- Email: [email protected]
Intrastriatal targets of projection fibers from the central lateral nucleus of the rat thalamus
Neuroscience Letters 302 (2001) 105±108
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Intrastriatal targets of projection ®bers from the central lateral nucleus of the rat thalamus Noritaka Ichinohe*, Hiroyasu Iwatsuki, Kazuhiko Shoumura Department of Anatomy, Hirosaki University, School of Medicine, 5 Zaifucho, Hirosaki, 036±8562, Japan Received 9 January 2001; received in revised form 23 February 2001; accepted 26 February 2001
Abstract We examined light and electron microscopically intrastriatal targets of projection ®bers from the central lateral thalamic nucleus (CL), which is a major relay of cerebello-striatal projections. The study was done in the rat by combining the anterograde tract-tracing with immunohistochemistry for parvalbumin (PV); an anterograde tracer (biotin dextran amine: BDA) was injected into the CL. In the striatum, 91% of BDA-labeled axon terminals made asymmetrical synapses on PV immunonegative dendritic spines (assumed to be those of striatal projection neurons); only 0.5% of BDA-labeled axon terminals made synapses on PV immunopositive dendritic shafts. The remaining BDA-labeled axon terminals were in synaptic contact with PV immunonegative dendritic shafts. The results suggest that the cerebello-striatal projections through the CL predominantly access to striatal projection neurons, with only minor access to PV immunopositive (assumed to be GABAergic) interneurons in the striatum. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Striatum; Central lateral nucleus of thalamus; Parvalbumin; Electron microscopy; Rat; Biotin dextran amine
The basal ganglia and the cerebellum are the major subcortical structures that are important for regulation and control of voluntary movement (for review, see Refs. [4,7]). Projections from the deep cerebellar nuclei to the motorrelated striatum, which receives inputs from the motor cortical areas [11,13], have been reported to be relayed at several thalamic nuclei, i.e. the ventral lateral nucleus pars caudalis, ventral posterior lateral nucleus pars oralis, area X and central lateral nucleus (CL) in the monkey, and the ventral lateral nucleus and CL in the rat [5,11,13]. Precise understanding of the function of these cerebello-thalamo-striatal projections requires detailed knowledge of intrastriatal targets of projection ®bers from the cerebello-recipient thalamic nuclei. In the previous study, we demonstrated that thalamostriatal neurons in the CL receive direct strong projections from the lateral cerebellar nucleus in the rat [11]. Thus, in the present, we examined the intrastriatal targets of projection ®bers from the CL. Special attention was paid to (1) whether or not projection ®bers from the CL make synapses on dendritic spines, which are a good marker for striatal projection neurons [2,8]; and (2) whether or not projection ®bers from the CL are in synaptic contact with * Corresponding author. Tel.: 181-172-33-5111; fax: 181-17233-4540. E-mail address: [email protected] (N. Ichinohe).
parvalbumin (PV) immunopositive striatal interneurons, which represent the major class of GABAergic interneurons in the striatum [3,8,9]. Ten male Wistar rats (280±340 g) were anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and placed in a stereotaxic head holder. All procedures of the experiments were approved by the Animal Research Committee, Hirosaki University. A 10% solution of biotin dextran amine (BDA) (Molecular Probes, Eugene, OR) in 0.01 M phosphate buffer (pH 7.2) was iontophoretically injected into the middle-third of the CL (Fig. 1), where the main relay occurs from the lateral cerebellar nucleus to the laterodorsal part of the striatum [11]. The injection was done through a glass micropipette (tip diameter: 15 mm) using a positive ejecting current (5±7 mA, 7 s on/7 s off) for 20±30 min. After a survival period of 7 days, the rats were re-anesthetized and perfused transcardially with 100 ml of saline followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). The brain was removed, post-®xed for 1 h in the same ®xative without glutaraldehyde and then stored overnight at 48C in PB. The following day, the brain was serially cut 50 mm thick in the frontal plane on a microslicer (Dosaka EM, Kyoto, Japan). The sections containing the striatum and thalamus were collected and washed in 0.1 M phosphate buffered saline (PBS). For electron micro-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 66 6- 4
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N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
Fig. 1. Photomicrograph of a section through the thalamus, showing a BDA injection site in the central lateral nucleus (CL). CM, central medial nucleus; Hb, habenular nuclei; LD, laterodorsal nucleus; MD, mediodorsal nucleus; Pc, paracentral nucleus; Po, posterior nucleus; VB, ventrobasal complex; VL, ventrolateral nucleus. Scale bar, 500 mm.
scopy, the sections were freeze-thawed in isopentane cooled in liquid nitrogen after equilibration in a cryoprotectant consisting of 25% (w/v) sucrose and 10% (v/v) glycerol in 0.05 M phosphate buffer (pH 7.4). To visualize injected and transported BDA, the sections were incubated in PBS containing avidin-biotin-peroxidase complex (ABC) (1:100±200 dilution; Vector, Burlingame, CA) for 2 h at room temperature. Triton X-100 was added (0.5%) for light microscopy only. The sections were again washed in PBS, and then incubated in 0.05 M Tris-buffer (pH 7.6) containing 0.1% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis, MO) and 0.003% H2O2. BDAlabeled axons were visualized as brown (Fig. 2). The
Fig. 2. Photomicrograph of a section through the striatum, showing PV immunopositive neurons (purple) and BDA-labeled axons (brown). BDA-labeled axons have varicosities (arrowheads). Scale bar, 20 mm.
sections which contained the striatum were then processed for PV immunohistochemistry using VIP (Vector) as a chromogen [16]. Following several washes in PBS, the sections were incubated with a mouse anti-PV monoclonal antibody (Chemicon, Temecula, CA) diluted at 1:1000 for 24 h at 48C. They were then incubated in a 1:200 biotinylated horse anti-mouse IgG antibody (Vector) for 2 h at room temperature, followed by an 1:100 ABC complex for 2 h at room temperature. After several washes in 0.01 M sodium PBS (pH 7.5), peroxidase activity was visualized using the VIP complex according to the instructions included in the VIP kit. PV immunopositive structures were visualized as purple (Fig. 2). The sections were thoroughly washed in PBS, then either mounted onto gelatin-coated glass-slides for light microscopic examination or further processed for electron microscopy. For electron microscopy, the sections were post®xed with a solution of 1% osmium tetroxide in PB for 30 min. After washing in PB, the sections were dehydrated in a graded series of dilutions of ethanol, cleared in propylene oxide and then ¯at-embedded in Epon. Striatal areas that contained many anterogradely labeled ®bers around PV immunopositive perikarya and dendritic processes were trimmed. Thin sections were serially cut on an ultramicrotome, collected on formvar-coated, single-slot grids, stained with lead citrate and uranyl acetate, and examined with an electron microscope (JEM 2000-EX; JEOL, Tokyo, Japan). In order to avoid counting the same pro®le at different levels, only one section in 10 was scanned for quanti®cation of data. However, when the classi®cation of structures which were contacted by BDAlabeled boutons was not obvious, we used serial sections to con®rm the identity of those structures. The degree of penetration from the surface of sections may vary among antibodies and ABC complex used in this study. Thus, to ensure that the examined area contained both DAB- and VIP-labeled elements, quantitative analysis was performed on DAB-labeled boutons within 5 mm apart from VIPlabeled neural elements and vise versa. To evaluate the relative frequency of the CL boutons to all of excitatory inputs on PV immunopositive interneurons, we recorded both BDA-labeled and unlabeled boutons which made asymmetrical synaptic specializations with PV immunopositive structures [14]. In three rats, the BDA injections were well localized within the middle-third of the CL (Fig. 1). At the light microscopic level, BDA-labeled ®bers were distributed mainly in the laterodorsal part of the striatum and intermingled with PV immunopositive neurons (Fig. 2). All BDA-labeled ®bers were small in diameter (0.1±0.3 mm) and exhibited an extended type of arborization with long, straight, relatively unbranched trajectories through neuropil. Varicosities were formed along these axons almost exclusively in an en passant fashion (Fig. 2). Some BDA-stained varicosities were in close apposition with PV immunopositive dendrites. However, no thalamic varicose axons entwined PV immunopositive dendrites.
N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
Fourteen ultrathin sections from three rats were examined under electron microscopy. PV immunopositive cell bodies, dendrites and axons contained electron-dense discrete granules, while BDA-labeled axon terminals were recognized by the presence of electron-dense amorphous reaction product (Fig. 3). BDA-labeled axon terminals were small in size (0.3±0.9 mm in the minor diameter), contained round or oval vesicles, and made asymmetrical synapses on their targets. Of 408 BDA-labeled terminals, 371 (90.9%) were in synaptic contact with PV immunonegative dendritic spines, 35 (8.6%) made synapses on PV immunonegative dendritic shafts, and only two (0.5%) formed synapses on PV immunopositive dendritic shafts (Fig. 3). In the same tissues, we observed 302 PV immunopositive dendrites. These dendrites received 350 (99.4%) unlabeled and two (0.6%) BDA-labeled asymmetrical synaptic contacts (Fig. 3B,C). In the present study, we demonstrated that a large proportion (about 91%) of thalamostriatal ®bers from the CL terminated on PV immunonegative dendritic spines, which were assumed to be those of striatal projection neurons [2,7]. This proportion is comparable to the proportion of projection neurons in all striatal neurons in the rat (90± 95%) [2,6]. Thus, the target preference of thalamostriatal
107
®bers from the CL seems not to have a signi®cant bias to interneurons over projection neurons in the striatum. However, for precise estimation of this target preference, whether or not PV immunonegative dendritic shafts which were contacted by BDA-labeled terminals are striatal projection neurons should be determined. Xu et al. [15] examined, in the rat, the targets of thalamostriatal ®bers from the paracentral and centromedial thalamic nuclei, which constituted the anterior intralaminar nuclei together with the CL. Thalamostriatal ®bers from these nuclei formed synapses mainly on dendritic spines. In contrast, dendritic shafts were observed to be the main targets of thalamostriatal ®bers from the parafascicular nucleus (Pf), a part of the posterior intralaminar nuclei [15]. These ®ndings suggest that the anterior and posterior intralaminar nuclei modulate striatal function in different ways. The present study showed that only 0.5% of axon terminals of thalamostriatal ®bers from the CL terminated on PV immunopositive dendrites. Even if considering the fact that PV immunopositive neurons account for 3±5% of all striatal neurons [3,9], this number is proportionally small. Caution is, however, required to a possible technical limitation that PV immunoreactive products may not identify the complete
Fig. 3. BDA-labeled terminals are in asymmetrical synaptic contact (arrowheads) with a PV-immunonegative dendritic spine (A), with a PV-immunopositive dendritic shaft (B) and with a PV immunonegative dendritic shaft (C). Unlabeled axon terminals are in asymmetrical synaptic contact with a PV immunopositive dendrite (C). D, dendritic shaft; S, dendritic spine; PV, PV immunopositive dendrites. Scale bar, 1 mm.
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N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
extent of the dendritic tree. Furthermore, this study showed that CL terminals comprised only 0.6% of all asymmetrical synapses on PV immunopositive dendrites. To interpret this number, one should take it into account that the BDA injection sites occupied only a part of the CL. According to Rudkin and Sadikot [14], however, only 1.4% of axon terminals of thalamostriatal ®bers from the rat Pf made synapses on PV immunopositive dendrites, and, even after a large BDA injection into the Pf, only 4% of axon terminals which were in asymmetrical synaptic contact with PV immunopositive dendrites were labeled with BDA. Thus, the thalamostriatal projections from the CL and Pf were not likely to be the major inputs to PV immunopositive interneurons, which were assumed to be GABAergic [3,8,9]. This is in contrast with the corticostriatal projections, which are the strong driving force to PV immunopositive interneurons [1,9,12]. In summary, the present results suggest that cerebelloCL-striatal projections have predominant access (probably excitatory) to striatal projection neurons, and also minor access to PV immunopositive striatal interneurons, which are a strong GABAergic inhibitory source to striatal projection neurons [10]. This study was supported by Karoji Memorial Foundation for Medical Research and a grant for Medical Research from Aomori Bank. The authors are grateful to Drs Paul Hollister and Kathleen S. Rockland for their helpful advice on English. [1] Bennett, B.D. and Bolam, J.P., Synaptic input and output of parvalbumin-immunoreactive neurons in the neostriatum of the rat, Neuroscience, 62 (1994) 707±719. [2] Bolam, J.P., Hanley, J.J., Booth, P.A. and Bevan, M.D., Synaptic organisation of the basal ganglia, J. Anat., 196 (2000) 527±542. [3] Cowan, R.L., Wilson, C.J., Emson, P.C. and Heizmann, C.W., Parvalbumin-containing GABAergic interneurons in the rat neostriatum, J. Comp. Neurol., 302 (1990) 197±205. [4] DeLong, M.R., The Basal Ganglia, In E.R. Kandel, J.H.
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Schwartz and T.M. Jessell (Eds.), Principles of Neural Science, 4th edition, McGraw-Hill, New York, 2000, pp. 853±872. Erro, E., Lanciego, J.L. and Gimenez-Amaya, J.M., Relationships between thalamostriatal neurons and pedunculopontine projections to the thalamus: a neuroanatomical tracttracing study in the rat, Exp. Brain Res., 127 (1999) 162±170. Graveland, G.A. and DiFiglia, M., The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum, Brain Res., 327 (1985) 307± 311. Ghez, C. and Thach, W.T., The Cerebellum, In E.R. Kandel, J.H. Schwartz and T.M. Jessell (Eds.), Principles of Neural Science, 4th edition, McGraw-Hill, New York, 2000, pp. 832± 852. Kawaguchi, Y., Wilson, C.J., Augood, S.J. and Emson, P.C., Striatal interneurones: chemical, physiological and morphological characterization, Trends Neurosci., 18 (1995) 527±535. Kita, H., GABAergic circuits of the striatum, Prog. Brain. Res., 99 (1993) 51±72. Koos, T. and Tepper, J.M., Inhibitory control of neostriatal projection neurons by GABAergic interneurons, Nat. Neurosci., 2 (1999) 467±472. Ichinohe, N., Mori, F. and Shoumura, K., A di-synaptic projection from the lateral cerebellar nucleus to the laterodorsal part of the striatum via the central lateral nucleus of the thalamus in the rat, Brain Res., 880 (2000) 191±197. Lapper, S.R., Smith, Y., Sadikot, A.F., Parent, A. and Bolam, J.P., Cortical input to parvalbumin-immunoreactive neurones in the putamen of the squirrel monkey, Brain Res., 580 (1992) 215±224. McFarland, N.R. and Haber, S.N., Convergent inputs from thalamic motor nuclei and frontal cortical areas to the dorsal striatum in the primate, J. Neurosci., 20 (2000) 3798±3813. Rudkin, T.M. and Sadikot, A.F., Thalamic input to parvalbumin-immunoreactive GABAergic interneurons: organization in normal striatum and effect of neonatal decortication, Neuroscience, 88 (1999) 1165±1175. Xu, Z.C., Wilson, C.J. and Emson, P.C., Restoration of thalamostriatal projections in rat neostriatal grafts: an electron microscopic analysis, J. Comp. Neurol., 303 (1991) 22±34. Zhou, M. and Grofova, I., The use of peroxidase substrate Vector VIP in electron microscopic single and double antigen localization, J. Neurosci. Methods, 62 (1995) 149±158.
www.elsevier.com/locate/neulet
Intrastriatal targets of projection ®bers from the central lateral nucleus of the rat thalamus Noritaka Ichinohe*, Hiroyasu Iwatsuki, Kazuhiko Shoumura Department of Anatomy, Hirosaki University, School of Medicine, 5 Zaifucho, Hirosaki, 036±8562, Japan Received 9 January 2001; received in revised form 23 February 2001; accepted 26 February 2001
Abstract We examined light and electron microscopically intrastriatal targets of projection ®bers from the central lateral thalamic nucleus (CL), which is a major relay of cerebello-striatal projections. The study was done in the rat by combining the anterograde tract-tracing with immunohistochemistry for parvalbumin (PV); an anterograde tracer (biotin dextran amine: BDA) was injected into the CL. In the striatum, 91% of BDA-labeled axon terminals made asymmetrical synapses on PV immunonegative dendritic spines (assumed to be those of striatal projection neurons); only 0.5% of BDA-labeled axon terminals made synapses on PV immunopositive dendritic shafts. The remaining BDA-labeled axon terminals were in synaptic contact with PV immunonegative dendritic shafts. The results suggest that the cerebello-striatal projections through the CL predominantly access to striatal projection neurons, with only minor access to PV immunopositive (assumed to be GABAergic) interneurons in the striatum. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Striatum; Central lateral nucleus of thalamus; Parvalbumin; Electron microscopy; Rat; Biotin dextran amine
The basal ganglia and the cerebellum are the major subcortical structures that are important for regulation and control of voluntary movement (for review, see Refs. [4,7]). Projections from the deep cerebellar nuclei to the motorrelated striatum, which receives inputs from the motor cortical areas [11,13], have been reported to be relayed at several thalamic nuclei, i.e. the ventral lateral nucleus pars caudalis, ventral posterior lateral nucleus pars oralis, area X and central lateral nucleus (CL) in the monkey, and the ventral lateral nucleus and CL in the rat [5,11,13]. Precise understanding of the function of these cerebello-thalamo-striatal projections requires detailed knowledge of intrastriatal targets of projection ®bers from the cerebello-recipient thalamic nuclei. In the previous study, we demonstrated that thalamostriatal neurons in the CL receive direct strong projections from the lateral cerebellar nucleus in the rat [11]. Thus, in the present, we examined the intrastriatal targets of projection ®bers from the CL. Special attention was paid to (1) whether or not projection ®bers from the CL make synapses on dendritic spines, which are a good marker for striatal projection neurons [2,8]; and (2) whether or not projection ®bers from the CL are in synaptic contact with * Corresponding author. Tel.: 181-172-33-5111; fax: 181-17233-4540. E-mail address: [email protected] (N. Ichinohe).
parvalbumin (PV) immunopositive striatal interneurons, which represent the major class of GABAergic interneurons in the striatum [3,8,9]. Ten male Wistar rats (280±340 g) were anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and placed in a stereotaxic head holder. All procedures of the experiments were approved by the Animal Research Committee, Hirosaki University. A 10% solution of biotin dextran amine (BDA) (Molecular Probes, Eugene, OR) in 0.01 M phosphate buffer (pH 7.2) was iontophoretically injected into the middle-third of the CL (Fig. 1), where the main relay occurs from the lateral cerebellar nucleus to the laterodorsal part of the striatum [11]. The injection was done through a glass micropipette (tip diameter: 15 mm) using a positive ejecting current (5±7 mA, 7 s on/7 s off) for 20±30 min. After a survival period of 7 days, the rats were re-anesthetized and perfused transcardially with 100 ml of saline followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). The brain was removed, post-®xed for 1 h in the same ®xative without glutaraldehyde and then stored overnight at 48C in PB. The following day, the brain was serially cut 50 mm thick in the frontal plane on a microslicer (Dosaka EM, Kyoto, Japan). The sections containing the striatum and thalamus were collected and washed in 0.1 M phosphate buffered saline (PBS). For electron micro-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 66 6- 4
106
N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
Fig. 1. Photomicrograph of a section through the thalamus, showing a BDA injection site in the central lateral nucleus (CL). CM, central medial nucleus; Hb, habenular nuclei; LD, laterodorsal nucleus; MD, mediodorsal nucleus; Pc, paracentral nucleus; Po, posterior nucleus; VB, ventrobasal complex; VL, ventrolateral nucleus. Scale bar, 500 mm.
scopy, the sections were freeze-thawed in isopentane cooled in liquid nitrogen after equilibration in a cryoprotectant consisting of 25% (w/v) sucrose and 10% (v/v) glycerol in 0.05 M phosphate buffer (pH 7.4). To visualize injected and transported BDA, the sections were incubated in PBS containing avidin-biotin-peroxidase complex (ABC) (1:100±200 dilution; Vector, Burlingame, CA) for 2 h at room temperature. Triton X-100 was added (0.5%) for light microscopy only. The sections were again washed in PBS, and then incubated in 0.05 M Tris-buffer (pH 7.6) containing 0.1% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis, MO) and 0.003% H2O2. BDAlabeled axons were visualized as brown (Fig. 2). The
Fig. 2. Photomicrograph of a section through the striatum, showing PV immunopositive neurons (purple) and BDA-labeled axons (brown). BDA-labeled axons have varicosities (arrowheads). Scale bar, 20 mm.
sections which contained the striatum were then processed for PV immunohistochemistry using VIP (Vector) as a chromogen [16]. Following several washes in PBS, the sections were incubated with a mouse anti-PV monoclonal antibody (Chemicon, Temecula, CA) diluted at 1:1000 for 24 h at 48C. They were then incubated in a 1:200 biotinylated horse anti-mouse IgG antibody (Vector) for 2 h at room temperature, followed by an 1:100 ABC complex for 2 h at room temperature. After several washes in 0.01 M sodium PBS (pH 7.5), peroxidase activity was visualized using the VIP complex according to the instructions included in the VIP kit. PV immunopositive structures were visualized as purple (Fig. 2). The sections were thoroughly washed in PBS, then either mounted onto gelatin-coated glass-slides for light microscopic examination or further processed for electron microscopy. For electron microscopy, the sections were post®xed with a solution of 1% osmium tetroxide in PB for 30 min. After washing in PB, the sections were dehydrated in a graded series of dilutions of ethanol, cleared in propylene oxide and then ¯at-embedded in Epon. Striatal areas that contained many anterogradely labeled ®bers around PV immunopositive perikarya and dendritic processes were trimmed. Thin sections were serially cut on an ultramicrotome, collected on formvar-coated, single-slot grids, stained with lead citrate and uranyl acetate, and examined with an electron microscope (JEM 2000-EX; JEOL, Tokyo, Japan). In order to avoid counting the same pro®le at different levels, only one section in 10 was scanned for quanti®cation of data. However, when the classi®cation of structures which were contacted by BDAlabeled boutons was not obvious, we used serial sections to con®rm the identity of those structures. The degree of penetration from the surface of sections may vary among antibodies and ABC complex used in this study. Thus, to ensure that the examined area contained both DAB- and VIP-labeled elements, quantitative analysis was performed on DAB-labeled boutons within 5 mm apart from VIPlabeled neural elements and vise versa. To evaluate the relative frequency of the CL boutons to all of excitatory inputs on PV immunopositive interneurons, we recorded both BDA-labeled and unlabeled boutons which made asymmetrical synaptic specializations with PV immunopositive structures [14]. In three rats, the BDA injections were well localized within the middle-third of the CL (Fig. 1). At the light microscopic level, BDA-labeled ®bers were distributed mainly in the laterodorsal part of the striatum and intermingled with PV immunopositive neurons (Fig. 2). All BDA-labeled ®bers were small in diameter (0.1±0.3 mm) and exhibited an extended type of arborization with long, straight, relatively unbranched trajectories through neuropil. Varicosities were formed along these axons almost exclusively in an en passant fashion (Fig. 2). Some BDA-stained varicosities were in close apposition with PV immunopositive dendrites. However, no thalamic varicose axons entwined PV immunopositive dendrites.
N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
Fourteen ultrathin sections from three rats were examined under electron microscopy. PV immunopositive cell bodies, dendrites and axons contained electron-dense discrete granules, while BDA-labeled axon terminals were recognized by the presence of electron-dense amorphous reaction product (Fig. 3). BDA-labeled axon terminals were small in size (0.3±0.9 mm in the minor diameter), contained round or oval vesicles, and made asymmetrical synapses on their targets. Of 408 BDA-labeled terminals, 371 (90.9%) were in synaptic contact with PV immunonegative dendritic spines, 35 (8.6%) made synapses on PV immunonegative dendritic shafts, and only two (0.5%) formed synapses on PV immunopositive dendritic shafts (Fig. 3). In the same tissues, we observed 302 PV immunopositive dendrites. These dendrites received 350 (99.4%) unlabeled and two (0.6%) BDA-labeled asymmetrical synaptic contacts (Fig. 3B,C). In the present study, we demonstrated that a large proportion (about 91%) of thalamostriatal ®bers from the CL terminated on PV immunonegative dendritic spines, which were assumed to be those of striatal projection neurons [2,7]. This proportion is comparable to the proportion of projection neurons in all striatal neurons in the rat (90± 95%) [2,6]. Thus, the target preference of thalamostriatal
107
®bers from the CL seems not to have a signi®cant bias to interneurons over projection neurons in the striatum. However, for precise estimation of this target preference, whether or not PV immunonegative dendritic shafts which were contacted by BDA-labeled terminals are striatal projection neurons should be determined. Xu et al. [15] examined, in the rat, the targets of thalamostriatal ®bers from the paracentral and centromedial thalamic nuclei, which constituted the anterior intralaminar nuclei together with the CL. Thalamostriatal ®bers from these nuclei formed synapses mainly on dendritic spines. In contrast, dendritic shafts were observed to be the main targets of thalamostriatal ®bers from the parafascicular nucleus (Pf), a part of the posterior intralaminar nuclei [15]. These ®ndings suggest that the anterior and posterior intralaminar nuclei modulate striatal function in different ways. The present study showed that only 0.5% of axon terminals of thalamostriatal ®bers from the CL terminated on PV immunopositive dendrites. Even if considering the fact that PV immunopositive neurons account for 3±5% of all striatal neurons [3,9], this number is proportionally small. Caution is, however, required to a possible technical limitation that PV immunoreactive products may not identify the complete
Fig. 3. BDA-labeled terminals are in asymmetrical synaptic contact (arrowheads) with a PV-immunonegative dendritic spine (A), with a PV-immunopositive dendritic shaft (B) and with a PV immunonegative dendritic shaft (C). Unlabeled axon terminals are in asymmetrical synaptic contact with a PV immunopositive dendrite (C). D, dendritic shaft; S, dendritic spine; PV, PV immunopositive dendrites. Scale bar, 1 mm.
108
N. Ichinohe et al. / Neuroscience Letters 302 (2001) 105±108
extent of the dendritic tree. Furthermore, this study showed that CL terminals comprised only 0.6% of all asymmetrical synapses on PV immunopositive dendrites. To interpret this number, one should take it into account that the BDA injection sites occupied only a part of the CL. According to Rudkin and Sadikot [14], however, only 1.4% of axon terminals of thalamostriatal ®bers from the rat Pf made synapses on PV immunopositive dendrites, and, even after a large BDA injection into the Pf, only 4% of axon terminals which were in asymmetrical synaptic contact with PV immunopositive dendrites were labeled with BDA. Thus, the thalamostriatal projections from the CL and Pf were not likely to be the major inputs to PV immunopositive interneurons, which were assumed to be GABAergic [3,8,9]. This is in contrast with the corticostriatal projections, which are the strong driving force to PV immunopositive interneurons [1,9,12]. In summary, the present results suggest that cerebelloCL-striatal projections have predominant access (probably excitatory) to striatal projection neurons, and also minor access to PV immunopositive striatal interneurons, which are a strong GABAergic inhibitory source to striatal projection neurons [10]. This study was supported by Karoji Memorial Foundation for Medical Research and a grant for Medical Research from Aomori Bank. The authors are grateful to Drs Paul Hollister and Kathleen S. Rockland for their helpful advice on English. [1] Bennett, B.D. and Bolam, J.P., Synaptic input and output of parvalbumin-immunoreactive neurons in the neostriatum of the rat, Neuroscience, 62 (1994) 707±719. [2] Bolam, J.P., Hanley, J.J., Booth, P.A. and Bevan, M.D., Synaptic organisation of the basal ganglia, J. Anat., 196 (2000) 527±542. [3] Cowan, R.L., Wilson, C.J., Emson, P.C. and Heizmann, C.W., Parvalbumin-containing GABAergic interneurons in the rat neostriatum, J. Comp. Neurol., 302 (1990) 197±205. [4] DeLong, M.R., The Basal Ganglia, In E.R. Kandel, J.H.
[5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
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