xylene and coverslipped. In control sections, the primary antibody was replaced with the antibody preincubated with the corresponding blocking peptide. The control peptides were sc-546p (5 mg/ml, Santa Cruz Biotech.), sc-545p (5 mg/ml, Santa Cruz Biotech.), sc-547p (10 mg/ml, Santa Cruz Biotech.) and sc-117p (5 mg/ml, Santa Cruz Biotech.) for BDNF, NT-4/5, NT-3 and TrkC proteins, respectively. The remainder of the procedure was the same as that in the presence of primary antibody.
3. Results 3.1. Levels of neurotrophins in the developing monkey cerebellar cortex In our ELISAs, we could detect low levels of neurotrophins without any cross-reactivities to the other neurotrophins (Fig. 1A, C and E). Extracts from the monkey cerebellar tissues paralleled the standard curves of each rh-neurotrophins. The recoveries of all exogenous rh-neurotrophins in these ELISAs were over 80% at each developmental stage. The concentrations of BDNF were low at E120 and increased in an age-dependent manner during embryonic stages. At P1d, BDNF concentrations reached adult levels, which were almost three times higher than at E120 (Fig. 1B). Similar to the changes of BDNF, NT-4/5 levels were low at E120, increased to a peak at postnatal 2 years (P2y) and then gradually decreased toward adulthood (Fig. 1D). In contrast to BDNF and NT-4/5, the highest levels of NT-3 were observed at E120. NT-3 levels then rapidly declined until P1d and subsequently gradually decreased toward adulthood (Fig. 1F). The levels of NT-3 at adult stages were more than three times lower than at E120.
3.2. Neurotrophin-IR in the developing monkey cerebellar cortex In the present study, no clear differences in immunoreactivity by species or fixation buffer or lobules were observed in any of the three neurotrophins or TrkC.
Intense BDNF-IR was observed in the cell bodies and dendrites of the Purkinje cells throughout all stages examined and the granule cells in the internal granule cell layer (IGL) were also found to be BDNF immunoreactive (Fig. 2A). In the molecular layer (ML), the stellate cells and basket cells showed BDNF-IR, and the density of BDNF-IR increased from P1d to adult (AD) (Fig. 2A and B). The entire ML was weakly BDNF immunopositive throughout development. There was no apparent BDNF-IR in the external granule cell layer (EGL) at P1d or postnatal 1 month (P1m) (Fig. 2A). The cell bodies and dendrites of the Golgi cells and unipolar brush cells were strongly BDNF immunopositive throughout all stages examined (Fig. 2C and D). Weak BDNF-IR was also observed in a few neurons in the white matter (Fig. 2E). As shown in Fig. 3, NT-4/5-IR was found in the Purkinje cells and granule cells throughout all stages. Weak NT-4/5-IR was present in the stellate and basket cells at P1d and P1m and the density of NT-4/5-IR in these cells increased toward AD. As in the case of BDNF, the entire ML showed weak NT-4/5-IR throughout postnatal development. The Golgi cells were NT-4/ 5 immunopositive throughout all stages examined (data not shown). NT-3-IR was observed in the Bergmann glial fibers throughout all stages. The intensity of NT-3-IR in the Bergmann glial fibers was high at P1d and P1m, and gradually decreased toward AD (Fig. 4A and B). Most of the Purkinje cells were NT-3 immunonegative, but some showed clear NT-3IR in their soma and dendrites (data not shown). At P1d and P1m, strong NT-3-IR was detected in the internal part of the EGL, and the entire ML showed NT-3-IR throughout postnatal development (Fig. 4A). The granule cells were also NT-3 immunopositive throughout all stages examined. Weak NT-3IR was observed in the stellate cells and basket cells, whereas the Golgi cells showed intense NT-3-IR throughout all stages (Fig. 4A and D arrows). As indicated in Fig. 4A, strong TrkC-IR was detected in the Purkinje cells throughout all stages examined. The granule cells showed a weak TrkC-IR and the stellate cells and basket cells were also TrkC immunopositive. At P1d and P1m, the external part of the EGL was TrkC immunonegative, but horizontal bipolar cells in the internal part of the EGL were TrkC immunopositive (Fig. 4A and C arrowheads). The relative IR for neurotrophins and TrkC are summarized in Tables 2 and 3. 4. Discussion 4.1. Levels and distributions of BDNF The results of BDNF ELISA showed an age-dependent increase in BDNF protein levels, which is consistent with previous results in rodent cerebellum (Katoh-Semba et al., 1997; Das et al., 2001). Although the levels of BDNF reached plateau at P1d, BDNF-IR in the stellate and basket cells increased during postnatal development. In previous studies (Ohira and Hayashi, 2003; Ohira et al., 2004), cells in the ML of the adult monkey cerebellum were immunopositive for TrkB,
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Fig. 1. Standard curves of ELISAs and developmental changes in protein levels of BDNF, NT-4/5 and NT-3 in monkey cerebellum. Extracts from monkey cerebellar tissue are parallel to the standard curves of BDNF (A), NT-4/5 (C) and NT-3 (E) in each ELISA. Levels of BDNF (B) and NT-4/5 (D) increased during development, while high levels of NT-3 occurred during embryonic stages and gradually declined until adulthood (F). The error bars indicate standard error (S.E.). OD, optical density.
the high affinity receptor for BDNF and NT-4/5. These results suggest that BDNF may play a role in maintaining or remodeling the neural circuits of those interneurons through the use of TrkB in the mature monkey cerebellum. We observed BDNF-IR in the Purkinje cells and the granule cells, which is in agreement with previous studies in rodent and monkey cerebellum (Friedman et al., 1998; Das et al., 2001; Dieni and Rees, 2002; Ohira and Hayashi, 2003; Ohira et al., 2004). In monkey cerebellar cortex, TrkB is expressed by almost all Purkinje cells throughout postnatal development
(Ohira et al., 2004). In situ hybridization studies in rodents (Rocamora et al., 1993; Neveu and Arenas, 1996) have demonstrated the expression of BDNF mRNA in granule cells. Because the granule cells are known to send their axons to the Purkinje cells, BDNF released from the granule cells may act on the Purkinje cells anterogradely. At early postnatal stages, the precursors of granule cells constituting the EGL were BDNF immunonegative. This was also reported in previous studies of guinea pig and rat cerebellum (Rocamora et al., 1993; Neveu and Arenas,
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Fig. 2. Distributions of BDNF immunoreactivities in the developing monkey cerebellar cortex. (A) BDNF immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. Note the intensity of BDNF immunoreactivity in cells in ML increased during development. Pre-absorption of antibody with synthetic peptide of BDNF eliminated the staining (Control). Panels display a part of lobelu VI. (B–E) High magnification of BDNF immunoreactivity in ML at AD (B), BDNF immunoreactive Golgi cell at P1m (C), BDNF immunoreactive unipolar brush cells (arrowheads) at P1m (D) and a BDNF immunoreactive neuron (arrow) in the white matter at P6m (E). EGL, external granule cell layer. IGL, internal granule cell layer. ML, molecular cell layer. PCL, Purkinje cell layer. Scale bars = 100 mm (A), 20 mm (B–E).
1996; Dieni and Rees, 2002). Otherwise, every type of cerebellar neuron was BDNF immunopositive throughout postnatal development. This finding is consistent with the idea that BDNF may be an important neurotrophic molecule for development and maintenance of not only the Purkinje and granule cells, but also various interneurons in primate
cerebellum. Among the four kinds of cerebellar interneurons, the Golgi cells and unipolar brush cells showed particularly prominent BDNF-IR throughout development. To our knowledge, this is the first demonstration of BDNFIR in these two cell types. The axons of Golgi cell form GABAergic synapses on to granule cell dendrites in the IGL
Fig. 3. Distributions of NT-4/5 immunoreactivities in the developing monkey cerebellar cortex. NT-4/5 immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. Purkinje cells were NT-4/5 immunopositive throughout development. Pre-absorption of antibody with synthetic peptide of NT-4/5 eliminated the staining (Control). Panels display a part of lobelu VI. Scale bar = 100 mm.
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Fig. 4. Distributions of NT-3 and TrkC immunoreactivities in the developing monkey cerebellar cortex. (A) NT-3 and TrkC immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. The Bergmann glial fibers were NT-3 immunopositive throughout development. The Golgi cells showed intense NT-3-IR (arrow). Preabsorption of antibody with synthetic peptide of NT-3 or TrkC eliminated the staining (Control). Panels display a part of lobelu VI. (B–D) High magnifications of NT3 immunoreactive Bergmann glial fibers at P1m (B), TrkC immunoreactive premigratory cells (arrowheads) at P1d (C) and NT-3 immunoreactive Golgi cells (arrows) at P1m (D). Scale bars = 100 mm (A), 25 mm (B and C), 50 mm (D).
and contribute to the glomeruli where mossy fibers establish synapses on granule cell dendrites. Studies using BDNF transgenic mice have indicated that BDNF may modulate the maturation of the Golgi cells and/or their GABAergic synapses (Bao et al., 1999; Richardson and Leitch, 2002). The present results confirmed the presence of BDNF in the Golgi cells at adult stages, suggesting that BDNF may also
act on mature Golgi cells. Unipolar brush cells have been shown to receive inputs from mossy fibers and their axons terminate on granule cell dendrites. Recently, this circuit has been postulated to produce a powerful feed forward excitatory network in the cerebellum (Dino et al., 2000; Nunzi et al., 2001). A possible inference from the presence of BDNF-IR in the unipolar brush cells might be that BDNF
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Table 2 Summary of immunoreactivity for BDNF and NT-4/5 in cerebellar cortex Neuronal type
EGL Stellate and Basket cell Purkinje cell Granule cell Golgi cell Unipolar brush cell
BDNF
NT-4/5
P1d
P1m
P6m
AD
P1d
P1m
P6m
AD
+ +++ ++ +++ +++
+ +++ ++ +++ +++
++ +++ ++ +++ +++
+++ +++ ++ +++ +++
+ +++ ++ +
+ +++ ++ +
++ +++ ++ +
++ +++ ++ +
Key: () none; (+) weak; (++) moderate; (+++) intense immunoreactivity.
is involved in the maintenance and/or plasticity of this cerebellar network.
4.2. Levels and distributions of NT-4/5 In the present study, NT-4/5 levels increased from embryonic stages to infantile stages (Fig. 1D) and NT-4/5-IR in the stellate and basket cells became stronger during postnatal development, as was observed for BDNF. These findings suggest that NT-4/5 may also participate in the development and maintenance of these interneurons through TrkB. Although intense NT-4/5-IR was found in the Purkinje cell body throughout the postnatal development, as has been reported in rats (Friedman et al., 1998), NT-4/5-IR in the dendrites of the Purkinje cells was weaker than BDNF-IR (Fig. 3). This may reflect the lower levels of NT-4/5 than those of BDNF (Fig. 1B and D). Since there have been no reports on distribution of NT-4/5 mRNA even in the rodent cerebellum, it is unclear whether NT-4/5 is synthesized in the Purkinje cells and/or in other cell types and transported. There exist other differences between BDNF and NT-4/5 immunoreactive structures in the monkey cerebellum. For example, no NT-4/5 immunopositive unipolar brush cells were detected. There is the possibility that we could not distinguish immunopositive unipolar brush cells from granule cells because the dendrites of unipolar brush cells, which are a unique morphological characteristic, were NT-4/5 immunonegative, whereas the somata of unipolar brush cells were immunopositive. Further precise anatomical studies will be required to resolve this issue.
4.3. Levels and distributions of NT-3 The level of NT-3 showed the opposite pattern of expression from BDNF and NT-4/5; the highest level was observed at embryonic stages and subsequently rapidly declined. This is consistent with previous results of studies on the developmental changes in NT-3 mRNA and protein in rat cerebellum (Maisonpierre et al., 1990; Das et al., 2001) and NT-3 protein in the various cerebral cortices of the developing monkey brain (Mori et al., 2002). These findings support the idea that NT-3 may take part in various important physiological processes, such as proliferation and migration of neuronal cells during early development. In rhesus monkeys, the precursors of granule cells are known to migrate along the Bergmann glial fibers from the EGL toward the IGL until about postnatal 3 months (Rakic, 1971). In the present study, strong NT-3-IR was observed in the internal part of the EGL and the Bergmann glial fibers at early postnatal stages, its intensity decreased toward adulthood (Fig. 4A and B). Although the localization of NT-3 in EGL has been reported (Das et al., 2001), the present study is the first finding of the localization of NT-3 in Bergmann glial fibers. NT-3 has been reported to stimulate the migration of the granule cells in vivo (Neveu and Arenas, 1996; Doughty et al., 1998). In addition, we found that the external part of the EGL was TrkC immunonegative, while the premigratory granule cells, which extend their cell bodies horizontally, were TrkC immunopositive. All these findings lend support to a model postulating that NT-3 in the Bergmann glial fibers may act on the premigratory granule cells to regulate their migration in the developing monkey cerebellum.
Table 3 Summary of immunoreactivity for NT-3 and TrkC in cerebellar cortex Neuronal type
EGL Stellate and Basket cell Bergmann glial fiber Purkinje cell Granule cell Golgi cell Unipolar brush cell
NT-3
TrkC
P1d
P1m
P6m
AD
P1d
P1m
P6m
AD
+++ + +++ + ++ +++
+++ + +++ + ++ +++
++ + + ++ +++
++ + + ++ +++
+ ++ ++ +
+ ++ ++ +
++ +++ ++
++ +++ ++
Key: () none; (+) weak; (++) moderate; (+++) intense immunoreactivity.
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Previous studies have shown that NT-3 protein (Friedman et al., 1998; Das et al., 2001), but no NT-3 mRNA (Lindholm et al., 1993a; Neveu and Arenas, 1996), is present in Purkinje cells in rat, suggesting the uptake of NT-3 by Purkinje cells. We found that the Purkinje cells were strongly TrkC immunopositive and the granule cells were NT-3 immunopositive throughout all postnatal stages (Fig. 4A). These findings are consistent with the hypothesis that NT-3 produced by granule cells may act on Purkinje cells through TrkC. In the present study, the stellate and basket cells also showed TrkC-IR throughout postnatal development. Because the granule cells are also known to send their axons to the stellate and basket cells in the ML, NT-3 released from the granule cells may act on those interneurons in an anterograde manner. However, there is also the possibility that NT-3 synthesized by stellate and basket act in a local autocrine manner, considering that NT-3-IR was observed in these cells. Further in situ hybridization study of NT-3 will be necessary to clarify the relationship between synthesis and transport of NT-3 in those interneurons in cerebellar cortex. Acknowledgements This work was supported by The Grant for the Biodiversity Research of the 21st Century COE (A14) and for Scientific Research on Priority Areas—Advanced Brain Science Project (no. 15016056) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.H.), and by a scholarship from Daiko Foundation (to T.M. and K.T.). We are grateful to Dr. C. Nakayama (Sumitomo Pharmaceutical) for kindly providing anti-BDNF antibody (RP43-01). References Allen, G., Buxton, R.B., Wong, E.C., Courchesne, E., 1997. Attentional activation of the cerebellum independent of motor involvement. Science 275, 1940–1943. Bao, S., Chen, L., Qiao, X., Thompson, R.F., 1999. Transgenic brain-derived neurotrophic factor modulates a developing cerebellar inhibitory synapse. Learn. Mem. 6, 276–283. Bates, B., Rios, M., Trumpp, A., Chen, C., Fan, G., Bishop, J.M., Jaenisch, R., 1999. Neurotrophin-3 is required for proper cerebellar development. Nat. Neurosci. 2, 115–117. Das, K.P., Chao, S.L., White, L.D., Haines, W.T., Harry, G.J., Tilson, H.A., Barone Jr., S., 2001. Differential patterns of nerve growth factor, brainderived neurotrophic factor and neurotrophin-3 mRNA and protein levels in developing regions of rat brain. Neuroscience 103, 739–761. Dieni, S., Rees, S., 2002. Distribution of brain-derived neurotrophic factor and TrkB receptor proteins in the fetal and postnatal hippocampus and cerebellum of the guinea pig. J. Comp. Neurol. 454, 229–240. Dino, M.R., Schuerger, R.J., Liu, Y., Slater, N.T., Mugnaini, E., 2000. Unipolar brush cell: a potential feedforward excitatory interneuron of the cerebellum. Neuroscience 98, 625–636. Doughty, M.L., Lohof, A., Campana, A., Delhaye-Bouchaud, N., Mariani, J., 1998. Neurotrophin-3 promotes cerebellar granule cell exit from the EGL. Eur. J. Neurosci. 10, 3007–3011. Friedman, W.J., Black, I.B., Kaplan, D.R., 1998. Distribution of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the postnatal rat brain: an immunocytochemical study. Neuroscience 84, 101–114.
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Gao, J.H., Parsons, L.M., Bower, J.M., Xiong, J., Li, J., Fox, P.T., 1996. Cerebellum implicated in sensory acquisition and discrimination rather than motor control. Science 272, 545–547. Gao, W.Q., Zheng, J.L., Karihaloo, M., 1995. Neurotrophin-4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) act at later stages of cerebellar granule cell differentiation. J. Neurosci. 15, 2656–2667. Hayashi, M., Mitsunaga, F., Itoh, M., Shimizu, K., Yamashita, A., 2000. Development of full-length Trk B-immunoreactive structures in the prefrontal and visual cortices of the macaque monkey. Anat. Embryol. (Berl.) 201, 139–147. Ito, M., 1984. The Cerebellum and Neural Control. Raven, New York. Katoh-Semba, R., Takeuchi, I.K., Semba, R., Kato, K., 1997. Distribution of brain-derived neurotrophic factor in rats and its changes with development in the brain. J. Neurochem. 69, 34–42. Kawamoto, Y., Nakamura, S., Nakano, S., Oka, N., Akiguchi, I., Kimura, J., 1996. Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain. Neuroscience 74, 1209–1226. Kubo, T., Nonomura, T., Enokido, Y., Hatanaka, H., 1995. Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture. Brain Res. Dev. Brain Res. 85, 249–258. Lindholm, D., Castren, E., Tsoulfas, P., Kolbeck, R., Berzaghi Mda, P., Leingartner, A., Heisenberg, C.P., Tessarollo, L., Parada, L.F., et al., 1993a. Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation. J. Cell Biol. 122, 443–450. Lindholm, D., Dechant, G., Heisenberg, C.P., Thoenen, H., 1993b. Brainderived neurotrophic factor is a survival factor for cultured rat cerebellar granule neurons and protects them against glutamate-induced neurotoxicity. Eur. J. Neurosci. 5, 1455–1464. Lindholm, D., Hamner, S., Zirrgiebel, U., 1997. Neurotrophins and cerebellar development. Perspect. Dev. Neurobiol. 5, 83–94. Maisonpierre, P.C., Belluscio, L., Friedman, B., Alderson, R.F., Wiegand, S.J., Furth, M.E., Lindsay, R.M., Yancopoulos, G.D., 1990. NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5, 501–509. Middleton, F.A., Strick, P.L., 1994. Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function. Science 266, 458–461. Mori, T., Shimizu, K., Hayashi, M., 2004. Differential expression patterns of TrkB ligands in the macaque monkey brain. Neuroreport 15, 2507–2511. Mori, T., Yamashita, D., Homma, K.J., Shimizu, K., Hayashi, M., 2002. Changes in NT-3 and TrkC in the primary visual cortex of developing macaques. Neuroreport 13, 1689–1694. Neveu, I., Arenas, E., 1996. Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. J. Cell Biol. 133, 631–646. Nonomura, T., Kubo, T., Oka, T., Shimoke, K., Yamada, M., Enokido, Y., Hatanaka, H., 1996. Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule cells. Brain Res. Dev. Brain Res. 97, 42–50. Nunzi, M.G., Birnstiel, S., Bhattacharyya, B.J., Slater, N.T., Mugnaini, E., 2001. Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex. J. Comp. Neurol. 434, 329–341. Ohira, K., Funatsu, N., Nakamura, S., Hayashi, M., 2004. Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum. Gene Expr. Patterns 4, 257–261. Ohira, K., Hayashi, M., 2003. Expression of TrkB subtypes in the adult monkey cerebellar cortex. J. Chem. Neuroanat. 25, 175–183. Palay, S.L., Chan-Palay, V., 1974. Cerebellar Cortex: Cytology and Organization. Springer, New York. Radka, S.F., Holst, P.A., Fritsche, M., Altar, C.A., 1996. Presence of brainderived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay. Brain Res. 709, 122–301. Raichle, M.E., Fiez, J.A., Videen, T.O., MacLeod, A.M., Pardo, J.V., Fox, P.T., Petersen, S.E., 1994. Practice-related changes in human brain functional anatomy during nonmotor learning. Cereb. Cortex 4, 8–26. Rakic, P., 1971. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus. J. Comp. Neurol. 141, 283–312.
220
K. Takumi et al. / Journal of Chemical Neuroanatomy 30 (2005) 212–220
Richardson, C.A., Leitch, B., 2002. Cerebellar Golgi, Purkinje, and basket cells have reduced gamma-aminobutyric acid immunoreactivity in stargazer mutant mice. J. Comp. Neurol. 453, 85–99. Rocamora, N., Garcia-Ladona, F.J., Palacios, J.M., Mengod, G., 1993. Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system. Brain Res. Mol. Brain Res. 17, 1–8. Schwartz, P.M., Borghesani, P.R., Levy, R.L., Pomeroy, S.L., Segal, R.A., 1997. Abnormal cerebellar development and foliation in BDNF/ mice reveals a role for neurotrophins in CNS patterning. Neuron 19, 269–281.
Segal, R.A., Takahashi, H., McKay, R.D., 1992. Changes in neurotrophin responsiveness during the development of cerebellar granule neurons. Neuron 9, 1041–1052. Shimizu, K., 1988. Ultrasonic assessment of pregnancy and fetal development in three species of macaque monkeys. J. Med. Primatol. 17, 247– 256. Tanaka, S., Sekino, Y., Shirao, T., 2000. The effects of neurotrophin-3 and brainderived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro. Neuroscience 97, 727–734.
Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex Ken Takumi, Takuma Mori, Keiko Shimizu, Motoharu Hayashi * Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan Received 5 April 2005; received in revised form 8 August 2005; accepted 12 August 2005 Available online 10 October 2005
Abstract Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells. # 2005 Elsevier B.V. All rights reserved. Keywords: Cerebellar cortex; Primate; BDNF; NT-3; TrkC; ELISA; Immunohistochemistry
1. Introduction The mammalian cerebellar cortex is comprised of six distinct neuronal cell types, Purkinje cells, granule cells, stellate cells, basket cells, Golgi cells and unipolar brush cells. The distributions of those cells and the synaptic connections between them have been thoroughly elucidated (Palay and Chan-Palay, 1974; Ito, 1984). In addition, several developmentally critical events, such as neurogenesis, neuroblast migration and neuronal differentiation occur extensively in postnatal stages in the cerebellum. The cerebellum has therefore been used as a model system to study the role of epigenetic factors in the development of the central nervous system (CNS). Despite the fact that the cerebellum has been considered to control mainly motor systems, recent studies have demonstrated that it may also be involved in higher cognitive
* Corresponding author. Tel.: +81 568 63 0572; fax: +81 568 63 0576. E-mail address: [email protected] (M. Hayashi). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.08.004
functions. For example, cerebellar output through thalamic neurons has been shown to extend to the prefrontal cortex (Middleton and Strick, 1994) which is of importance for planning and working memory. Several functional neuroimaging studies have also demonstrated activation of the cerebellum during various cognitive tasks, such as verbal tasks, passive sensory tasks and visual attention shifting (Raichle et al., 1994; Gao et al., 1996; Allen et al., 1997). Neurotrophins are a family of structurally related proteins, which includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5) and neurotrophin-3 (NT-3). Neurotrophins and their receptors have been reported to be expressed widely in the developing and adult cerebellum of various experimental animals (Maisonpierre et al., 1990; Rocamora et al., 1993; Kawamoto et al., 1996; Lindholm et al., 1997; Friedman et al., 1998; Das et al., 2001; Dieni and Rees, 2002; Ohira and Hayashi, 2003; Ohira et al., 2004). Studies using primary cultures of rat cerebellar neurons have demonstrated promotion of survival, movement and neurite extension by neurotrophins (Segal et al., 1992;
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Lindholm et al., 1993b; Gao et al., 1995; Kubo et al., 1995; Nonomura et al., 1996; Tanaka et al., 2000). Furthermore, genetargeting studies have shown that BDNF (Schwartz et al., 1997) and NT-3 (Bates et al., 1999) are required for normal foliation. Although developmental changes in levels and localizations of neurotrophins have been investigated intensively in the rodent cerebellum (Lindholm et al., 1997), less is known about the primate cerebellum. In view of the above, we considered it worthwhile to investigate developmental patterns in the levels and cellular distributions of neurotrophins in the primate cerebellum. In this study, we examined developmental changes in the protein levels of BDNF, NT-4/5 and NT-3 by enzyme-linked immunosorbent assays (ELISAs) and the distributions of those three neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellar cortex of macaque monkeys. Most previous reports have focused on the localizations of neurotrophins in the Purkinje cells and granule cells, and precise studies on the cerebellar interneurons have not been performed. Therefore, we also analyzed the localizations of neurotrophins in inhibitory interneurons, such as stellate cells, basket cells, Golgi cells and unipolar brush cells, which are of importance in the cerebellar circuitry.
2. Materials and methods 2.1. Experimental animals and tissue preparation Twenty-three crab-eating monkeys (Macaca fascicularis) and six rhesus monkeys (Macaca mulatta) from embryonic day 120 (E120) to 15 years old were the subjects for ELISAs and twelve rhesus monkeys and three crab-eating monkeys from postnatal day 1 (P1d) to 15 years old were used for immunohistochemical analysis (Table 1). The method used to determine the embryonic day of age of fetal monkeys was previously described by Shimizu (Shimizu, 1988). Embryonic monkeys were obtained by Caesarian section under anesthesia with ketamine hydrochloride (10 mg/kg, i.m.) and maintained with halothane (1%)–N2O–oxygen inhalation. For non-survival procedures, all monkeys were pretreated with ketamine hydrochloride (10 mg/kg, i.m.) and deeply anaesthetized with pentobarbital sodium (25 mg/kg, i.v.). For ELISAs, monkeys were killed by bloodletting from Table 1 Monkeys used in this study Age (abbreviation)
ELISAs Embryonic day 120 (E120) Embryonic day 140 (E140) Postnatal day 1 (P1d) Postnatal 2 months (P2m) Postnatal 2 years (P2y) Postnatal 4 years (P4m) Postnatal 7–15 years (AD) Immunohistochemistry Postnatal day 1 (P1d) Postnatal 1 month (P1m) Postnatal 6 months (P6m) Postnatal 5–15 years (AD)
Macaca facicularis
Macaca mulatta
Male
Female
3 3 2 3 1
1 1 2 1
3
1
Male
Female
1 2
1 1
2 1 1
2
2 2 3 2
1
2
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the carotid artery. The skulls were quickly opened and dissection of cerebellar tissue was performed on ice. The dissection of cerebellum included cerebellar cortex of lobule VI to VIII, but not cerebellar nuclei. The dissected tissues were stored at 80 8C until use. For immunohistochemistry, monkeys were perfused through the heart with 0.15 M NaCl followed by one of the fixation buffers (1) 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4 (PB), (2) 2% PFA and 0.5% glutaraldehyde (GA) in PB, (3) 4% PFA in PB or (4) 4% PFA and 0.1% GA in PB. After perfusion, the cerebellum were immediately removed and immersed in 2% PFA and 5% sucrose in PB at 4 8C for 24 h, followed by successive immersion in 10% sucrose in 0.1 M phosphate-buffered saline, pH 7.4 (PBS), 20% sucrose in PBS and 30% sucrose in PBS containing 0.02% sodium azide. The cerebellum was sagittally cut into blocks 5 mm thick and mounted in Tissue-Tek (Miles, Elkhart, IN, USA) and frozen rapidly in a dry-ice/acetone bath, and stored at 80 8C until dissection. All procedures were carried out in accordance with The Guide for the Care and Use of Laboratory Animals established by the NIH (1985) and The Guide for the Care and Use of Laboratory Primates established by the Primate Research Institute of Kyoto University (2002).
2.2. Antibodies For ELISAs, we used each of three kinds of antibodies as the coating antibody: anti-BDNF mouse monoclonal IgG, R43-01 (Radka et al., 1996), antiNT-4 mouse monoclonal IgG (Promega) and anti-NT-3 mouse monoclonal IgG (Promega). For the primary antibody, we used the following: anti-BDNF chick polyclonal IgY (Promega), anti-NT-4 chick polyclonal IgY (Promega), anti-NT3 chick polyclonal IgY (Promega) and normal chick IgY (Promega). Horseradish peroxidase (HRP) conjugated anti-chick IgY antibody (Promega) was used as secondary antibody. For immunohistochemistry, we used anti-BDNF rabbit polyclonal IgG (1:1600, sc-546, Santa Cruz Biotech., Calif., USA), NT-4/5 (1:3200, sc-545, Santa Cruz Biotech.), NT-3 (1:3200, sc-547, Santa Cruz Biotech.) and TrkC (1:400, sc-117, Santa Cruz Biotech.) as the primary antibody. As the secondary antibody, we used biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA).
2.3. ELISAs ELISA methods we used in this study were based on the procedure described by Mori et al. (2002, 2004). The monkey cerebellar tissue was homogenized in 10 volumes of extraction buffer (1 mM EDTA, 1 M guanidine hydrochloride, 10 mg/ml aprotinin, 0.2 mM benzethonium chloride, 2 mM benzamidine and 1 mM PMSF in 0.1 M phosphate buffer, pH 7.2). The homogenate was centrifuged for 30 min at 8000 g at 4 8C, the supernatants (first extraction) were collected and the sediments were rehomogenized and centrifuged as described above. The supernatant (second extraction) were collected, added to the first extraction and stored at 80 8C until use. Hundred microliters of the supernatant was used to determine the concentrations of neurotrophins. To estimate the recovery of exogenous neurotrophins in our ELISA methods, we added recombinant human (rh) neurotrophins (200 pg/ml of BDNF, 50 pg/ml of NT-3 and 50 pg/ml of NT-4; Peprotech, London, UK) into the cerebellar extract. Ninety six-well plates (Nunc, Roskilde, Denmark) were coated with 100 ml of the coating antibody in coating buffer (50 mM sodium carbonate–bicarbonate buffer, pH 9.6) and incubated overnight at 4 8C. After aspiration of excess antibody and three washes with washing buffer (50 mM Tris–HCl, 0.6 M NaCl and 0.1% Triton X-100), 300 ml of coating buffer containing 1% BSA was added to each well and the plates were incubated at room temperature for 1 h. The standard amounts of rh-neurotrophins were diluted in sample buffer (1 M guanidine hydrochloride and 1% BSA in washing buffer). After aspiration and three washes with washing buffer, 100 ml of standards or duplicated test samples were added per well and wells were incubated overnight at 4 8C. The plates were then washed three times with washing buffer, followed by incubation with 100 ml of the primary antibody in washing buffer overnight at 4 8C. After three washes, 100 ml of a 1:1000 dilution of the secondary antibody in washing buffer were added per well, and the plates were incubated for 6 h at 4 8C. After further washing, the reaction was developed for 15 min with 100 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB; Kirkegaard and Perry Laboratories, Gaithersburg, MD) and
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stopped with 100 ml of 1 M phosphoric acid. The optical density of the plates was read on a multilabel counter (Wallac 1420 ARVOsx; Wallac, Turku, Finland) at 460 nm.
2.4. Immunohistochemistry The immunohistochemical methods used in this study were similar to those described by Hayashi et al. (2000) (Ohira and Hayashi, 2003). The sections were cut at 40 mm thickness with a cryostat (HM500-M, Micron, Walldorf, Germany). Free-floating sections were washed for 30 min in PBS and incubated with PBS containing 0.1% Triton X-100, 1% bovine serum albumin and 4% normal goat serum (PBS-GB) for at least 1 h. The sections were then incubated at 4 8C for 48 h with one of the primary antibodies in PBS-GB. After several washes with PBS, the sections were incubated with the secondary antibody at 4 8C overnight. The immunoreactive sites were visualized by the avidin–biotin complex peroxidase method using the ABC Elite kit (Vector Laboratories). A solution of 0.3% H2O2, 20 mg/ml 3,30 -diamino benzidine tetrahydrochloride and 0.3% nickel ammonium sulfate in 0.05 M Tris–HCl, pH 7.6, was used as the substrate for peroxidase. The sections were mounted on slide glasses that had been precoated with VECTABOND (Vector Laboratories), and allowed to dry overnight. Adjacent sections were stained with cresyl violet (Merck, Darmstadt, Germany). They were then dehydrated in ethanol, cleared in xylene and coverslipped. In control sections, the primary antibody was replaced with the antibody preincubated with the corresponding blocking peptide. The control peptides were sc-546p (5 mg/ml, Santa Cruz Biotech.), sc-545p (5 mg/ml, Santa Cruz Biotech.), sc-547p (10 mg/ml, Santa Cruz Biotech.) and sc-117p (5 mg/ml, Santa Cruz Biotech.) for BDNF, NT-4/5, NT-3 and TrkC proteins, respectively. The remainder of the procedure was the same as that in the presence of primary antibody.
3. Results 3.1. Levels of neurotrophins in the developing monkey cerebellar cortex In our ELISAs, we could detect low levels of neurotrophins without any cross-reactivities to the other neurotrophins (Fig. 1A, C and E). Extracts from the monkey cerebellar tissues paralleled the standard curves of each rh-neurotrophins. The recoveries of all exogenous rh-neurotrophins in these ELISAs were over 80% at each developmental stage. The concentrations of BDNF were low at E120 and increased in an age-dependent manner during embryonic stages. At P1d, BDNF concentrations reached adult levels, which were almost three times higher than at E120 (Fig. 1B). Similar to the changes of BDNF, NT-4/5 levels were low at E120, increased to a peak at postnatal 2 years (P2y) and then gradually decreased toward adulthood (Fig. 1D). In contrast to BDNF and NT-4/5, the highest levels of NT-3 were observed at E120. NT-3 levels then rapidly declined until P1d and subsequently gradually decreased toward adulthood (Fig. 1F). The levels of NT-3 at adult stages were more than three times lower than at E120.
3.2. Neurotrophin-IR in the developing monkey cerebellar cortex In the present study, no clear differences in immunoreactivity by species or fixation buffer or lobules were observed in any of the three neurotrophins or TrkC.
Intense BDNF-IR was observed in the cell bodies and dendrites of the Purkinje cells throughout all stages examined and the granule cells in the internal granule cell layer (IGL) were also found to be BDNF immunoreactive (Fig. 2A). In the molecular layer (ML), the stellate cells and basket cells showed BDNF-IR, and the density of BDNF-IR increased from P1d to adult (AD) (Fig. 2A and B). The entire ML was weakly BDNF immunopositive throughout development. There was no apparent BDNF-IR in the external granule cell layer (EGL) at P1d or postnatal 1 month (P1m) (Fig. 2A). The cell bodies and dendrites of the Golgi cells and unipolar brush cells were strongly BDNF immunopositive throughout all stages examined (Fig. 2C and D). Weak BDNF-IR was also observed in a few neurons in the white matter (Fig. 2E). As shown in Fig. 3, NT-4/5-IR was found in the Purkinje cells and granule cells throughout all stages. Weak NT-4/5-IR was present in the stellate and basket cells at P1d and P1m and the density of NT-4/5-IR in these cells increased toward AD. As in the case of BDNF, the entire ML showed weak NT-4/5-IR throughout postnatal development. The Golgi cells were NT-4/ 5 immunopositive throughout all stages examined (data not shown). NT-3-IR was observed in the Bergmann glial fibers throughout all stages. The intensity of NT-3-IR in the Bergmann glial fibers was high at P1d and P1m, and gradually decreased toward AD (Fig. 4A and B). Most of the Purkinje cells were NT-3 immunonegative, but some showed clear NT-3IR in their soma and dendrites (data not shown). At P1d and P1m, strong NT-3-IR was detected in the internal part of the EGL, and the entire ML showed NT-3-IR throughout postnatal development (Fig. 4A). The granule cells were also NT-3 immunopositive throughout all stages examined. Weak NT-3IR was observed in the stellate cells and basket cells, whereas the Golgi cells showed intense NT-3-IR throughout all stages (Fig. 4A and D arrows). As indicated in Fig. 4A, strong TrkC-IR was detected in the Purkinje cells throughout all stages examined. The granule cells showed a weak TrkC-IR and the stellate cells and basket cells were also TrkC immunopositive. At P1d and P1m, the external part of the EGL was TrkC immunonegative, but horizontal bipolar cells in the internal part of the EGL were TrkC immunopositive (Fig. 4A and C arrowheads). The relative IR for neurotrophins and TrkC are summarized in Tables 2 and 3. 4. Discussion 4.1. Levels and distributions of BDNF The results of BDNF ELISA showed an age-dependent increase in BDNF protein levels, which is consistent with previous results in rodent cerebellum (Katoh-Semba et al., 1997; Das et al., 2001). Although the levels of BDNF reached plateau at P1d, BDNF-IR in the stellate and basket cells increased during postnatal development. In previous studies (Ohira and Hayashi, 2003; Ohira et al., 2004), cells in the ML of the adult monkey cerebellum were immunopositive for TrkB,
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Fig. 1. Standard curves of ELISAs and developmental changes in protein levels of BDNF, NT-4/5 and NT-3 in monkey cerebellum. Extracts from monkey cerebellar tissue are parallel to the standard curves of BDNF (A), NT-4/5 (C) and NT-3 (E) in each ELISA. Levels of BDNF (B) and NT-4/5 (D) increased during development, while high levels of NT-3 occurred during embryonic stages and gradually declined until adulthood (F). The error bars indicate standard error (S.E.). OD, optical density.
the high affinity receptor for BDNF and NT-4/5. These results suggest that BDNF may play a role in maintaining or remodeling the neural circuits of those interneurons through the use of TrkB in the mature monkey cerebellum. We observed BDNF-IR in the Purkinje cells and the granule cells, which is in agreement with previous studies in rodent and monkey cerebellum (Friedman et al., 1998; Das et al., 2001; Dieni and Rees, 2002; Ohira and Hayashi, 2003; Ohira et al., 2004). In monkey cerebellar cortex, TrkB is expressed by almost all Purkinje cells throughout postnatal development
(Ohira et al., 2004). In situ hybridization studies in rodents (Rocamora et al., 1993; Neveu and Arenas, 1996) have demonstrated the expression of BDNF mRNA in granule cells. Because the granule cells are known to send their axons to the Purkinje cells, BDNF released from the granule cells may act on the Purkinje cells anterogradely. At early postnatal stages, the precursors of granule cells constituting the EGL were BDNF immunonegative. This was also reported in previous studies of guinea pig and rat cerebellum (Rocamora et al., 1993; Neveu and Arenas,
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Fig. 2. Distributions of BDNF immunoreactivities in the developing monkey cerebellar cortex. (A) BDNF immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. Note the intensity of BDNF immunoreactivity in cells in ML increased during development. Pre-absorption of antibody with synthetic peptide of BDNF eliminated the staining (Control). Panels display a part of lobelu VI. (B–E) High magnification of BDNF immunoreactivity in ML at AD (B), BDNF immunoreactive Golgi cell at P1m (C), BDNF immunoreactive unipolar brush cells (arrowheads) at P1m (D) and a BDNF immunoreactive neuron (arrow) in the white matter at P6m (E). EGL, external granule cell layer. IGL, internal granule cell layer. ML, molecular cell layer. PCL, Purkinje cell layer. Scale bars = 100 mm (A), 20 mm (B–E).
1996; Dieni and Rees, 2002). Otherwise, every type of cerebellar neuron was BDNF immunopositive throughout postnatal development. This finding is consistent with the idea that BDNF may be an important neurotrophic molecule for development and maintenance of not only the Purkinje and granule cells, but also various interneurons in primate
cerebellum. Among the four kinds of cerebellar interneurons, the Golgi cells and unipolar brush cells showed particularly prominent BDNF-IR throughout development. To our knowledge, this is the first demonstration of BDNFIR in these two cell types. The axons of Golgi cell form GABAergic synapses on to granule cell dendrites in the IGL
Fig. 3. Distributions of NT-4/5 immunoreactivities in the developing monkey cerebellar cortex. NT-4/5 immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. Purkinje cells were NT-4/5 immunopositive throughout development. Pre-absorption of antibody with synthetic peptide of NT-4/5 eliminated the staining (Control). Panels display a part of lobelu VI. Scale bar = 100 mm.
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Fig. 4. Distributions of NT-3 and TrkC immunoreactivities in the developing monkey cerebellar cortex. (A) NT-3 and TrkC immunoreactivity in sagittal sections at P1d, P1m, P6m and AD. The Bergmann glial fibers were NT-3 immunopositive throughout development. The Golgi cells showed intense NT-3-IR (arrow). Preabsorption of antibody with synthetic peptide of NT-3 or TrkC eliminated the staining (Control). Panels display a part of lobelu VI. (B–D) High magnifications of NT3 immunoreactive Bergmann glial fibers at P1m (B), TrkC immunoreactive premigratory cells (arrowheads) at P1d (C) and NT-3 immunoreactive Golgi cells (arrows) at P1m (D). Scale bars = 100 mm (A), 25 mm (B and C), 50 mm (D).
and contribute to the glomeruli where mossy fibers establish synapses on granule cell dendrites. Studies using BDNF transgenic mice have indicated that BDNF may modulate the maturation of the Golgi cells and/or their GABAergic synapses (Bao et al., 1999; Richardson and Leitch, 2002). The present results confirmed the presence of BDNF in the Golgi cells at adult stages, suggesting that BDNF may also
act on mature Golgi cells. Unipolar brush cells have been shown to receive inputs from mossy fibers and their axons terminate on granule cell dendrites. Recently, this circuit has been postulated to produce a powerful feed forward excitatory network in the cerebellum (Dino et al., 2000; Nunzi et al., 2001). A possible inference from the presence of BDNF-IR in the unipolar brush cells might be that BDNF
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Table 2 Summary of immunoreactivity for BDNF and NT-4/5 in cerebellar cortex Neuronal type
EGL Stellate and Basket cell Purkinje cell Granule cell Golgi cell Unipolar brush cell
BDNF
NT-4/5
P1d
P1m
P6m
AD
P1d
P1m
P6m
AD
+ +++ ++ +++ +++
+ +++ ++ +++ +++
++ +++ ++ +++ +++
+++ +++ ++ +++ +++
+ +++ ++ +
+ +++ ++ +
++ +++ ++ +
++ +++ ++ +
Key: () none; (+) weak; (++) moderate; (+++) intense immunoreactivity.
is involved in the maintenance and/or plasticity of this cerebellar network.
4.2. Levels and distributions of NT-4/5 In the present study, NT-4/5 levels increased from embryonic stages to infantile stages (Fig. 1D) and NT-4/5-IR in the stellate and basket cells became stronger during postnatal development, as was observed for BDNF. These findings suggest that NT-4/5 may also participate in the development and maintenance of these interneurons through TrkB. Although intense NT-4/5-IR was found in the Purkinje cell body throughout the postnatal development, as has been reported in rats (Friedman et al., 1998), NT-4/5-IR in the dendrites of the Purkinje cells was weaker than BDNF-IR (Fig. 3). This may reflect the lower levels of NT-4/5 than those of BDNF (Fig. 1B and D). Since there have been no reports on distribution of NT-4/5 mRNA even in the rodent cerebellum, it is unclear whether NT-4/5 is synthesized in the Purkinje cells and/or in other cell types and transported. There exist other differences between BDNF and NT-4/5 immunoreactive structures in the monkey cerebellum. For example, no NT-4/5 immunopositive unipolar brush cells were detected. There is the possibility that we could not distinguish immunopositive unipolar brush cells from granule cells because the dendrites of unipolar brush cells, which are a unique morphological characteristic, were NT-4/5 immunonegative, whereas the somata of unipolar brush cells were immunopositive. Further precise anatomical studies will be required to resolve this issue.
4.3. Levels and distributions of NT-3 The level of NT-3 showed the opposite pattern of expression from BDNF and NT-4/5; the highest level was observed at embryonic stages and subsequently rapidly declined. This is consistent with previous results of studies on the developmental changes in NT-3 mRNA and protein in rat cerebellum (Maisonpierre et al., 1990; Das et al., 2001) and NT-3 protein in the various cerebral cortices of the developing monkey brain (Mori et al., 2002). These findings support the idea that NT-3 may take part in various important physiological processes, such as proliferation and migration of neuronal cells during early development. In rhesus monkeys, the precursors of granule cells are known to migrate along the Bergmann glial fibers from the EGL toward the IGL until about postnatal 3 months (Rakic, 1971). In the present study, strong NT-3-IR was observed in the internal part of the EGL and the Bergmann glial fibers at early postnatal stages, its intensity decreased toward adulthood (Fig. 4A and B). Although the localization of NT-3 in EGL has been reported (Das et al., 2001), the present study is the first finding of the localization of NT-3 in Bergmann glial fibers. NT-3 has been reported to stimulate the migration of the granule cells in vivo (Neveu and Arenas, 1996; Doughty et al., 1998). In addition, we found that the external part of the EGL was TrkC immunonegative, while the premigratory granule cells, which extend their cell bodies horizontally, were TrkC immunopositive. All these findings lend support to a model postulating that NT-3 in the Bergmann glial fibers may act on the premigratory granule cells to regulate their migration in the developing monkey cerebellum.
Table 3 Summary of immunoreactivity for NT-3 and TrkC in cerebellar cortex Neuronal type
EGL Stellate and Basket cell Bergmann glial fiber Purkinje cell Granule cell Golgi cell Unipolar brush cell
NT-3
TrkC
P1d
P1m
P6m
AD
P1d
P1m
P6m
AD
+++ + +++ + ++ +++
+++ + +++ + ++ +++
++ + + ++ +++
++ + + ++ +++
+ ++ ++ +
+ ++ ++ +
++ +++ ++
++ +++ ++
Key: () none; (+) weak; (++) moderate; (+++) intense immunoreactivity.
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Previous studies have shown that NT-3 protein (Friedman et al., 1998; Das et al., 2001), but no NT-3 mRNA (Lindholm et al., 1993a; Neveu and Arenas, 1996), is present in Purkinje cells in rat, suggesting the uptake of NT-3 by Purkinje cells. We found that the Purkinje cells were strongly TrkC immunopositive and the granule cells were NT-3 immunopositive throughout all postnatal stages (Fig. 4A). These findings are consistent with the hypothesis that NT-3 produced by granule cells may act on Purkinje cells through TrkC. In the present study, the stellate and basket cells also showed TrkC-IR throughout postnatal development. Because the granule cells are also known to send their axons to the stellate and basket cells in the ML, NT-3 released from the granule cells may act on those interneurons in an anterograde manner. However, there is also the possibility that NT-3 synthesized by stellate and basket act in a local autocrine manner, considering that NT-3-IR was observed in these cells. Further in situ hybridization study of NT-3 will be necessary to clarify the relationship between synthesis and transport of NT-3 in those interneurons in cerebellar cortex. Acknowledgements This work was supported by The Grant for the Biodiversity Research of the 21st Century COE (A14) and for Scientific Research on Priority Areas—Advanced Brain Science Project (no. 15016056) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.H.), and by a scholarship from Daiko Foundation (to T.M. and K.T.). We are grateful to Dr. C. Nakayama (Sumitomo Pharmaceutical) for kindly providing anti-BDNF antibody (RP43-01). References Allen, G., Buxton, R.B., Wong, E.C., Courchesne, E., 1997. Attentional activation of the cerebellum independent of motor involvement. Science 275, 1940–1943. Bao, S., Chen, L., Qiao, X., Thompson, R.F., 1999. Transgenic brain-derived neurotrophic factor modulates a developing cerebellar inhibitory synapse. Learn. Mem. 6, 276–283. Bates, B., Rios, M., Trumpp, A., Chen, C., Fan, G., Bishop, J.M., Jaenisch, R., 1999. Neurotrophin-3 is required for proper cerebellar development. Nat. Neurosci. 2, 115–117. Das, K.P., Chao, S.L., White, L.D., Haines, W.T., Harry, G.J., Tilson, H.A., Barone Jr., S., 2001. Differential patterns of nerve growth factor, brainderived neurotrophic factor and neurotrophin-3 mRNA and protein levels in developing regions of rat brain. Neuroscience 103, 739–761. Dieni, S., Rees, S., 2002. Distribution of brain-derived neurotrophic factor and TrkB receptor proteins in the fetal and postnatal hippocampus and cerebellum of the guinea pig. J. Comp. Neurol. 454, 229–240. Dino, M.R., Schuerger, R.J., Liu, Y., Slater, N.T., Mugnaini, E., 2000. Unipolar brush cell: a potential feedforward excitatory interneuron of the cerebellum. Neuroscience 98, 625–636. Doughty, M.L., Lohof, A., Campana, A., Delhaye-Bouchaud, N., Mariani, J., 1998. Neurotrophin-3 promotes cerebellar granule cell exit from the EGL. Eur. J. Neurosci. 10, 3007–3011. Friedman, W.J., Black, I.B., Kaplan, D.R., 1998. Distribution of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the postnatal rat brain: an immunocytochemical study. Neuroscience 84, 101–114.
219
Gao, J.H., Parsons, L.M., Bower, J.M., Xiong, J., Li, J., Fox, P.T., 1996. Cerebellum implicated in sensory acquisition and discrimination rather than motor control. Science 272, 545–547. Gao, W.Q., Zheng, J.L., Karihaloo, M., 1995. Neurotrophin-4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) act at later stages of cerebellar granule cell differentiation. J. Neurosci. 15, 2656–2667. Hayashi, M., Mitsunaga, F., Itoh, M., Shimizu, K., Yamashita, A., 2000. Development of full-length Trk B-immunoreactive structures in the prefrontal and visual cortices of the macaque monkey. Anat. Embryol. (Berl.) 201, 139–147. Ito, M., 1984. The Cerebellum and Neural Control. Raven, New York. Katoh-Semba, R., Takeuchi, I.K., Semba, R., Kato, K., 1997. Distribution of brain-derived neurotrophic factor in rats and its changes with development in the brain. J. Neurochem. 69, 34–42. Kawamoto, Y., Nakamura, S., Nakano, S., Oka, N., Akiguchi, I., Kimura, J., 1996. Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain. Neuroscience 74, 1209–1226. Kubo, T., Nonomura, T., Enokido, Y., Hatanaka, H., 1995. Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture. Brain Res. Dev. Brain Res. 85, 249–258. Lindholm, D., Castren, E., Tsoulfas, P., Kolbeck, R., Berzaghi Mda, P., Leingartner, A., Heisenberg, C.P., Tessarollo, L., Parada, L.F., et al., 1993a. Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation. J. Cell Biol. 122, 443–450. Lindholm, D., Dechant, G., Heisenberg, C.P., Thoenen, H., 1993b. Brainderived neurotrophic factor is a survival factor for cultured rat cerebellar granule neurons and protects them against glutamate-induced neurotoxicity. Eur. J. Neurosci. 5, 1455–1464. Lindholm, D., Hamner, S., Zirrgiebel, U., 1997. Neurotrophins and cerebellar development. Perspect. Dev. Neurobiol. 5, 83–94. Maisonpierre, P.C., Belluscio, L., Friedman, B., Alderson, R.F., Wiegand, S.J., Furth, M.E., Lindsay, R.M., Yancopoulos, G.D., 1990. NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5, 501–509. Middleton, F.A., Strick, P.L., 1994. Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function. Science 266, 458–461. Mori, T., Shimizu, K., Hayashi, M., 2004. Differential expression patterns of TrkB ligands in the macaque monkey brain. Neuroreport 15, 2507–2511. Mori, T., Yamashita, D., Homma, K.J., Shimizu, K., Hayashi, M., 2002. Changes in NT-3 and TrkC in the primary visual cortex of developing macaques. Neuroreport 13, 1689–1694. Neveu, I., Arenas, E., 1996. Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. J. Cell Biol. 133, 631–646. Nonomura, T., Kubo, T., Oka, T., Shimoke, K., Yamada, M., Enokido, Y., Hatanaka, H., 1996. Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule cells. Brain Res. Dev. Brain Res. 97, 42–50. Nunzi, M.G., Birnstiel, S., Bhattacharyya, B.J., Slater, N.T., Mugnaini, E., 2001. Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex. J. Comp. Neurol. 434, 329–341. Ohira, K., Funatsu, N., Nakamura, S., Hayashi, M., 2004. Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum. Gene Expr. Patterns 4, 257–261. Ohira, K., Hayashi, M., 2003. Expression of TrkB subtypes in the adult monkey cerebellar cortex. J. Chem. Neuroanat. 25, 175–183. Palay, S.L., Chan-Palay, V., 1974. Cerebellar Cortex: Cytology and Organization. Springer, New York. Radka, S.F., Holst, P.A., Fritsche, M., Altar, C.A., 1996. Presence of brainderived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay. Brain Res. 709, 122–301. Raichle, M.E., Fiez, J.A., Videen, T.O., MacLeod, A.M., Pardo, J.V., Fox, P.T., Petersen, S.E., 1994. Practice-related changes in human brain functional anatomy during nonmotor learning. Cereb. Cortex 4, 8–26. Rakic, P., 1971. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus. J. Comp. Neurol. 141, 283–312.
220
K. Takumi et al. / Journal of Chemical Neuroanatomy 30 (2005) 212–220
Richardson, C.A., Leitch, B., 2002. Cerebellar Golgi, Purkinje, and basket cells have reduced gamma-aminobutyric acid immunoreactivity in stargazer mutant mice. J. Comp. Neurol. 453, 85–99. Rocamora, N., Garcia-Ladona, F.J., Palacios, J.M., Mengod, G., 1993. Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system. Brain Res. Mol. Brain Res. 17, 1–8. Schwartz, P.M., Borghesani, P.R., Levy, R.L., Pomeroy, S.L., Segal, R.A., 1997. Abnormal cerebellar development and foliation in BDNF/ mice reveals a role for neurotrophins in CNS patterning. Neuron 19, 269–281.
Segal, R.A., Takahashi, H., McKay, R.D., 1992. Changes in neurotrophin responsiveness during the development of cerebellar granule neurons. Neuron 9, 1041–1052. Shimizu, K., 1988. Ultrasonic assessment of pregnancy and fetal development in three species of macaque monkeys. J. Med. Primatol. 17, 247– 256. Tanaka, S., Sekino, Y., Shirao, T., 2000. The effects of neurotrophin-3 and brainderived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro. Neuroscience 97, 727–734.