Morphological changes in immunopositive cells of ionotropic glutamate receptor subunits during the development of transplanted fetal ventral mesencephalic neurons

Brain Research 940 (2002) 79–85 www.elsevier.com / locate / bres

Research report

Morphological changes in immunopositive cells of ionotropic glutamate receptor subunits during the development of transplanted fetal ventral mesencephalic neurons Yasushi Ishida a , *, Kazunari Todaka a , Hiroyuki Hashiguchi a , Ryuichiro Takeda a , Yoshio Mitsuyama a , Toshikazu Nishimori b a

Department of Psychiatry, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki 889 -1692, Japan b Division of Biology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki 889 -1692, Japan Accepted 25 February 2002

Abstract To elucidate the morphological changes in immunopositive cells of ionotropic glutamate receptors within intrastriatal ‘developing’ grafts of fetal ventral mesencephalon (VM) in 6-hydroxydopamine-lesioned rats, immunohistochemistry was performed to detect cells expressing N-methyl-D-aspartate (NMDA) receptor subunit 1 (NR1), the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR1, GluR2 / 3, and GluR4), or tyrosine hydroxylase (TH) in the intrastriatal VM grafts at 1, 4, and 12 weeks following transplantation. One week after transplantation, TH-positive cells were detected without any immunoreactivity of the NMDA and AMPA receptor subunits in the grafts. Four weeks after transplantation, TH-positive cells, distributed homogeneously in the grafts, appeared to be multipolar and larger compared to those at 1 week post-grafting. At this stage, we could observe immunopositive cells of NMDA and AMPA receptors distributed homogeneously in the grafts. Twelve weeks after transplantation, the numbers of NR1- and GluR1-positive cells were smaller than that at 4 weeks post-grafting, whereas TH-positive cells appeared to be more matured in shape and size. On the other hand, the numbers of GluR2 / 3- and GluR4-positive cells were not changed as compared with those at 4 weeks post-grafting. These results suggest that the ionotropic glutamate receptors have differential roles during the developmental period of the intrastriatal VM grafts.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Transplantation Keywords: Dopaminergic transplant; Glutamate receptor; Immunohistochemistry; Neurodevelopment; Parkinson’s disease

1. Introduction Glutamate is the major fast excitatory neurotransmitter in the mammalian central nervous system (CNS) [12,34]. Glutamate neurotransmission is involved in numerous physiological brain functions, including neuronal plasticity, neural development, memory, and learning [11,18,22,28,32]. However, under certain conditions, excessive exposure to glutamate can produce an over-stimulation of excitatory amino acid (EAA) receptors and lead to neuronal injury [6,16,30]. *Corresponding author. Tel.: 181-985-852-969; fax: 181-985-855475. E-mail address: [email protected] (Y. Ishida).

Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal dopamine (DA) pathway develop contralateral sensory neglect, as well as postural and motor asymmetry, which is characterized by drug-induced rotation of the animal. These motor and sensorimotor deficits of the rat model of hemi-parkinsonism are improved by intrastriatal transplants of DA-rich fetal ventral mesencephalon (VM) [4]. In the 6-OHDA-lesioned rats, intrastriatal VM grafts partially restore DA content and DA release, and the graft-derived axon terminals establish synaptic contacts with striatal neurons [36]. Factors affecting the developmental organization and toxic vulnerability of dopaminergic brain regions are of interest in view of the central role that DA neurons are postulated to play in a number of diseases such as

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02595-7

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Parkinson’s disease and schizophrenia. Ventral mesencephalic brain regions containing DA cell bodies (e.g. the substantia nigra and ventral tegmental area), as well as dopaminergic terminal field regions (e.g. the striatum, nucleus accumbens, and cerebral cortex), are known to receive major EAA inputs. Recent evidence indicates that some EAA receptors are localized directly on DA neurons [29], where they act as modulators of DA release [19,25,35]. Electrophysiological data have also suggested the presence of both N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of the glutamate receptor on DA neurons in the rat midbrain [9,33]. Thus, these EAA receptors could play a role in regulating dopaminergic function and / or development in the CNS [8], and also in mediating glutamate neurotoxicity on dopaminergic neurons [6,30,42]. In the rat model of hemi-parkinsonism, we previously confirmed that tyrosine hydroxylase (TH) immunoreactivity, as an index of dopaminergic neurons, and the numbers of neurons immunopositive for ionotropic glutamate receptors, including NMDA receptor subunit NR1 and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR1 and GluR2 / 3 more so than GluR4), were simultaneously decreased in the substantia nigra pars compacta ipsilateral to the lesions with 6-OHDA [46,47]. Further, we previously reported that a number of cells immunopositive for NR1, GluR1, and GluR2 / 3 were observed within the established VM grafts, and a portion of the TH-positive cells in the grafts was also found immunopositive for NR1, and GluR1 or GluR2 / 3 in previous immunohistochemical studies [46,48]. These data suggested that both dopaminergic neurons and nondopaminergic neurons in the VM grafts appear to be modified functionally by glutamatergic afferents via various glutamate receptors including NR1, GluR1, and GluR2 / 3. In the present study, to elucidate the morphological changes in immunopositive cells of ionotropic glutamate receptors within intrastriatal ‘developing’ grafts of fetal VM in a rat with unilateral 6-OHDA lesions of the mesostriatal DA pathway, immunohistochemistry of NMDA (NR1) and AMPA receptor subunits (GluR1, GluR2 / 3, GluR4) was performed at 1, 4, and 12 weeks following transplantation. TH immunohistochemistry was also performed in order to reveal the morphological relationship between dopaminergic neurons and immunopositive cells of the various glutamate receptor subunits in the intrastriatal VM grafts.

2. Materials and methods

cycle with free access to food and water. The experimental protocols used in this study were approved by the ethical committee of animal experimentation at Miyazaki Medical College.

2.2. 6 -OHDA lesion, transplantation surgery, and behavioral testing The rats were anesthetized with pentobarbital (50 mg / kg, i.p.), and unilateral lesions of the left medial forebrain bundle were made by injection of 8 mg 6-OHDA hydrobromide (Sigma, St. Louis, MO, USA) in 4 ml saline containing 0.05% ascorbic acid. Stereotaxic coordinates for the lesions were as follows: AP 3.3 mm rostral to the interaural line, L 1.4 mm left of midline, and V 7.0 mm ventral to the dural surface, with the incisor bar set 2.4 mm below the level of the ear bars [23]. Two weeks after the 6-OHDA lesions were made, motor disturbance was assessed by counting full rotations per min in a cylindrical container (30 cm diameter) at 10-min intervals for the first 60 min after the administration of methamphetamine (3 mg / kg, i.p.). Animals accomplishing no fewer than 7 turns / min on methamphetamine challenge were included in the study [17]. Three weeks after the unilateral 6-OHDA lesions, neural transplantation was performed from fetal VM tissue according to a cell suspension method [2], with some modification. The VM tissue from rat embryos on embryonic day 15 was incubated in 0.1% trypsin / 0.05% DNase / Dulbecco’s modified Eagle’s medium (DMEM) (trypsin: Sigma, type II, crude; DNase: Sigma, type I, DN-25; both obtained from Sigma, St. Louis, MO, USA) at 37 8C for 20 min, then rinsed four or five times in 0.05% DNase / DMEM. The tissue was then mechanically dissociated to obtain homogenous single-cell suspension. Recipient rats were anesthetized with pentobarbital (50 mg / kg, i.p.) and placed in a stereotaxic apparatus (Narishige, Tokyo, Japan). Six microlitres of the suspension, containing 5.0310 5 cells, were injected at a rate of 1 ml / min into two sites of the denervated striatum: (1) AP 1.5 mm rostral to the bregma, L 2.5 mm left of midline, V 5.0 mm ventral to the dural surface; and (2) AP 0.5 mm rostral to the bregma, L 3.2 mm left of midline, V 5.0 mm ventral to the dural surface [23]. The grafted rats were allocated into three groups: rats to be examined behaviorally and histologically at 1 week post-grafting (n56); rats to be examined at 4 weeks post-grafting (n56); and rats to be examined at 12 weeks post-grafting (n56). Thus, the grafted rats were tested a second time for rotational asymmetry at 1, 4, or 12 weeks after transplantation at the time of histological examination.

2.1. Animals Female Wistar rats (Japan SLC, Hamamatsu, Japan) weighing 120–130 g at the beginning of the experiment were used. They were housed under a 12 h light / dark

2.3. Immunohistochemistry of TH and ionotropic glutamate receptor subunits Immediately after the last (second) behavioral testing,

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the rats were injected with an overdose of pentobarbital and perfused transcardially first with cold saline, followed by cold freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brains were removed immediately from the skulls, postfixed for 1 h in the same fixative, then immersed successively in PB containing 10% (1 h) and 30% sucrose (overnight). The brains were subsequently cut on a freezing microtome into 40-mm coronal sections. Several numbers of parallel series were collected from the area of the intrastriatal grafts and the substantia nigra in phosphate buffered saline (PBS; pH 7.4). Subsequently, immunohistochemistry was performed to detect immunopositive cells of TH and four glutamate receptor subunits (NR1, GluR1, GluR2 / 3, and GluR4). Free-floating sections were preincubated in 10% normal horse (for TH) or goat (for NR1, GluR1, GluR2 / 3, and GluR4) serum in 0.1 M PBS for 60 min at room temperature, then incubated at 4 8C overnight (for TH) or two overnights (for NR1, GluR1, GluR2 / 3, and GluR4) in primary antibodies to TH (diluted 1:20,000), NR1 (1:75), GluR1 (1:75), GluR2 / 3 (1:75), or GluR4 (1:75), respectively. A monoclonal antibody for TH [13] was kindly provided by Dr. Hatanaka at Osaka University. Anti-NR1 and all anti-GluR antisera were rabbit polyclonal antibodies raised against peptides corresponding to C-terminal portions of NR1, GluR1, GluR2 / 3, and GluR4 (Chemicon International Inc., Temecula, CA, USA). All of these antibodies against glutamate receptors have been thoroughly characterized and widely used for immunohistochemical localization of glutamate receptors with light and electron microscopic immunohistochemistry [26,38–40,46–48,51]. After several rinses with PBS, the sections were reacted with biotinylated horse anti-mouse IgG (for TH: 1:200; Vector Laboratories, Burlingame, CA, USA) or biotinylated goat anti-rabbit IgG (for NR1, GluR1, GluR2 / 3, and GluR4: 1:200; Vector Laboratories) for 60 min, then visualized using an avidin–biotin–peroxidase complex system (Vectastain Elite Kit, Vector Laboratories). Staining was followed by incubation in 0.3 mg / ml 39,3diaminobenzidine tetrahydrochloride and 0.01% H 2 O 2 in PBS. The sections were mounted on gelatin-coated glass slides, air-dried, dehydrated, and coverslipped. Immunoreactivity in selected sections including the VM grafts was examined under light microscopy. The cellular immunoreactivities of NR1, GluR1, GluR2 / 3, and GluR4 were quantified by counting the number of immunopositive cells in a unit area within the VM grafts (0.1530.15 mm 2 ) using an eyepiece micrometer. Numbers obtained from 4–6 sections were averaged to give the numbers for each animal and each antibody.

2.4. Statistical analysis Behavioral and histological data were analyzed nonparametrically by the Wilcoxon signed rank test and Mann–Whitney U-test, respectively. P values of ,0.05 were regarded as significant.

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3. Results

3.1. Methamphetamine-induced rotation Before transplantation, methamphetamine (3 mg / kg, i.p.) induced strong ipsilateral rotation in 6-OHDAlesioned rats. In the lesioned animals that did not receive VM grafts (n56), methamphetamine-induced rotation continued to be observed extensively at 15 weeks following the lesion generation, a time point corresponding to 12 weeks post-grafting in the grafted animals. In the grafted animals, this behavior was not altered the first week but was reversed the fourth week after transplantation of the VM grafts, and continued throughout 12 weeks postgrafting. Some of the grafted animals showed contralateral rotation (Fig. 1).

3.2. Distribution of TH-immunopositive cells in the grafts At 1 week post-grafting, many cell bodies showed THlike immunoreactivity in the graft. The immature-shaped TH-positive cells of smaller size (10–20 mm) distributed in a homogenous fashion within the grafts. TH-positive processes were also visible mostly within the grafts (Fig. 2A). By the fourth week post-grafting, the morphology of TH-positive cells showed progressive maturation. By this time, TH-positive cells in the grafts resembled those seen in the normal mesencephalon; they were multipolar, large (15–30 mm) cells with long neurites. However, smaller rounded TH-positive cells with fewer processes were usually also present among this TH-positive population (Fig. 2B). At 12 weeks post-grafting, a relatively dense innervation could be seen extending toward the surrounding host striatum from the grafts. By this time, modifications of the distribution of TH-positive cells within the grafts were apparent. The TH-positive cells were found aligned along the graft–host interface (Fig. 2C). Similar findings regarding morphological changes in the TH-positive cells within the VM grafts have been previously reported [1,3].

3.3. Distribution of immunopositive cells of ionotropic glutamate receptor subunits in the grafts Immunoreactivity of each glutamate receptor subunit was very low, and few immunopositive cells could be observed within the VM graft at 1 week post-grafting in common (Fig. 2D and G). Thus, quantification of the cellular immunoreactivity of each glutamate receptor subunit was not performed at this stage. At 4 weeks post-grafting, immunopositive cells of NR1 (Fig. 2E), GluR1 (Fig. 2H), and GluR2 / 3 were observed within the VM grafts. Weak GluR4-positive cells were seen in the grafts at 4 and 12 weeks post-grafting, although in some cases the staining was too faint to detect any immunopositive cells. At 12 weeks post-grafting, the numbers of

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Fig. 1. The mean numbers of rotations (6S.E.M.) per min over a 60-min test period in response to methamphetamine (3 mg / kg, i.p.) are shown for unilaterally 6-OHDA-lesioned rats with or without intrastriatal grafts (n56 in each group). ‘ipsi (1)’ and ‘contra (2)’ refer to the direction of rotation to lesion and transplantation side. The decrease in ipsilateral rotation scores found in the group receiving fetal VM grafts compared with pre-graft scores was significant at 4 and 12 weeks after transplantation (*P,0.05; Wilcoxon signed rank test).

NR1- and GluR1-positive cells tended to decrease as compared to those at 4 weeks post-grafting (Table 1, Fig. 2F and I). Compared with the morphological features of TH-positive cells in the grafts, the immunopositive cells of the glutamate receptor subunits tended to locate homogeneously within the grafts and were composed of various cell sizes and shapes, although these were mainly mediumsized (10–20 mm in diameter) aspiny cells. Representative photographs of the light microscopy are presented in Fig. 2 to show the results of TH-, NR1-, and GluR1-immunostaining.

4. Discussion In the rat brain, both TH- and DA-immunopositive neurons are present at embryonic day 13–13.5, and the innervation of the ganglionic eminence takes place 2 days later [43,50]. At postnatal day 1, the morphology of dopaminergic neurons is almost mature [45]. At postnatal day 2, patches with dense dopaminergic fibers are distributed throughout the striatum. From postnatal day 8–20, dopaminergic fibers become more diffuse in the striatum, and the number of varicose dopaminergic fibers increases dramatically, reaching almost adult levels at the end of the third week after birth [50], a time point which corresponds approximately to 4 weeks post-grafting in this study. The ability of the VM grafts to markedly influence behavior

took 4 weeks to develop, a timing correlated with the development and maturation of TH-positive cells in the VM grafts. Here we report a temporal increase in immunoreactivity of the NR1- and GluR1-positive cells in the fetal dopaminergic grafts implanted in the striatum in 6-OHDA rats. One week after transplantation, TH-positive cells were detected without any immunoreactivity of the NMDA and AMPA receptor subunits in the grafts. Four weeks after transplantation, TH-positive cells, distributed homogeneously in the grafts, appeared to be multipolar and larger compared to those at 1 week post-grafting. At this stage, we could more easily observe immunopositive cells of NR1, GluR1, and GluR2 / 3 than those of GluR4 distributing homogeneously in the grafts. Twelve weeks after transplantation, the numbers of NR1- and GluR1-positive cells decreased as compared with those at 4 weeks postgrafting, whereas TH-positive cells appeared to have matured in shape and size. On the other hand, the numbers of GluR2 / 3- and GluR4-positive cells had not changed in comparison with those at 4 weeks post-grafting. These results suggest that the ionotropic glutamate receptors have differential roles during the developmental period of the VM grafts. The pharmacological and physiological properties of GluR1–4 have been investigated previously using the subunit expression system with Xenopus oocytes or cultured mammalian cells. These studies revealed that each

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Fig. 2. Coronal sections throughout the VM graft in the 6-OHDA-lesioned striatum taken from three animals representing the three groups examined at 1 (A, D, G), 4 (B, E, H), and 12 weeks post-grafting (C, F, I). These consecutive sections show the differential distributions of immunopositive cells of TH (A–C), and NR1 (D–F) or GluR1 (G–I) within the VM grafts. A temporal hyperexpression of NR1- and GluR1-immunoreactivity of cells in the VM grafts was observed at 4 weeks post-grafting. Arrows indicate graft–host interfaces. Bar, 100 mm.

expression-induced subunit has its own response pattern, as have their combinations [14,20,49]. For example, a subunit composition lacking GluR2 forms Ca 21 -permeable channels, showing a marked inward rectification, whereas that including GluR2 apparently forms Ca 21 -impermeable channels which show a linear current–voltage relation over a wide range. These results suggest that GluR2 is a key subunit in the production of an AMPA-type response, Table 1 Quantification of immunopositive cells of NR1 and GluR (GluR1, GluR2 / 3, and GluR4) (number / 0.1530.15 mm 2 ) detected within the VM grafts Cellular character

Weeks post-grafting 4

12

NR1 GluR1 GluR2 / 3 GluR4

54.4564.86 33.0362.40 34.8161.82 7.0960.44

38.3163.93* 19.4261.30** 35.1661.47 7.0161.09

Results are means6S.E.M. (n56 in each group). Asterisks indicate significant difference from the results in the corresponding area at 4 weeks post-grafting (*P,0.05; **P,0.01; Mann–Whitney U-test).

although homo-oligomers of GluR2 could not respond to glutamate [15,49]. The present study has shown a lack of decline in the number of GluR2 / 3-positive cells during a period of 12 weeks post-grafting. During this period, a decline in the numbers of NR1- and GluR1-positive cells was observed; thus, the differential expression of glutamate receptors in the ‘developing’ grafts might be related to the above-mentioned pharmacological and physiological character of GluR2. Our previous studies using a double-labeling immunofluorescence method have revealed that a portion of the NR1-, GluR1-, and GluR2 / 3-positive cells in the established VM grafts were dopaminergic [46,48]. Electrophysiological, neurochemical, and neuroanatomical studies have revealed that glutamatergic neurotransmission seems to play an intrinsically widespread role in the functions of the substantia nigra pars compacta [5,21,24,41]. DA autoregulation and negative feedback are thought to be important physiological mechanisms for tonic regulation of dopaminergic graft function, and this regulation may be sufficient for the reinstatement of motor and sensorimotor behavior after the introduction of intrastriatal VM grafts

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[44]. In a recent study using co-cultures, the striatal target cells were suggested to induce a differential expression of the different glutamate receptor subtypes in VM cells [30]. These findings suggest that the glutamate receptors in the fetal VM grafts may play a role in establishing and / or maintaining functions such as amelioration of motor and sensorimotor deficits in the 6-OHDA rats. The present results, which showed a lack of decline in the number of GluR2 / 3-positive cells during the period 12 weeks postgrafting, suggest that GluR2 and / or GluR3 might be the key subunit(s) to maintain the functional recovery caused by the intrastriatal grafting. The present study has shown that a number of cells immunopositive for ionotropic glutamate receptors and of various sizes and shapes are distributed homogeneously within the intrastriatal VM grafts. Neuroanatomical studies have revealed that VM grafts are heterogeneous, in that grafts contain nondopaminergic cells such as GABA-, substance P-, and serotonin-containing neurons [10,27]. Both dopaminergic and nondopaminergic neurons in the VM grafts appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including NR1, GluR1, and GluR2 / 3. Results of double immunostaining partially support this interpretation, in that a portion of the dopaminergic neurons in the grafts was also immunopositive for NR1, GluR1, and GluR2 / 3 [46,48]. The rotational behavior induced by dopaminergic agonists observed in 6-OHDA-lesioned rats might be sensitive to glutamatergic modulation through NMDA receptors [37]. Cenci et al. indicated that in 6-OHDA-lesioned VMgrafted rats, the increased responsiveness to amphetamine, which manifests itself as an exaggerated striatal Fos expression in the striatal host tissue and, in most cases, also as a rotational bias away from the lesion-grafted side, is dependent on an intact corticostriatal input [7]. This study proposed that such increased responsiveness might reflect abnormal functional interactions between the innate corticostriatal glutamatergic input and the new dopaminergic striatal innervation provided by the transplanted neurons. The existence of NR1 and / or GluR on the cells within the grafts might be related to the interaction mentioned above. There is emerging evidence that EAAs play an important role in the control of neuronal differentiation and synaptic organization during early postnatal development [31]. A transient increase of EAA receptor binding sites in rat VM has been reported beyond adult levels; this increase peaked at postnatal day 7 (P7) for [ 3 H]glutamate binding to NMDA receptors, and at P14 for [ 3 H]AMPA and [ 3 H]kainate binding [8]. In the first 2 weeks of neonatal life in the rat, the NMDA subtype of glutamate receptor undergoes a period of hypersensitivity, during which neurons bearing NMDA receptors are rendered highly sensitive to excitotoxic degeneration [16]. These findings raise the possibility that the transient overexpression of EAA receptors in the VM may affect the developmental

fate of dopaminergic and other neurons in this region. A role similar to that of the EAA receptors in the developing CNS may be played, at least in part, by the NR1 and GluR1 receptors, which showed a temporary increase in cellular immunoreactivity in the VM grafts in our study.

Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. We are grateful to Ms. Fumiko Tsuda for technical assistance.

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