Neurobiology of Disease 39 (2010) 381–392
Contents lists available at ScienceDirect
Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
A direct projection from the subthalamic nucleus to the ventral thalamus in monkeys
Alberto J. Rico a,b, Pedro Barroso-Chinea a,b, Lorena Conte-Perales a,b, Elvira Roda a,b, Virginia Gómez-Bautista a,b, Miriam Gendive a, José A. Obeso a,b, José L. Lanciego a,b,⁎ a Area de Neurociencias, Centro de Investigación Médica Aplicada (CIMA), Facultad de Medicina, Universidad de Navarra, Pamplona, Spain b Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Pamplona, Spain article info abstract
Article history: The current basal ganglia model considers the internal division of the globus pallidus and the substantia Received 25 March 2010 nigra pars reticulata as the sole sources of basal ganglia output to the thalamus. However, following the Revised 26 April 2010 delivery of retrograde tracers into the ventral anterior/ventral lateral thalamic nuclei, a moderate number of Accepted 1 May 2010 labeled neurons were found within the subthalamic nucleus (STN) in control cases, MPTP-treated monkeys Available online 7 May 2010 and animals with levodopa-induced dyskinesias. Furthermore, dual tracing experiments showed that subthalamo-thalamic and subthalamo-pallidal projections arise from different subpopulations of STN Keywords: Basal ganglia efferent neurons. Moreover, upregulated expression of the mRNA coding the vesicular glutamate transporter MPTP 2 (vGlut2) was found in retrogradely-labeled STN neurons in MPTP-treated monkeys. By contrast, there is a Hyperdirect pathway reduction in vGlut2 mRNA expression in subthalamo-thalamic neurons in dyskinetic monkeys. In conclusion, Retrograde tracing our findings support the presence of a direct projection from the STN to the ventral thalamus that appears to Parkinson's disease be functionally modulated by dopaminergic activity. © 2010 Elsevier Inc. All rights reserved.
Introduction In recent years, certain anatomical evidence has appeared that has led to the reconsideration of the position of the STN nucleus within The classic model of the basal ganglia (Albin et al., 1989; DeLong, the basal ganglia indirect pathway. For instance, it is well known that 1990), considers the internal division of the globus pallidus (GPi) and STN efferents are highly branched and therefore most STN neurons the substantia nigra, pars reticulata (SNr) as the only basal ganglia collateralize to simultaneously innervate the GPe, GPi and SNr (Van output nuclei. Information processed within basal ganglia circuits der Kooy and Hattori, 1980; Kita et al., 1983; Kita and Kitai, 1987; converges in GPi/SNr neurons to be further funneled to the thalamus Plenz and Kitai, 1999; Sato et al., 2000; Parent et al., 2000; Castle et al., through the pallido- and nigrothalamic pathways. At the thalamic 2005). Moreover, STN neurons are also known to receive strong level, the ventral anterior (VA) and ventral lateral (VL) nuclei are glutamatergic innervation from the caudal intralaminar nuclei known to be the major recipients of basal ganglia output in monkeys (reviewed in Lanciego et al., 2009) as well as from the cerebral (Sidibé et al., 1997; Illinsky et al., 1997; Kultas-Illinsky et al., 1997). cortex, the latter also known as the “hyperdirect” pathway (Nambu, Under circumstances of dopaminergic depletion, basal ganglia output 2004; Nambu et al., 2002). Finally, it is worth noting that a direct neurons become hyperactive, leading to excessive GABAergic outflow projection arising from the STN that reaches the cerebral cortex has reaching thalamic targets, which impairs thalamo-cortical connectiv- also been reported (Jackson and Crossman, 1981; Degos et al., 2008). ity and leads to the appearance of the cardinal motor symptoms that In an attempt to better elucidate whether STN efferents gain direct characterize Parkinson's disease. Although it is widely accepted that access to the VA/VL ventral thalamic motor nuclei in monkeys, we GABA is the only neurotransmitter involved in basal ganglia output, have carried out single- and double-retrograde tract-tracing studies. the presence of sparse projections arising from the STN that reach the Our findings confirm and expand earlier observations (Nauta and VA/VL thalamic nuclei has been reported in monkeys and cats (Nauta Cole, 1978) by showing that the STN nucleus is composed of at least and Cole, 1978). Nevertheless, since these findings were not two different subtypes of projection neurons, one subtype innervating confirmed in later reports (Smith et al., 1990; Sato et al., 2000) they the GPi; and the other involving a moderate number of neurons that have since been neglected. directly project to the VA/VL nuclei, presumably providing an excitatory input to these thalamic targets.
⁎ Corresponding author. Area de Neurociencias, Centro de Investigación Médica Methods Aplicada (CIMA), Universidad de Navarra, Pio XII Ave. 55, 31008 Pamplona, Spain. Fax: +34 948 194 715. E-mail address: [email protected] (J.L. Lanciego). A total of 8 adult male Macaca fascicularis monkeys (body weight Available online on ScienceDirect (www.sciencedirect.com). ranging from 3.8 to 4.5 kg) were used in this study. At all times the
0969-9961/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2010.05.004 382 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 animals were handled in accordance with the European Council Dual tracing experiments were performed in 2 control monkeys Directive 86/609/EEC as well as in agreement with the Society for through the delivery of CTB in VA/VL (as described above) coupled Neuroscience Policy on the Use of Animals in Neuroscience Research. with the injection of 0.3 µL of Fluoro-Gold (FG, Fluorochrome Inc.) The experimental design was approved by the Ethical Committee for into the internal division of the globus pallidus (GPi). FG was Animal Testing of the University of Navarra (ref: 018/2008). pressure-delivered in pulses of 0.1 µL every 2 min as a 0.2% solution in 0.05 M cacodylate buffer pH 7.2 by the same procedure as that MPTP intoxication employed for the injection of CTB. The chosen stereotaxic coordinates to target the GPi were 4 mm caudal to ac, 7.5 mm lateral to the In 4 monkeys, the neurotoxin MPTP (Sigma Chemical Co., St. Louis, midline, and 2 mm ventral to the ac–pc line. MO) was administrated intravenously as a 0.2% solution in saline (0.2 mg/kg, injected once weekly) until a stable parkinsonian Tissue processing (I): single- and double-retrograde tract-tracing syndrome was reached. The severity of the MPTP-induced parkinso- nian syndrome was evaluated using Kurlan's scale (Kurlan et al., Two weeks after surgery, the animals were anesthetized with an 1991), which rates parkinsonian motor symptoms in a cumulative overdose of chloral hydrate and perfused transcardially. The perfusates scale where the highest score is 29, reflecting maximum severity. Monkeys reached a stable score above 21 points that was maintained over a MPTP washout period of 2 months (with an estimated variation of 21 points ±2). Subsequently, 2 MPTP-intoxicated monkeys received daily oral treatment with levodopa administered at a dose of 25 mg/kg (Madopar levodopa/benserazide, 200:50; Roche, France). The response to levodopa was measured by the magnitude of the changes in the Kurlan's scale from the “off” medication motor condition to the “on” motor state and the “on” duration. Levodopa- induced dyskinesias (LIDs) were rated as 1 (mild), when presented only occasionally under stress; 2 (moderate) for LIDs present during most of the “on” period without interfering with voluntary move- ments; and 3 (severe) when LIDs were continuous, generalized and they perturbed motor behavior. This scoring system matches the scale of Obeso included in the Core Assessment Program for Intracerebral Transplantation for Parkinson's disease (Langston et al., 1992), which was subsequently modified and validated for routine assessment of dyskinesias in patients (Goetz et al., 1994).
Stereotaxic surgery for tracer delivery
Surgical anesthesia was induced by intramuscular injection of a mixture of ketamine (5 mg/kg) and midazolam (0.5 mg/kg), produc- ing deep anesthesia for 2 to 3 h. Just before surgery, local anesthesia was produced with a 10% solution of lidocain. Analgesia was achieved with an intramuscular injection of 1 mL of Finadyne (5 mg/kg) once surgery was completed, which was repeated 24 and 48 h post-surgery. A similar schedule of antibiotic administration was implemented (ampicilin, 0.5 mL/day) and animals were kept under constant control in single cages with “ad libitum” access to food and water. Stereotaxic coordinates were taken from the atlas of Martin and Bowden (1997) and target selection was assisted by ventriculography. Each ventriculography consisted of the delivery of 0.3 mL of a Rx contrast (Omnigraph 300) into the frontal horn of the lateral ventricle to completely fill the ventricular system. Once the Rx plate was developed, both the anterior (ac) and the posterior (pc) commissures were localized. Subsequently, the ac–pc line was measured and used as a reference to position the monkey's head parallel to the horizontal plane, as well as to calibrate the stereotaxic reference atlas. Six monkeys (2 control animals, 2 animals treated with MPTP and 2 dyskinetic monkeys) received a single pressure-injection of 5 µL of unconjugated cholera toxin B subunit (CTB, List Biological Laborato- ries, Campbell, CA) through a Hamilton syringe (final concentration of 5 mg/mL in 0.01 M phosphate buffer, pH 7.5). The retrograde tracer CTB was delivered into a thalamic area containing the ventral anterior (VA) and ventral lateral (VL) thalamic nuclei. The stereotaxic coordinates selected to target the VA/VL were 4.5 mm caudal to the center of the anterior commissure (ac), 4 mm lateral to the midline, and 2 mm dorsal to the ac–pc line. Tracer delivery was accomplished Fig. 1. Extent of MPTP-induced dopaminergic depletion. The levels of tyrosine hydroxylase (TH) in the striatum and substantia nigra pars compacta (SNc) that in pulses of 1 µL every 2 min and once completed, the microsyringe typically characterizes a control primate (panel A) are clearly reduced in both the was left in place for 15 min before withdrawal to minimize tracer MPTP-treated monkeys (B) as well as in the parkinsonian monkeys showing levodopa- reflux and uptake through the injection tract. induced dyskinesias (panel C). Scale bar is 2500 µm for all panels. A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 383 consisted on a saline Ringer solution followed by 3000 mL of a fixative 0.125 M PB, pH 7.4. For the monkeys that received a single injection of solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in unconjugated CTB (2 controls, 2 MPTP-treated and 2 dyskinetic 0.125 M phosphate buffer (PB), pH 7.4. Perfusion was continued with monkeys), one series of sections was used for the immunohistochemical 1000 mL of a cryoprotectant solution containing 10% of glycerin and 1% detection of tyrosine hydroxylase (TH) in order to assess the extent of of dimethylsulphoxide (DMSO) in 0.125 M PB, pH 7.4. Once perfusion the dopaminergic lesion induced by MPTP treatment. Another series of was completed, the skull was opened and the brain removed and stored sections were used for the immunohistochemical detection of trans- for 48 h at 4 °C in a cryoprotectant solution containing 20% glycerin and ported CTB and finally, a further series of sections were devoted to the 2% DMSO in 0.125 M PB, pH 7.4. Finally, frozen serial sagittal sections simultaneous fluorescent visualization of transported CTB together with (40 µm-thick) were obtained on a sliding microtome and collected in in situ hybridization for vGlut2 mRNA (see below). In the remaining 2
Fig. 2. Basal ganglia neurons innervating the VA/VL thalamic nuclei in control monkeys. A large CTB deposit that was placed in the VA/VL nuclei without tracer spread to adjacent structures such as the neighboring thalamic nuclei or the internal capsule. Panels A and B also show the lack of tracer uptake along the injection tract. Following CTB delivery into the VA/VL, most retrograde-labeled neurons were observed in basal ganglia output nuclei, such as the GPi (C) and SNr (D). Furthermore, a moderate number of CTB-labeled neurons were observed in the STN nucleus, as illustrated in panels E and F. Scale bar is 3500 µm in panels A and B, 500 µm in panels C and D (120 µm in insets), 600 µm in panel E and 85 µm in F. Abbreviations: ac: anterior commissure; CL: central lateral thalamic nucleus; CM-Pf: centromedian-parafascicular thalamic nucleus; GPe: globus pallidus, external division; GPi: globus pallidus, internal division; ic: internal capsule; LP: lateral posterior thalamic nucleus; MD: mediodorsal thalamic nucleus; ot: optic tract; Pul: pulvinar thalamic nucleus; SNr: substantia nigra, pars reticulata; STN: subthalamic nucleus; VA: ventral anterior thalamic nucleus; VL: ventral lateral thalamic nucleus; VPM: ventral posteromedial thalamic nucleus. 384 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 control monkeys that received two tracers (CTB in the VA/VL and FG in diaminobenzidine (DAB, Sigma). The immunohistochemical detection the GPi), the detection of retrograde-labeled cells was accomplished by of transported CTB was carried out by incubating the sections with a immunofluorescent visualization under epifluorescent illumination. primary antibody against CTB (1:2000; overnight at 4 °C; rabbit anti- Sagittal sections throughout the entire mediolateral extent of the CTB antibody from GenWay, San Diego, CA), followed by a substantia nigra and striatum were processed for the immunohisto- biotinylated donkey anti-rabbit IgG (1:600, 2 h at room temperature; chemical detection of TH. Accordingly, sections were incubated antibody from Jackson Immunoresearch). Sections were then incu- overnight at 4 °C with a mouse anti-TH primary antibody (1:1000; bated in HRP-conjugated streptavidin (1:5000; 90 min at room Sigma Chemical Co., St. Louis, MO), and then for 2 h at room temperature; Sigma) and finally visualized in brown with DAB temperature with a biotinylated goat anti-mouse IgG (1:600; Jackson (Sigma). Immunoresearch). Next, sections were incubated for 90 min at room Sections were mounted on glass slides using a 2% gelatin solution temperature in 1:5000 HRP-conjugated streptavidin (Sigma) and the (Merck, Darmstadt, Germany) in 0.05 M Tris/HCl pH 7.6 and they TH staining was then visualized by incubation in a regular solution of were dried at room temperature. Sections for TH staining were then
Fig. 3. Basal ganglia neurons innervating the VA/VL thalamic nuclei in MPTP-treated monkeys. A large CTB deposit that was performed in the VA/VL nuclei without tracer spread to adjacent structures such as the neighboring thalamic nuclei or the internal capsule. Panels A and B also show the lack of tracer uptake along the injection tract. Following CTB delivery into the VA/VL, most retrograde-labeled neurons were observed in basal ganglia output nuclei such as the GPi (C) and SNr (D). Furthermore, a moderate number of CTB-labeled neurons were observed in the STN nucleus, as illustrated in panels E and F. Scale bar is 3500 µm in panels A and B, 500 µm in panels C and D (120 µm in insets), 600 µm in panel E and 85 µm in F. Abbreviations: ac: anterior commissure; CL: central lateral thalamic nucleus; CM-Pf: centromedian-parafascicular thalamic nucleus; cp: cerebral peduncle; GPe: globus pallidus, external division; GPi: globus pallidus, internal division; ic: internal capsule; LP: lateral posterior thalamic nucleus; MD: mediodorsal thalamic nucleus; ot: optic tract; Pul: pulvinar thalamic nucleus; SNc: substantia nigra, pars compacta; SNr: substantia nigra, pars reticulata; STN: subthalamic nucleus; VA: ventral anterior thalamic nucleus; VL: ventral lateral thalamic nucleus; VPM: ventral posteromedial thalamic nucleus. A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 385
Fig. 4. Basal ganglia neurons innervating the VA/VL thalamic nuclei in dyskinetic monkeys. In both dyskinetic monkeys, CTB was deposited in medially-located thalamic areas, mainly comprising the VA nucleus with very little involvement of the VL nucleus. Panels A and B illustrate the lack of tracer uptake along the injection tract. Besides a large number of pallidothalamic- and nigrothalamic-projecting neurons (panels C and D, respectively), STN neurons innervating the VA nucleus were also found consistently (panels E and F). Scale bar is 3500 µm in panels A and B, 500 µm in panels C and D (120 µm in insets), 1100 µm in panel E and 45 µm in F. Abbreviations: ac: anterior commissure; AM/AV: anteromedial and anteroventral thalamic nuclei; CM-Pf: centromedian-parafascicular thalamic nucleus; GPe: globus pallidus, external division; GPi: globus pallidus, internal division; ic: internal capsule; MD: mediodorsal thalamic nucleus; ot: optic tract; PC: paracentral thalamic nucleus; SNr: substantia nigra, pars reticulata; STN: subthalamic nucleus; VA: ventral anterior thalamic nucleus; VL: ventral lateral thalamic nucleus; VPM: ventral posteromedial thalamic nucleus. 386 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 387 dehydrated in ascending series of ethanol, cleared in xylene and staining. For each animal, the densitometry analysis was performed coverslipped with Entellan (Merck). The sections used for CTB on a total number of 20 CTB-labeled neurons. Only CTB-labeled immunohistochemistry were then counterstained with thionin to neurons in which the nucleus was clearly visible were considered as accurately delineate the nuclear boundaries of the brain structures appropriate ROIs for densitometric analysis. Student's t test was used showing CTB labeling. After the Nissl staining was completed, the to assess the statistical significance of the data obtained. sections were coverslipped with Entellan. All antisera used in these immunohistochemical procedures were diluted in 0.05 M Tris- Results buffered saline, pH 8.0, with 0.5% of Triton X-100 (TBS-Tx; Tx from Sigma). The incubations in the primary antisera were implemented MPTP-induced dopaminergic lesion with 2% of bovine serum albumin (Merck). Extensive washing with 0.05 M TBS-Tx pH 8.0 was carried out throughout the procedure and All 4 monkeys intoxicated with MPTP developed a stable several rinses with 0.05 M Tris/HCl pH 7.6 were carried out prior to parkinsonian syndrome between 5 and 8 months after the initiation and after the reaction with the DAB chromogen solution. of MPTP administration, scoring 21 to 25 points in the accumulative The immunofluorescent visualization of CTB was carried out by Kurlan scale (Kurlan et al., 1991). The two monkeys showing the firstly incubating the sections with a primary antibody against CTB highest scores (24 and 25) received levodopa treatment and raised in rabbit (1:2,000; overnight at 4 °C; GenWay) followed by an developed mild dyskinesias (LIDs' level 1) from the end of the first Alexa546-coupled donkey anti-rabbit IgG (1:200; Molecular Probes). month under daily levodopa treatment. In both monkeys, the severity There is no need to conduct further staining to visualize the of LIDs increased to level 3 (overt dyskinetic symptoms) 1 month later transported FG, since this tracer “auto-fluoresces” in the UV range. and they remained stable until the injection of CTB. The immunocy- Sections were then mounted in gelatin-coated glass slides, dried in tochemical detection of tyrosine hydroxylase (TH) confirmed the the dark, quickly dehydrated in toluene and coverslipped with extent of the nigrostriatal damage induced in the 4 monkeys treated Entellan (Merck). The material obtained was inspected by epifluor- with MPTP, when compared with the TH stain in the control monkeys escent illumination in a Nikon Eclipse 800 microscope. (Fig. 1).
Tissue processing (II): single retrograde tract-tracing combined with in Single retrograde tract-tracing experiments situ hybridization for vGlut2 mRNA Large deposits of CTB were placed in VA/VL in the VA/VL of the 6 Since a detailed explanation of the procedure to the simulta- monkeys single-injected with CTB (2 control animals, 2 MPTP-treated neously detect a given transcript by fluorescent in situ hybridization and 2 dyskinetic monkeys), although in the 2 dyskinetic monkeys, the (ISH) within retrograde-labeled neurons is already available else- injection sites were located more medially and therefore, they mainly where (Pérez-Manso et al., 2006; Barroso-Chinea et al., 2007; Conte- affected the VA thalamic nucleus with minimal tracer spread towards Perales et al., 2010), only a brief summary is given below. the VL nucleus. Tracer leakage through the injection tract was not Fluorescent ISH for vGlut2 mRNA was carried out using a seen in any of the injected animals and moreover, no tracer spread digoxigenin-labeled riboprobe. Sagittal sections comprising the entire towards the adjacent internal capsule was present (illustrative mediolateral extent of the subthalamic nucleus (STN) were first examples of the injection sites are shown in Figs. 2–4). incubated for 90 min at room temperature with an anti-digoxigenin In all cases studied, a large number of CTB retrograde-labeled antibody raised in sheep (1:1000; Roche Diagnostics GmbH). neurons were found in both the ipsilateral GPi and substantia nigra Subsequently, the vGlut2 transcripts were then visualized using 2- pars reticulata (SNr; see Figs. 2–5). Furthermore, a large number of hydroxy-3-naphtoic acid-2′-phenylanilide phosphate (red HNPP CTB-labeled neurons were also observed bilaterally at the level of the fluorescence detection kit, Roche). Once the fluorescent ISH protocol pedunculopontine nucleus (PPN, albeit with an ipsilateral predomi- was completed, the immunofluorescent detection of transported CTB nance). In addition, there were a moderate number of labeled neurons was accomplished by incubating the sections with a primary antibody within the ventral part of the dentate nucleus of the contralateral against CTB raised in rabbit (1:2000; overnight at 4 °C; GenWay) cerebellar hemisphere. Most importantly, the delivery of CTB into the followed by an Alexa488-coupled donkey anti-rabbit IgG (1:200; VA/VL thalamic nuclei led to the appearance of a moderate number of Molecular Probes). The sections were then mounted on gelatine- retrograde-labeled neurons in the ipsilateral STN nucleus (Figs. 2–5). coated glass slides, dried in the dark, dehydrated rapidly in toluene CTB-labeled neurons were distributed throughout the STN in all 6 and coverslipped with Entellan (Merck). The sections were inspected monkeys single-injected with CTB, although labeled neurons on a Zeiss 510 Meta confocal microscope. To ensure the appropriate appeared to be discretely and preferentially located within medial visualization of labeled elements and to avoid false positive results, STN territories. By contrast, lateral STN territories consistently emission from the argon laser at 488 nm was filtered through a band- contained fewer CTB-positive neurons (Fig. 5). Finally, it is worth pass filter of 505–530 nm and color-coded in green. The emission noting that both dyskinetic monkeys had fewer labeled neurons in the following excitation with the helium laser at 543 nm was filtered STN, probably reflecting the more medial localization of the injection through a band-pass filter of 560–615 nm and color-coded in red. sites when compared to those performed in control animals as well as The intensity of vGlut2 mRNA expression in CTB-labeled neurons in the MPTP-treated monkeys (Figs. 2–5). was measured with the bi-dimensional densitometry software available for the Zeiss 510 Meta confocal microscope. A flat projection The expression of vGlut2 mRNA in subthalamo-thalamic projecting of each confocal stack obtained with the x63 oil-immersion lens was neurons generated for the channel showing vGlut2 mRNA labeling, and the number of pixels within a given region of interest (ROIs) were counted Isoform 2 of the vesicular glutamate transporter (vGlut2) is that at the single-cell level and normalized against the background typically expressed by STN neurons and indeed, fluorescent ISH
Fig. 5. Camera lucida drawings showing the number and localization of CTB-labeled neurons. Each dot represents one CTB-labeled neuron. As expected, most projection neurons were found in basal ganglia output nuclei such as the GPi and SNr. In comparison, fewer STN neurons innervated the VA/VL. It is also worth noting that subthalamo-thalamic projecting neurons are distributed throughout the territories of the STN nucleus, although with a somewhat preferential localization for medial STN areas. Abbreviations: ac: anterior commissure; cp: cerebral peduncle; GPe: globus pallidus, external division; GPi: globus pallidus, internal division; ic: internal capsule; ot: optic tract; SNr: substantia nigra, pars reticulata; STN: subthalamic nucleus. 388 ..Rc ta./Nuoilg fDsae3 21)381 (2010) 39 Disease of Neurobiology / al. et Rico A.J. – 392
Fig. 6. Changes in vGlut2 mRNA expression within STN neurons innervating the VA/VL in all the experimental conditions. The “red” channel shows the expression of vGlut2 mRNA (panels A, E and I), whereas CTB-labeled neurons innervating VA/VL are shown in the “green” channel (panels B, F and J). Bi-dimensional densitometry was performed for each confocal stack as illustrated in panels C, G and K and the mean intensity of vGlut2 mRNA expression was measured within projection neurons at the single-cell level. Scale bar is 16 µm in all panels. The obtained values are represented in the histogram showing the marked upregulation of vGlut2 mRNA expression levels in MPTP-treated monkeys when compared with control animals (pb0.001). Furthermore, CTB-projecting neurons in dyskinetic animals are typically characterized by the downregulation of vGlut2 mRNA expression when compared to both control (pb0.001) and MPTP-treated animals (pb0.001). A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 389 demonstrated the presence of vGlut2 transcripts in all STN neurons. earlier observations from tracing studies carried out by our group in When combined with the immunofluorescent detection of trans- rats showed that on average, 80% of STN efferent neurons project to ported CTB, notable vGlut2 mRNA expression was consistently found both the SNr and GPe (Castle et al., 2005). However, this general rule within the cytoplasm of CTB-positive neurons (Fig. 6). Furthermore, as to the highly branched nature of STN efferents does not appear to the densitometric analysis carried out showed a number of changes in apply when considering STN projections to brain structures other vGlut2 mRNA expression within the 3 different experimental groups than the basal ganglia such as the pedunculopontine nucleus (PPN). (controls, MPTP-treated and dyskinetic monkeys). When compared to Earlier observations suggested that the STN innervation reaching the the expression of vGlut2 mRNA observed in control animals, there is PPN nucleus arises from a subpopulation of STN neurons other than an approximately twofold increase in vGlut2 mRNA expression in the those innervating basal ganglia-related structures like the striatum, subthalamo-thalamic projecting neurons of the MPTP-treated ani- GPe and SNr (Takada et al., 1988). In this regard, it is critical to mals, together with a marked downregulation in CTB-positive ascertain whether or not STN neurons projecting “inside” the basal neurons from the dyskinetic monkeys (Fig. 6). All these reported ganglia are the same as those projecting “outside” the basal ganglia. changes in the patterns of gene expression for vGlut2 mRNA seem to The data provided here support the view that STN neurons projecting be a general property of all STN neurons, irrespective the brain nuclei to the VA/VL thalamic nuclei belong to a subtype of STN efferent to be innervated by STN efferents (as observed in the CTB-unlabeled neurons other than those innervating the GPi nucleus. neurons illustrated in panels C and G, Fig. 6). This is consistent with The first description of the presence of direct projections arising the increased activity of STN efferent neurons in the dopamine- from the STN nucleus reaching the VA/VL thalamic nuclei was depleted state, and the reduction of such activity when treated with provided through anterograde tract-tracing with tritiated amino acids levodopa (Crossman, 1987). (Nauta and Cole, 1978). With these techniques, sparse projections were seen in VA/VL following the delivery of tritiated amino acids in Dual retrograde tract-tracing experiments the STN of cats and monkeys. Later on, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into discrete regions In a single surgical session, two control monkeys were injected of the STN (Smith et al., 1990), although the earlier findings reported with CTB into the VA/VL thalamic nuclei and Fluoro-Gold (FG) into by Nauta and Cole (1978) were not reproduced. In addition, Sato et al. the GPi. These injections were carried out precisely within the VA/VL (2000) reconstructed the terminal arborizations from 75 single axons or GPi boundaries without any noticeable tracer spread towards labeled after very small injections of biotinylated dextran amine into neighboring areas (Fig. 7). CTB-labeled STN neurons (subthalamo- the primate STN and they reported up to 5 different subtypes of STN thalamic) were intermingled with neurons containing FG (subtha- efferent neurons according to their different patterns of collateraliza- lamo-pallidal). Moreover, no double-labeled neurons were found in tion into the striatum, GPe, GPi and SNr. However, once more, no any of the sections inspected. Hence, STN neurons projecting to the projections of STN neurons reaching the VA/VL or the PPN nucleus thalamus appear to represent a distinct subpopulation, different from were reported. This can be explained by the data reported here, along neurons projecting to the globus pallidus (Fig. 7). with other observations (Takada et al., 1988), considering that STN neurons projecting outside the basal ganglia are likely to belong to a Discussion different pool of efferent neurons than those projecting within the basal ganglia. In this regard, it is worth noting that we never found The presence of a direct glutamatergic connection from the STN neurons double-labeled with CTB (subthalamo-thalamic) and FG nucleus to the motor thalamus has not been considered either in the (subthalamo-pallidal). In other words, if STN efferents innervating the classic model of the basal ganglia (Crossman, 1987; Albin et al., 1989; VA/VL thalamic nuclei were collaterals of subthalamo-pallidal DeLong, 1990) or in later updates of this model (Mink, 1996; Obeso et neurons, then most of the retrogradely-labeled neurons observed in al., 2000; Wichmann and DeLong, 2003; DeLong and Wichmann, STN should have been double-labeled neurons. 2009). However, the data reported here confirm and expand earlier observations (Nauta and Cole, 1978) that (i) there is a subpopulation A functional correlate for the subthalamo-thalamic pathway of efferent STN neurons that directly innervate the ventral motor thalamic nuclei VA/VL (here referred as the “hyperindirect” path- The presence of a monosynaptic pathway that originates in the way); (ii) the innervation received by the VA/VL arises from a STN and reaches the same thalamic ventral nuclei targeted by the subpopulation of STN efferent neurons other than those projecting to basal ganglia output opens new possibilities within the functional the GPi nucleus; (iii) the subthalamo-thalamic projecting neurons are organization of the basal ganglia. The findings reported here need to glutamatergic neurons expressing vGlut2 mRNA; and (iv) upregula- be further defined and particularly, the physiological impact of this tion of vGlut2 mRNA expression occurs within STN neurons projection. Admittedly, when compared to the GABAergic pallido- and innervating thalamic targets following MPTP treatment, together nigrothalamic-projecting neurons, there are fewer STN projection with a reduced expression of vGlut2 transcripts in monkeys that neurons innervating the VA/VL nuclei. However, it is worth noting develop levodopa-induced dyskinesias. A schematic representation of that under circumstances of dopaminergic depletion the increases in these main findings in provided in Fig. 8. vGlut2 mRNA expression reported here probably would suggest enhanced glutamatergic outflow reaching thalamic territories that are STN neurons innervate the ventral thalamus also due to receive more GABA from hyperactive basal ganglia output structures (GPi and SNr). Therefore, it is tempting to speculate how a The main known targets for STN efferent neurons are the two projection of this kind could help to understand some relevant subdivisions of the globus pallidus (GPe and GPi) as well as the physiological and pathophysiological observations. There is still substantia nigra pars reticulata (SNr). The STN output is highly certain debate as to which mechanisms, excitation or inhibition, collateralized, as confirmed in several anatomical studies indicating underlie and sustain the remarkable clinical motor benefit induced in that most STN neurons send axons to both the SNr and GPi (Van der parkinsonian patients that undergo high-frequency deep brain Kooy and Hattori, 1980; Kita et al., 1983; Kita and Kitai, 1987; Plenz stimulation (HFS-DBS) of the STN nucleus (reviewed in Vitek, and Kitai, 1999). More recently, single-axon tracing studies reported 2002). In this regard, HFS of the STN nucleus of rats increased the presence of up to five distinct types of STN efferent neurons, each neuronal activity in the ventromedial nucleus (VM) of the thalamus in subtype being characterized by a distinctive pattern of axonal a frequency-dependent manner. Indeed, this increase was sustained collateralization (Sato et al., 2000; Parent et al., 2000). Accordingly, by enhanced glutamatergic outflow reaching the VM nucleus 390 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392
Fig. 7. Dual tracing experiments. To elucidate whether or not the thalamic innervation received from STN neurons arises from the same population of STN neurons innervating the GPi nucleus, 2 control monkeys received one injection of the retrograde tracer FG into the GPi (panel A) together with an injection of CTB into the VA/VL (panel B). Selected sagittal sections through the injection sites were immunohistochemically converted to a regular DAB precipitate to better appreciate the placement of the tracer deposits. Scale bar is 1000 µm in A and B. Under epifluorescent illumination STN neurons with either FG or CTB labeling were plotted, as illustrated in panel C. Each labeled neuron is represented by a single colored dot (light blue for FG-containing neurons and red for CTB-labeled neurons). It is evident that in all mediolateral STN levels examined, FG+ and CTB+ neurons are intermingled with each other. As expected, CTB+ neurons (subthalamo-thalamic) represented fewer projection neurons than the FG-labeled neurons (subthalamo-pallidal). Furthermore, it should also be noted that no double-labeled neurons were found from either of the animals inspected. This is also exemplified in panels D and E (taken from medial and lateral STN levels, respectively). Under dual epifluorescent illumination it became clear that STN neurons projecting to the VA/VL (CTB-labeled, magenta-red) represent a population of STN efferent neurons different from those innervating the GPi nucleus (labeled with FG, light blue). Scale bar is 25 µm in panels D and E. Abbreviations: ac: anterior commissure; al: ansa lenticularis; CL: central lateral thalamic nucleus; CM-Pf: centromedian-parafascicular thalamic nucleus; GPe: globus pallidus, external division; GPi: globus pallidus, internal division; ic: internal capsule; MD: mediodorsal thalamic nucleus; ot: optic tract; SNr: substantia nigra, pars reticulata; STN: subthalamic nucleus; VA: ventral anterior thalamic nucleus; VL: ventral lateral thalamic nucleus; VPM: ventral posteromedial thalamic nucleus. following STN-HFS (Gao et al., 1997). These findings cannot be easily through the “classical” multi-synaptic subthalamo-pallido-thalamo- explained “in the absence of evidence for a direct neuronal pathway cortical pathway and therefore, the antidromic excitation of the from STN to VM”. Moreover, experiments carried out in patients with cortico-subthalamic pathway offers the most likely explanation Parkinson's disease subjected to surgery, showed that single pulse (Hanajima et al., 2004). In this regard, we believe that a di-synaptic stimulation of the STN through the implanted DBS electrode facilitates subthalamo-thalamo-cortical pathway is a better potential anatom- the primary motor cortex (M1) with a very short latency of 3 ms ical substrate underlying such short latency cortical facilitation. This is (Hanajima et al., 2004). Such a short latency can hardly be explained also in keeping with more recent observations showing that the ..Rc ta./Nuoilg fDsae3 21)381 (2010) 39 Disease of Neurobiology / al. et Rico A.J. – 392
Fig. 8. Schematic representation of basal ganglia output to the thalamus. In keeping with existing knowledge, GABAergic pallidothalamic and nigrothalamic projections represent the bulk of the basal ganglia output reaching the thalamic ventral nuclei. Thalamic areas receiving pallidal and nigral inputs are the same ones as those also innervated by glutamatergic projections arising from the STN nucleus. As predicted by the basal ganglia model, STN efferent neurons are overactive in parkinsonian conditions, and this also applies to STN efferent neurons innervating the ventral thalamus (these neurons have increased expression of vGlut2 mRNA). Furthermore, when considering parkinsonian animals showing levodopa-induced dyskinesias, vGlut2 mRNA expression is downregulated in the STN neurons projecting to VA/VL. 391 392 A.J. Rico et al. / Neurobiology of Disease 39 (2010) 381–392 responses recorded in VA/VL neurons following effective STN Illinsky, I.A., Yi, H., Kultas-Illinsky, K., 1997. Mode of termination of pallidal afferents to the thalamus: a light and electron microscopic study with anterograde tracers and stimulation differed from the baseline in Macaca mulatta, character- immunocytochemistry in Macaca mulatta. J. Comp. Neurol. 386, 601–612. ized by an early excitation peak at short latencies (between 1 and Jackson, A., Crossman, A.R., 1981. Subthalamic nucleus efferent projection to the 1.5 ms after STN stimulation at 135 Hz) and followed by a smaller but cerebral cortex. Neuroscience 6, 2367–2377. Kita, H., Kitai, S.T., 1987. Efferent projections of the subthalamic nucleus in the rat: light prolonged period of inhibition. This change begins at 2 ms after the and electron microscopic analysis with the PHA-L method. J. Comp. Neurol. 260, stimulation pulse and lasts until the next stimulation pulse (Xu et al., 435–452. 2008). Thus, the direct subthalamo-thalamic projection described Kita, H., Chang, H.T., Kitai, S.T., 1983. The morphology of intracellularly labelled rat – here may provide fast excitatory input to the thalamus and hence, subthalamic neurons: a light microscopic analysis. J. Comp. Neurol. 215, 245 257. Kultas-Illinsky, K., Reising, L., Yi, H., Illinsky, I.A., 1997. Pallidal afferent territory of the sustain a positive feedback loop with the motor cortex that is engaged Macaca mulatta thalamus: neuronal and synaptic organization of the VAdc. J. Comp. in the maintenance of sequential and repetitive movements. In the Neurol. 386, 573–600. – parkinsonian state, this projection is overactive, which could enable Kurlan, R., Kim, M.H., Gash, D.M., 1991. Oral levodopa dose response study in MPTP- fi induced hemiparkinsonian monkeys: assessment with a new rating scale for us to understand the unexplained nding of increased neuronal monkey parkinsonism. Mov. Disord. 6, 111–118. activity in the primary motor cortex concomitant with hypoactive Lanciego, J.L., López, I.P., Rico, A.J., Aymerich, M.S., Pérez-Manso, M., Conte, L., Combarro, pre-motor and supplementary motor areas (Lefaucheur, 2005; Lin et C., Roda, E., Molina, C., Gonzalo, N., Castle, M., Tuñón, T., Erro, E., Barroso-Chinea, P., 2009. The search for a role of the caudal intralaminar nuclei in the pathophysiology al., 2008). Finally, the anatomical correlate explaining how striatal of Parkinson's disease. Brain Res. Bull. 78, 55–59. dopamine deficiency in Parkinson's disease leads to parkinsonian Langston, J.W., Widner, H., Goetz, C.G., Brooks, D., Fahn, S., Freeman, T., Watts, R., 1992. tremor remains a challenge (Rodríguez-Oroz et al., 2009). It is Core assessment program for intracerebral transplantations (CAPIT). Mov. Disord. 7, 2–13. tempting to speculate that the subthalamo-thalamic pathway repre- Lefaucheur, J.P., 2005. Motor cortex dysfunction revealed by cortical excitability studies sents the basal ganglia output route whereby the 4–6 Hz activity can in Parkinson's disease: influence of antiparkinsonian treatment and cortical gain access to the motor system. stimulation. Clin. Neurophysiol. 116, 244–253. Lin, T.P., Carbon, M., Tang, C., Mogilner, A.Y., Sterio, D., Beric, A., Dhawan, V., Eidelberg, D., 2008. Metabolic correlates of subthalamic nucleus activity in Parkinson's disease. Brain 131, 1373–1380. Acknowledgments Martin, R.F., Bowden, D.M., 1997. Template Atlas of the Primate Brain. University of Washington, Seattle, WA. This study was supported by Ministerio de Educación y Ciencia Mink, J.W., 1996. The basal ganglia: focused selection and inhibition of competing motor programs. Prog. Neurobiol. 50, 381–425. (BFU2006-06744 and BFU2009-08351), CIBERNED (CB06/05/0006), Nambu, A., 2004. A new dynamic model of the cortico-basal ganglia loop. Prog. Brain Departamento de Salud del Gobierno de Navarra and by the UTE- Res. 143, 461–466. fi project/Foundation for Applied Medical Research (FIMA). Nambu, A., Tokuno, H., Takada, M., 2002. Functional signi cance of the cortico- subthalamo-pallidal ‘hyperdirect’ pathway. Neurosci. Res. 43, 111–117. Nauta, H.J.W., Cole, M., 1978. Efferent projections of the subthalamic nucleus: an autoradiographic study in monkey and cat. J. Comp. Neurol. 180, 1–16. References Obeso, J.A., Rodríguez-Oroz, M.C., Rodríguez, M., Lanciego, J.L., Artieda, J., Gonzalo, N., Olanow, C.W., 2000. Pathophysiology of the basal ganglia in Parkinson's disease. Albin, R.L., Young, A.B., Penney, J.B., 1989. The functional anatomy of basal ganglia Trends Neurosci. 23, S8–S19. disorders. Trends Neurosci. 12, 366–375. Parent, A., Sato, F., Wu, Y., Gauthier, J., Lévesque, M., Parent, M., 2000. Organization of Barroso-Chinea, P., Aymerich, M.S., Castle, M.M., Pérez-Manso, M., Tuñón, T., Erro, E., the basal ganglia: the importance of axonal collateralization. Trends Neurosci. 23, Lanciego, J.L., 2007. Detection of two different mRNAs in a single section by dual in S20–27. situ hybridization: a comparison between colorimetric and fluorescent detection. Pérez-Manso, M., Barroso-Chinea, P., Aymerich, M.S., Lanciego, J.L., 2006. ‘Functional’ J. Neurosci. Meth. 162, 119–128. neuroanatomical tract-tracing: analysis of changes in gene expression of brain Castle, M., Aymerich, M.S., Sánchez-Escobar, C., Gonzalo, N., Obeso, J.A., Lanciego, J.L., circuits of interest. Brain Res. 1072, 91–98. 2005. Thalamic innervation of the direct and indirect basal ganglia pathways in the Plenz, D., Kitai, S.T., 1999. A basal ganglia pacemaker formed by the subthalamic nucleus rat: ipsi- and contralateral projections. J. Comp. Neurol. 483, 143–153. and external globus pallidus. Nature 400, 677–682. Conte-Perales, L, Barroso-Chinea, P., Rico, A.J., Gómez-Bautista, V., López, I.P., Roda, E., Rodríguez-Oroz, M.C., Jahanshahi, M., Krack, P., Litvan, I., Macias, R., Bezard, E., Obeso, Wouterlood, F.G., Lanciego, J.L., 2010. Neuroanatomical tracing combined with in J.A., 2009. Initial clinical manifestations of Parkinson's disease: features and situ hybridization: analysis of gene expression patterns within brain circuits of pathophysiological mechanisms. Lancet Neurol. 8, 1128–1139. interest. J. Neurosci. Meth. doi:10.1016/j.neurometh.2009.11.021. Sato, F., Parent, M., Lévesque, M., Parent, A., 2000. Axonal branching pattern of neurons Crossman, A.R., 1987. Primate models of dyskinesia: the experimental approach to the of the subthalamic nucleus in primates. J. Comp. Neurol. 424, 142–152. study of basal ganglia-related involuntary movement disorders. Neuroscience 21, Sidibé, M., Bevan, M.D., Bolam, J.P., Smith, Y., 1997. Efferent connections of the internal 1–40. globus pallidus in the squirrel monkey: I. Topography and synaptic organization of Degos, B., Deniau, J.M., Le Cam, J., Mailly, P., Maurice, N., 2008. Evidence for a direct the pallidothalamic projection. J. Comp. Neurol. 382, 323–347. subthalamo-cortical loop circuit in the rat. Eur. J. Neurosci. 27, 2599–2610. Smith, Y., Hazrati, L.N., Parent, A., 1990. Efferent projections of the subthalamic nucleus DeLong, M., 1990. Primate models of movement disorders of basal ganglia origin. in the squirrel monkey as studied by the PHA-L anterograde tracing method. Trends Neurosci. 13, 281–285. J. Comp. Neurol. 294, 306–323. DeLong, M., Wichmann, T., 2009. Update on models of basal ganglia function and Takada, M., Nishihama, M.S., Nishihama, C.C., Hattori, T., 1988. Two separate neuronal dysfunction. Parkin. Rel. Disord. 15S3, S237–S240. populations of the rat subthalamic nucleus project to the basal ganglia and Gao, D., Benazzouz, A., Bressand, K., Piallat, B., Benabid, A.L., 1997. Roles of GABA, pedunculopontine tegmental region. Brain Res. 442, 72–80. glutamate, acetylcholine and STN stimulation on thalamic VM in rats. NeuroReport Van der Kooy, D., Hattori, T., 1980. Single subthalamic nucleus neurons project to both 8, 2601–2605. the globus pallidus and substantia nigra in rat. J. Comp. Neurol. 192, 751–768. Goetz, C.G., Stebbins, G.T., Shale, H.M., Lang, A.E., Chernik, D.A., 1994. Utility of an Vitek, J.L., 2002. Mechanisms of deep brain stimulation: excitation or inhibition. Mov. objective dyskinesia rating scale for Parkinson's disease: inter- and intrarater Disord. 17, S69–72. reliability assessment. Mov. Disord. 9, 390–394. Wichmann, T., DeLong, M.R., 2003. Functional neuroanatomy of the basal ganglia in Hanajima, R., Ashby, P., Lozano, A.M., Lang, A.E., Chen, R., 2004. Single pulse stimulation Parkinson's disease. Adv. Neurol. 91, 9–18. of the human subthalamic nucleus facilitates the motor cortex at short intervals. Xu, W., Russo, G.S., Hashimoto, T., Zhang, J., Vitek, J.L., 2008. Subthalamic nucleus J. Neurophysiol. 92, 1937–1943. stimulation modulates thalamic neuronal activity. J. Neurosci. 28, 11916–11924.