Skip to main content
NCBI home page
Brain logoLink to Brain
. 2015 Feb 12;138(4):946–962. doi: 10.1093/brain/awv018

Reduced cortical innervation of the subthalamic nucleus in MPTP-treated parkinsonian monkeys

Abraham Mathai 1,2, Yuxian Ma 1,2, Jean-Francois Paré 1,2, Rosa M Villalba 1,2, Thomas Wichmann 1,2,3, Yoland Smith 1,2,3,✉
PMCID: PMC5014077  PMID: 25681412

Mathai et al. provide anatomical and electrophysiological evidence for a partial degeneration of the glutamatergic corticosubthalamic system in parkinsonian monkeys. The data demonstrate a loss of cortical terminals in the subthalamic nucleus of MPTP-treated monkeys, suggesting altered integration and transmission of cortical information to the basal ganglia in Parkinson’s disease.

Keywords: Parkinson’s disease, vesicular glutamate transporter, cortico-subthalamic, hyperdirect, synaptic plasticity

Abstract

The striatum and the subthalamic nucleus are the main entry points for cortical information to the basal ganglia. Parkinson’s disease affects not only the function, but also the morphological integrity of some of these inputs and their synaptic targets in the basal ganglia. Significant morphological changes in the cortico-striatal system have already been recognized in patients with Parkinson’s disease and in animal models of the disease. To find out whether the primate cortico-subthalamic system is also subject to functionally relevant morphological alterations in parkinsonism, we used a combination of light and electron microscopy anatomical approaches and in vivo electrophysiological methods in monkeys rendered parkinsonian following chronic exposure to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the light microscopic level, the density of vesicular glutamate transporter 1-positive (i.e. cortico-subthalamic) profiles in the dorsolateral part of the subthalamic nucleus (i.e. its sensorimotor territory) was 26.1% lower in MPTP-treated parkinsonian monkeys than in controls. These results were confirmed by electron microscopy studies showing that the number of vesicular glutamate transporter 1-positive terminals and of axon terminals forming asymmetric synapses in the dorsolateral subthalamic nucleus was reduced by 55.1% and 27.9%, respectively, compared with controls. These anatomical findings were in line with in vivo electrophysiology data showing a 60% reduction in the proportion of pallidal neurons that responded to electrical stimulation of the cortico-subthalamic system in parkinsonian monkeys. These findings provide strong evidence for a partial loss of the hyperdirect cortico-subthalamic projection in MPTP-treated parkinsonian monkeys.

Introduction

The basal ganglia receive topographically organized projections from functionally diverse regions of the cerebral cortex via two main input stations, the striatum and the subthalamic nucleus (STN) (Parent and Hazrati, 1995; Nambu et al., 1996; Mathai and Smith, 2011; Haynes and Haber, 2013). While the cortico-subthalamic projection is less extensive than the cortico-striatal system, it is a powerful source of excitation to STN neurons through which cortical inputs can rapidly regulate the activity of downstream basal ganglia output nuclei, the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr) (Nambu et al., 2000). Cortico-subthalamic axons target dendritic spines and distal dendritic shafts of STN neurons in rats (Bevan et al., 1995), but their pattern of synaptic innervation has not been characterized in primates.

In the parkinsonian state, neuronal activity patterns are abnormal in the STN and other basal ganglia nuclei (Wichmann et al., 2011; Wichmann and Dostrovsky, 2011). In the STN, neurons display a higher firing rate and a more bursty firing pattern compared to the normal state (Bergman et al., 1994; Bevan et al., 2002; Wichmann and DeLong, 2003). These changes probably occur in large part because of the loss of dopamine in the basal ganglia which alters transmission at γ-aminobutyric acidergic (GABAergic) synapses from the external pallidal segment (GPe) (Baufreton et al., 2005; Bevan et al., 2006; Fan et al., 2012), and in glutamatergic afferents from the cerebral cortex to the STN (Orieux et al., 2002; Mallet et al., 2008; Baudrexel et al., 2011; Shimamoto et al., 2013).

In addition to the (principally reversible) activity changes that reflect the lack of dopamine in the basal ganglia, there is also emerging evidence that the parkinsonian state is associated with ultrastructural remodelling of synaptic connections which may contribute to activity changes in the basal ganglia in parkinsonian animals. Such changes have, thus far, been documented to occur in the cortico-striatal and pallido-subthalamic projections (Ingham et al., 1989; Meshul et al., 2000; Villalba et al., 2009; Villalba and Smith, 2011, 2013; Fan et al., 2012). In this study, we investigated whether the motor cortico-subthalamic projection also undergoes ultrastructural changes, and whether such changes affect the transmission of cortical information to the sensorimotor territory of the globus pallidus in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys.

We used light and electron microscopy anatomical approaches to compare the density of cortical terminals (labelled with antibodies against the vesicular glutamate transporter 1, vGluT1) (Fremeau et al., 2001; Raju et al., 2008) and their pattern of dendritic innervation of neurons in the dorsolateral region (i.e. motor-related territory) of the STN between normal and MPTP-treated, parkinsonian, monkeys. Subsequently, we characterized the functional integrity of the motor cortico-subthalamo-pallidal system with in vivo electrophysiological techniques in monkeys under normal and parkinsonian condition. Our anatomical and electrophysiological studies revealed a partial loss of the motor cortico-subthalamic projection and related functional changes in the cortico-subthalamo-pallidal system in parkinsonian monkeys. Preliminary results of this study have been presented in abstract form (Mathai et al., 2010, 2011).

Materials and methods

Animals

All experiments were performed in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (Garber et al., 2010), and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals (amended 2002). The studies were approved by the Biosafety Committee and the Animal Care and Use Committee of Emory University.

Twenty adult rhesus monkeys (Macaca mulatta, 4.4–15.0 kg, 2–11 years old, eight males, 12 females) were used. Table 1 provides a listing of the animals and their use for the different experiments performed in this study. The monkeys were raised in the breeding colony of the Yerkes National Primate Research Centre. The animals had ad libitum access to food and water. Nine normal and nine MPTP-treated monkeys were used in the anatomical studies (Table 1). The remaining two monkeys (Monkeys I and O) were used for the electrophysiology studies. Prior to experimentation, these animals were acclimatized to the laboratory, and trained to permit handling by the experimenter, and to sit quietly in a primate restraint chair, using positive reinforcement techniques (McMillan et al., 2010). Electrophysiology data in the normal state were then collected from the left hemispheres in both animals, after which the monkeys were injected systemically with MPTP until they displayed stable parkinsonian motor signs (see below). This was followed by additional electrophysiological recordings from the right hemisphere of the animals. This sampling scheme ensured that intact brain tissue was used for the recordings in both the normal and the parkinsonian state.

Table 1.

Monkeys used for the different experiments described in the study

Animal number Animal groups
 Experimental procedures
Control MPTP- treated STN volume measurement vGluT1 LM vGluT1 EM Electrophysiology
MR129 X X
MR135 X X
MR136 X X
MR121 X X
MR141 X X
MR172 X X
MR153 X X
MR155 X X
MR162 X X
MR140 X X
MR157 X X
MR167 X X
MR116 X X
MR117 X X
MR125 X X
MR138 X X
MR148 X X
MR150 X X
Monkey I X X X
Monkey O X X X
Open in a new tab

LM = light microscopy; EM = electron microscopy.

Induction of parkinsonism

All parkinsonian monkeys used in this study were treated with MPTP according to the same regimen, and displayed comparable parkinsonian motor signs at the time of euthanasia. Briefly, they were rendered parkinsonian by weekly administration of MPTP (0.2–0.8 mg/kg/week i.m.; Sigma-Aldrich; cumulative doses, 3.2–19.4 mg/kg; treatment time range, 1–8 months) until moderate parkinsonian motor signs developed. As described in previous studies (Wichmann et al., 2001; Kliem et al., 2010; Masilamoni et al., 2011; Hadipour-Niktarash et al., 2012), the animal’s behaviour was observed in a cage in which one of the side panels was made of Plexiglas to facilitate an unobstructed view of the monkey. The cage was equipped with eight infrared beams, which allowed us to assess the severity and stability of the MPTP-induced motor disability by recording the monkey’s spontaneous movements using an automated infrared beam break counting system (infrared emitters and receivers made by Banner Engineering Corp). In addition, video records of the animal’s behaviour were scored using a parkinsonism rating scale that assessed gross motor activity, balance, posture, arm bradykinesia, arm hypokinesia, leg bradykinesia, leg hypokinesia, arm tremor and leg tremor. Each item was scored between 0 and 3 (normal to severe), for a maximum of 27 points. Monkeys were considered to be parkinsonian if the movement counts in the activity monitoring cage were reduced by more than 60% compared to the animal’s pretreatment baseline, and if the score in the parkinsonian rating scale was > 10 for a period of at least 6 weeks after the last administration of MPTP. The extent of MPTP-induced loss of the nigrostriatal dopamine system was later assessed using tyrosine hydroxylase (TH) immunohistochemistry (see below) (Fig. 1).

Figure 1.

Figure 1

Open in a new tab

Extent of dopaminergic denervation in MPTP-treated monkeys. (A–C) Light microscopic images of coronal brain sections showing immunostaining for tyrosine hydroxylase (TH) in the post-commissural putamen of a control animal (A) and the two MPTP-treated monkeys (B and C) that were used in the electrophysiological studies (Monkeys I and O). The pattern of tyrosine hydroxylase immunoreactivity in the striatum of the MPTP-treated animals used in the anatomical studies was comparable to that shown in Monkey O. Cd = caudate nucleus; IC = internal capsule; Put = putamen. Scale bars: A–C = 1 mm. (D) Normalized densitometry measurements of tyrosine hydroxylase immunoreactivity in the post-commissural putamen of normal (n = 2) and MPTP-treated (n = 8) monkeys. ***Significant difference from normal; P < 0.001; Student’s t-test).

Animal euthanasia and tissue fixation

At the time of euthanasia, the monkeys were deeply anaesthetized with an overdose of pentobarbital (100 mg/kg, i.v.) and then transcardially perfused with cold, oxygenated Ringer’s solution. Following this, the animals were perfused with 2 litres of a fixative—either 4% paraformaldehyde +0.1% glutaraldehyde in phosphate buffer (PB; 0.1 M, pH 7.4) or 2% paraformaldehyde + 3.75% acrolein in PB. After fixation, the brains were removed from the skull, cut into 10-mm thick blocks in the frontal plane and immersed in fixative (2% or 4% paraformaldehyde in PB, 0.1 M, pH 7.4) overnight at 4°C. Both fixative recipes and post-fixation procedures provided adequate ultrastructural preservation and antibody penetration in the tissue for both light and electron microscopic observations. There was no significant difference in vGluT1 labelling between animals of the same group perfused with either fixative solution.

Anatomical experiments

Tissue processing

Sixty-micrometre thick coronal sections were cut from the tissue blocks in cold phosphate-buffered saline (PBS; 0.01 M, pH 7.4) using a vibrating microtome. These sections were stored in an anti-freeze solution (30% ethylene glycol and 30% glycerol in PB) at −20°C until ready for immunohistochemistry. Prior to light or electron microscopy immunohistochemical processing, sections were treated with sodium borohydride (1% in PBS) for 20 min, followed by washes in PBS.

STN volume measurements

We used Cavalieri’s principle to estimate the volume of the STN from Nissl-stained sections in three normal and three age-matched parkinsonian monkeys (Gundersen and Osterby, 1981; West et al., 1991; West, 1999; Schmitz and Hof, 2005). The delineation of the STN and the unbiased volume estimation was performed at low magnification by multiplying the sum of the STN areas by the distance between sections. Because of tissue shrinkage, the mean section thickness was estimated from at least six measurements/section obtained by moving the focus from the top to the bottom surface of the tissue at each microscopic field with the Z-axis position encoder of the stereology system. Measurements were made from 1 of 12 serial sections (i.e. 5–7 sections per animal) through the rostrocaudal extent of the STN. This design resulted in a coefficient of error of 0.023 and 0.027 (Gundersen, m = 1) (Gundersen and Osterby, 1981), respectively for measurements made in normal versus MPTP-treated animals.

Pre-embedding immunoperoxidase staining of vGluT1 at the light microscopic level

Brain tissue from three normal and three parkinsonian animals was used in these light microscopy immunostaining experiments. Sections containing the STN were pre incubated for 1 h at room temperature in PBS containing 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.3% Triton™ X-100. These sections were then incubated for 24 h at room temperature in PBS containing 1% NGS, 1% BSA and 0.3% Triton™ X-100, and anti-vGluT1 (raised in guinea pig; 1:5000 dilution; EMD Millipore). The specificity of the anti-vGluT1 antibodies has been discussed in detail in a previous study (Raju et al., 2008). After thorough rinsing with PBS, the sections were further incubated for 1.5 h at room temperature in PBS containing 1% NGS, 1% BSA and 0.3% Triton™ X-100, and the secondary antibody, biotinylated anti-guinea pig IgGs (raised in goat; 1:200 dilution; Vector Laboratories). After three rinses with PBS, the sections were incubated for 1.5 h at room temperature in avidin-biotin peroxidase complex solution (1:100; Vectastain standard ABC kit; Vector Laboratories) in PBS, followed by rinses in PBS and Tris buffer (50 mM; pH 7.6). Sections were then treated with a solution containing 0.025% 3,3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 10 mM imidazole, and 0.005% hydrogen peroxide in Tris buffer for 10 min at room temperature. Later, they were thoroughly rinsed with PBS, placed onto gelatin-coated slides, and coverslipped with Permount™.

Quantitative analysis of vGluT1 immunostaining at the light microscopic level

At high magnification (>×40), a large number of immunolabelled pleomorphic processes, most likely corresponding to vGluT1-containing axons and terminal profiles, were homogenously distributed throughout the STN (Fig. 2B). To quantify the density of labelled profiles at the light microscopic level, a 500 µm × 500 µm region of interest was randomly chosen in the dorsolateral STN (Fig. 2A) in one section/animal that included the central core of the nucleus (interaural ∼ 10.65 mm according to Paxinos et al., 1999). In this material, the dorsolateral STN was defined as a 1 mm2 STN tissue area that extends medially from the dorsal and lateral edges of the central core of the nucleus (Fig. 2A). A total of 36–40 virtual grids (consisting of 80 × 80 µm square elements) were then placed in the region of interest with StereoInvestigator’s Optical Fractionator probe. Under a ×100 oil immersed objective, we counted all labelled processes in each counting frame (10 × 10 µm) placed in the top left corner of the different grids. We made sure that each labelled process was counted and that no labelled element was overlooked or counted twice, by gently moving the focal plane back and forth through the full thickness of the section. The region of interest was sampled using an automated software-based control of the microscope’s X-Y stage. The density of immunolabelled varicosities was calculated by dividing the number of counted varicosities by the tissue volume corresponding to the counting frames.

Figure 2.

Figure 2

Open in a new tab

vGluT1 immunostaining in the monkey STN. (A) Light microscopic view of a coronal section through the monkey STN showing immunostaining for vGluT1. The grey square indicates the size and approximate position of the region of interest in the dorsolateral territory of the STN that was used to quantify the density of vGluT1-positive varicose processes. Scale bar = 0.5 mm. ZI = zona incerta; IC = internal capsule. (B) Light micrograph showing vGluT1-immunopositive pleomorphic varicose processes in the dorsolateral STN of a normal monkey. Scale bar = 10 µm. (C) Comparison of the average STN volume ( ± SEM; n = 3) between normal and parkinsonian monkeys. (D) Average density ( ± SEM; n = 3) of vGluT1-immunoreactive varicosities in the dorsolateral STN of normal and parkinsonian monkeys. *P = 0.012; Student’s t-test of difference between the normal and parkinsonian animals.

Double-immuno electron microscopy to visualize vGluT1 and vGluT2

Glutamatergic inputs to the STN originate from the cerebral cortex (Monakow et al., 1978; Nambu et al., 1996; Haynes and Haber, 2013), the thalamus (Sadikot et al., 1992), the brainstem pedunculopontine tegmental nucleus (Lavoie and Parent, 1994) and local axon collaterals of STN neurons (Kita et al., 1983). In light of mRNA data and immunohistochemical findings from the striatum and other brain regions, glutamatergic inputs from the cerebral cortex can be distinguished from other sources of glutamatergic afferents based on their selective expression for vGluT1 and the lack of vGluT2 immunoreactivity (Fremeau et al., 2001, 2004; Kaneko and Fujiyama, 2002; Raju et al., 2008). To confirm that vGluT1 and vGluT2 proteins are also segregated in different populations of glutamatergic terminals in the STN, double immuno-electron microscopy techniques were used to assess the extent of vGluT1/vGluT2 co-localization in the monkey STN.

Brain sections containing the STN from three normal and three parkinsonian monkeys were processed according to standard double immuno-electron microscopy procedures used in our laboratory (Mitrano et al., 2010; Gonzales et al., 2013; Unal et al., 2014). After standard pre incubations, the sections were incubated for 24 h at room temperature in TBS-gelatin buffer containing 1% dry milk and a cocktail of the primary antibodies, anti-vGluT1 (raised in guinea pig; 1:5000 dilution; EMD Millipore) and anti-human vGluT2 (raised in rabbit; 1:5000 dilution; Mab Technologies) (see Raju et al., 2008 for specificity tests and characterization). Through the use of appropriate secondary antibodies, vGluT1 labelling was then visualized using an immunoperoxidase protocol, while vGluT2 labelling was visualized with an immunogold protocol, or vice versa. This double labelling approach allowed us to examine the degree of co-expression of vGluT1 and vGluT2 within the same terminals. Control sections were incubated in solutions in which either of the primary antibodies was omitted. Sections were then prepared for electron microscopy according to standard osmium post-fixation, dehydration and resin embedding procedures (see Mitrano et al., 2010; Gonzales et al., 2013; Unal et al., 2014 for details). Blocks of tissue from the dorsolateral STN (Fig. 2A) were cut out from the resin-embedded sections, glued on top of resin blocks with cyanoacrylate glue, cut into 60-nm thick ultrathin sections with an ultramicrotome (Ultracut T2; Leica), and collected on single-slot Pioloform-coated copper grids. The sections were then stained with lead citrate for 5 min and viewed under an electron microscope (JEM-1011; JEOL). Digital micrographs were collected with a CCD camera (Model 785; Gatan Inc) controlled by Digital Micrograph 3.11.1 software (Gatan).

Analysis of electron microscopy material

Studies of the co-localization of vGluT1 and vGluT2

Superficial sections from the dorsolateral STN tissue were sampled to assess whether vGluT1 and vGluT2 immunoreactivities were co-expressed. Immunoperoxidase-labelled boutons were tested for the presence of immunogold labelling in normal or parkinsonian animals. The proportion of single and double labelled boutons was expressed as percentage of the total number of labelled terminal profiles.

Density of vGluT1-containing terminals

At the electron microscopy level, vGluT1-immunoreactive terminals were defined as labelled profiles of various shapes filled with synaptic vesicles, while labelled unmyelinated axons were small (∼0.1–0.2 µm in diameter), circular in shape, and devoid of synaptic vesicles (Peters et al., 1991). To quantify the relative density of vGluT1-immunopositive terminals (revealed with immunoperoxidase) in tissue from normal and parkinsonian animals, we used the double immunolabelled material that was analysed for the vGluT1/vGluT2 co-localization studies. We sequentially imaged 100 viewing fields of ultrathin sections taken from the surface of STN tissue at ×25 000 (Fig. 3A). This strategy ensured that only superficial tissue layers where the antibody penetration was optimal were sampled. In each micrograph we counted every vGluT1-immunopositive terminal, regardless of whether it established a synaptic contact in the plane of the ultrathin section. The density of vGluT1-immunopositive terminals was calculated as the ratio of the total number of terminals counted across the 100 micrographs and the total surface area sampled within these micrographs.

Figure 3.

Figure 3

Open in a new tab

Changes in vGluT1-immunoreactive terminals density in the STN of parkinsonian monkeys. (A) Electron micrographs showing vGluT1-positive (v1-immunoperoxidase) and vGluT2-labelled (v2-immunogold) terminals in the dorsolateral STN. Scale bar = 0.5 µm. (B and C) Electron micrographs depicting an asymmetric synapse (putatively glutamatergic) in consecutive ultrathin serial sections of the dorsolateral monkey STN. Asymmetric synapse indicated by arrowheads. Scale bars: B and C = 0.2 µm. (D) Average density ( ± SEM; n = 3) of vGluT1-immunopositive terminals in the dorsolateral STN of normal and parkinsonian monkeys. *P = 0.02; Student’s t-test of difference between normal and parkinsonian animals. Total surface area analysed: 3960 µm2/animal. (E) Average density ( ± SEM; n = 3) of asymmetric synapses in the dorsolateral STN of normal and parkinsonian monkeys. *P = 0.029; Student’s t-test of difference between normal and parkinsonian animals. Total surface area analysed: 1980 µm2/animal.

Density of terminals forming asymmetric synapses

Because changes in the expression of vGluT1 protein in the parkinsonian state could affect our ability to identify cortico-subthalamic terminals in these experiments, we also measured the density of terminals forming asymmetric synapses in the dorsolateral STN, independent of their vGluT immunoreactivity. We assumed that if changes in the number of vGluT1-immunoreactive terminals within the STN were due to a genuine change in the number of cortico-subthalamic terminals (rather than altered expression of vGluT1 protein), it may be reflected by a change in the number of terminals forming asymmetric synapses in the STN.

The density of terminals forming clear asymmetric synapses was quantified in 50 viewing fields within the dorsolateral STN (same as those used to quantify number of vGluT1-positive terminals; total area 1980 µm2). To ensure that only terminals forming asymmetric synapses were counted, the synaptic specialization of each terminal was confirmed through analysis in pairs of adjacent ultrathin sections (Fig. 3B and C). The resulting count was expressed as terminal density by dividing it by the total surface area sampled.

Post-synaptic targets of vGluT1-positive terminals

To assess changes in the synaptic microcircuitry of vGluT1-positive terminals in parkinsonian animals, we compared the distribution of postsynaptic targets contacted by vGluT1-immunopositive boutons between normal and MPTP-treated monkeys. To do so, we collected digital images of STN tissue at ×60 000, and sampled only those vGluT1-immunopositive terminals whose synapse could be clearly seen in the plane of section (Fig. 4A and B). The postsynaptic targets of these glutamatergic terminals were identified based on their ultrastructural features (Peters et al., 1991). If the postsynaptic target was a dendritic shaft, it was further classified based on its width at the point where it received the synaptic innervation by the terminal. These diameters were measured using NIH’s Image J software as the shortest diameter passing through the approximate centre of its 2D representation on the electron microscopy image (Fig. 4A). Based on these diameter measurements, dendrites were categorized as large (>1.0 µm), medium (0.5–1.0 µm) or small (<0.5 µm).

Figure 4.

Figure 4

Open in a new tab

Post-synaptic targets of vGluT1-immunopositive terminals in the dorsolateral STN. (A and B) Electron micrographs showing vGluT1-containing terminals (v1) forming asymmetric synapses (indicated by arrowheads) with a dendritic shaft (dend) (A) and a dendritic spine (sp) (B). The black line with double-ended arrows on the dendritic shaft (A) illustrates the measurement of its cross-sectional diameter, defined as the shortest diameter passing through its centre. Scale bars: A and B = 0.2 µm. (C) Post-synaptic targets of vGluT1-immunopositive terminals in the dorsolateral STN. No significant difference was found in the proportion of vGluT1-immunoreactive terminals forming asymmetric synapses with dendritic shafts and spines in normal versus parkinsonian monkeys (left). The right panel shows the size distribution of all postsynaptic dendritic shafts contacted by vGluT1-immunoreactive terminals, which is not significantly different between normal and parkinsonian animals. Lg = large; Md = medium; Sm = small. Columns represent means ± SEM across three normal and three parkinsonian monkeys.

Tyrosine hydroxylase immunostaining

To assess the degree of nigrostriatal dopamine denervation in monkeys used in the anatomical studies, series of tissue sections from the striatum or the ventral midbrain were immunostained with antibodies against tyrosine hydroxylase (mouse anti-TH; 1:1000 dilution; EMD Millipore) (Fig. 1). The intensity of tyrosine hydroxylase immunoreactivity in the post-commissural putamen (corresponding to the sensorimotor striatum) was measured in one representative section from each animal using NIH’s Image J software, as described in our previous studies (Galvan et al., 2011; Masilamoni et al., 2011; Villalba et al., 2013b). Similarly, the intensity of post-commissural putamenal tyrosine hydroxylase immunostaining was measured in three coronal sections (spread apart by 0.2–0.4 mm) from each of the animals used in the electrophysiological studies (n = 2). The intensity of striatal tyrosine hydroxylase labelling in these MPTP-treated monkeys was compared with that measured in brain sections of control animals (n = 2) from our laboratory’s monkey brain tissue bank.

Electrophysiological experiments

General strategy

We recorded the responses of pallidal neurons to the stimulation of the cortico-subthalamic tract in normal and parkinsonian monkeys, using a subcortical stimulation location in the internal capsule. Short-latency excitatory components of responses of pallidal neurons to activation of the hyperdirect pathway are dependent on cortico-subthalamic transmission (Nambu et al., 2000). We elected to record pallidal neurons instead of STN neurons, because such recordings are more stable and less influenced by stimulation artefacts than recordings in the STN.

Surgery

Under aseptic conditions and isofluorane anaesthesia (1–3%), stainless steel cylindrical recording chambers (16 mm i.d., Crist Instrument Co) were fastened to the skull over trephine holes. The chambers were stereotaxically targeted to the putamen/pallidum and STN regions with a 36° angle from the vertical in the coronal plane in one monkey, and with a 40° angle in the other. The chambers were affixed to the skull with dental acrylic and stainless steel screws. Metal head holding bolts (Crist Instrument) were also embedded in the acrylic. Post-surgery, the monkeys were given prophylactic antibiotic treatment, and were allowed to recuperate for at least 1 week before the start of the electrophysiological experiments.

Electrophysiological recordings

All recording sessions were conducted while the monkeys were awake and quietly seated in a primate chair with the head restrained. We first electrophysiologically mapped the locations of the internal capsule, putamen, GPe, GPi and STN, with standard tungsten microelectrodes (impedance 0.5–1 MΩ at 1 kHz; Frederic Haer Co). Thereafter, a concentric bipolar stimulation electrode (SNEX120, impedance 40–50 kΩ at 1 kHz; inter-contact separation 1 mm; Rhodes Medical) was positioned in the posterior limb of the internal capsule (Fig. 5), followed by insertion of a tungsten microelectrode into the ventrolateral portion (i.e. sensorimotor territory) of GPe and GPi (Fig. 5). Spiking activity of pallidal cells was accepted for recording if the signal to noise ratio was ≥ 3. Baseline signals were recorded for 60 s, followed by recordings during internal capsule stimulation with 100 randomly spaced single biphasic square wave pulses (500 µA, 200 µs/phase, minimum spacing between consecutive pulses 250 ms, monopolar stimulation). We estimate that stimulation with these parameters affected myelinated axons up to 1.7 mm away from the stimulation site (Ranck, 1975). The stimulation did not induce movements in the animals.

Figure 5.

Figure 5

Open in a new tab

Approximate locations of the placements of the stimulation electrodes projected onto coronal representations of the monkey brain. Experiments in the normal condition were conducted in the left hemisphere, whereas those in the parkinsonian states were performed on the right side. Thick black lines indicate the paths of the stimulation electrode with the red tips marking the positions of the electrical contact points of the electrode. Antero-posterior coordinates were determined by comparing the brain sections with a standard monkey atlas (Paxinos et al., 1999). Put = putamen; Cd = caudate; Thal = thalamus; Ctx = cerebral cortex; SN = substantia nigra.

At the beginning of each experimental session, the dura was punctured with a guide tube. This was followed by lowering of the stimulation and recording electrodes into the brain using a multi-probe microdrive (NAN Instruments Ltd). Neuronal activity was recorded using standard extracellular recording methods, filtered (fourth order Butterworth band-pass filter; 400 Hz–6 kHz) and amplified (DAM80 preamplifier, WPI Inc, and model 3364 filter and amplifier, Krohn-Hite). The resulting signal was digitized using an A-D interface (sampling rate, 50 kHz; Power1401/Spike2; Cambridge Electronic Design) and stored to computer disk for off-line processing. The analogue signal was also displayed on a digital oscilloscope (DL 1540; Yokogawa) and audio-amplified. The timing and amplitude of stimuli were controlled via the Power1401 interface, and the stimuli were generated by a constant-current stimulus isolation unit (A395R, WPI).

Analysis of electrophysiological data

The electrophysiological data were preprocessed with a Matlab (Mathworks) based stimulus artefact subtraction program (Fig. 6A). Because of clipping of stimulation artefacts, the neuronal activity occurring within 2 ms of each stimulus could not be reliably recovered. The artefact-subtracted records were subjected to waveform matching spike-sorting in Spike2. Inter-spike interval (ISI) distribution histograms were generated for each cell to examine the quality of spike sorting.

Figure 6.

Figure 6

Open in a new tab

Response of a pallidal neuron to internal capsule (IC) stimulation. (A) Six overlaid traces of neuronal activity show that the neuron often fires around 6–8 ms post-stimulation. The arrowhead points to a neuronal spike occurring along with the stimulation artefact. The neuronal spike was successfully recognized after processing the data with a stimulus artefact removal algorithm. (B) Peri-stimulus histogram depicting the firing rate of the neuron during the first 20 ms post-stimulation shows an early excitation response (red) to internal capsule stimulation. Peri-stimulus histogram generated from 100 randomly spaced consecutive stimuli (bin size = 1 ms), as described in the ‘Materials and methods’ section. The neuronal traces (A) and peri-stimulus histograms (B) are aligned to identical time axes.

All subsequent analysis steps were carried out in the Matlab environment. We generated peri-stimulus histograms (PSTHs) (bin size, 1 ms; pre stimulus period, 100 ms; post-stimulus period, 20 ms) (Fig. 6B). The mean and standard deviation (SD) of the number of events in peri-stimulus histogram bins in the pre stimulus period were calculated, and a threshold (mean + 2SD) was established to identify excitation events. Based on previous findings about the responses of pallidal neurons to activation of the primary motor cortex (Nambu et al., 2000), neurons were identified as showing an early excitatory response if at least two consecutive post-stimulation peri-stimulus histogram bins were above the threshold, with the first bin occurring no later than 10 ms after the stimulus and the last bin no later than 13 ms (Fig. 6B). The length of the epoch occupied by the consecutive supra-threshold peri-stimulus histogram bins was considered as the duration of the early excitatory response. If there were multiple excitatory responses (as defined) within the early excitation detection window, only the first one was considered. Finally, we compared the proportion of pallidal neurons showing an early excitatory response to internal capsule stimulation between normal and parkinsonian states (Fig. 7A). Long latency responses (i.e. those starting more than 10 ms after the stimulus) were not considered in this analysis.

Figure 7.

Figure 7

Open in a new tab

Electrophysiological responses of pallidal neurons to internal capsule stimulation. (A) Proportion of pallidal neurons responding with a characteristic early excitation to internal capsule stimulation in normal and parkinsonian monkeys. *P = 0.046 (Student’s t-test of difference between normal and parkinsonian state). Means ± SEM across two animals. (B) Intensity of early excitatory events in pallidal neurons showing an early excitatory response in normal and parkinsonian states (see ‘Materials and methods’ section for details). (C) Latency of early excitation responses in pallidal neurons in the normal and parkinsonian conditions. (D) Percent failure rate of spike occurrence during the early excitation response epoch after individual stimuli. Columns B–D represent means ± SEM across neurons with an early excitation response.

We also calculated Z-scores of the frequency of firing associated with each peri-stimulus histogram bin with respect to the pre stimulus period mean and standard deviation. For neurons with early excitatory responses, the mean Z-score of all bins constituting the excitation was calculated (Fig. 7B). The latencies of the early excitatory events were also compared between the normal and parkinsonian condition (Fig. 7C). Moreover we assessed the likelihood of a neuron showing an early excitation response to fire in response to individual stimuli. For this, we counted the number of stimulation trials during which there was no neuronal spike in the timeframe encompassed by the early excitation event and divided it by the total number of sweeps, thus computing the ‘failure rate’ of the neuron’s early excitation response (Fig. 7D).

Post-mortem identification of electrode tracks

After conclusion of the recording experiments, the monkeys were euthanized with an overdose of pentobarbital, and their brains perfused, fixed and processed, as stated previously. In this case, brain sections were cut into 50-µm thick coronal sections with a freezing microtome. Out of every 12 sections, one was Nissl-stained and another was immunolabelled to reveal the neuronal marker microtubule associated protein 2 (mouse anti-MAP2; 1:1000 dilution; EMD Millipore) for light microscopy observations, using protocols described previously (Galvan et al., 2010, 2011). The slides were scanned with a ScanScope CS scanning light microscope system (Aperio Technologies) at × 20 magnification, and their digital representations were analysed. Appropriate coordinates (with respect to the interaural line) were ascribed to the sections after confirming their antero-posterior locations by comparing the images with standard monkey atlas coronal plates (Paxinos et al., 1999). The relatively thick stimulation electrode tracks were easily reconstructed (Fig. 5). The recording sites were within the ventrolateral two-thirds of the GPe and GPi, corresponding to the sensorimotor territory of these nuclei. Most of the recording electrode tracks that were formed by the recording electrodes could be identified.

Statistical analyses

For each set of anatomical or electrophysiological data, we assessed the normality using either the Shapiro-Wilk test or the Kolmogorov-Smirnov test. Because all the different data sets were compatible with a normal distribution, we used the parametric Student’s t-test to assess the significance of differences between the normal and parkinsonian state.

Results

Parkinsonian motor signs and nigrostriatal dopaminergic pathology in MPTP-treated monkeys

All MPTP-treated monkeys in this study showed moderate to severe parkinsonism, as assessed with beam break counts in a behavioural observation cage and with the parkinsonism rating scale (score range at the time of euthanasia between 13–20), as used in many of our previous studies (Kliem et al., 2010; Hadipour-Niktarash et al., 2012; Bogenpohl et al., 2013). Densitometry measurements of tyrosine hydroxylase immunoreactivity showed that all parkinsonian animals had >80% dopaminergic denervation of the post-commissural putamen, compared to material from normal animals (Fig. 1). There was no major difference in the behavioural and anatomical scores between monkeys that were used in the light and electron microscopy studies and the animals used in electrophysiology experiments.

Lack of vGluT1 and vGluT2 co-localization in the STN

To examine whether the segregation of vGluT1 and vGluT2 described in the striatum (Fujiyama et al., 2004, 2006; Raju et al., 2006, 2008) and other forebrain regions (Hur and Zaborszky, 2005; Kubota et al., 2007; Liguz-Lecznar and Skangiel-Kramska, 2007) also applies to the STN, we carried out electron microscopy studies of the co-localization of vGluT1 and vGluT2 in single terminals, using immunoperoxidase or immunogold labelling for either vGluTs (Fig. 3A). Of 255 vGluT1-immunoreactive terminals sampled in the STN, none displayed vGluT2 immunostaining, thus showing that vGluT1 and vGluT2 are largely segregated in different populations of glutamatergic terminals originating from distinct sources. Together with evidence that the cerebral cortex is the main source of vGluT1-containing terminals to the STN (Fremeau et al., 2001, 2004; Fujiyama et al., 2006; Raju et al., 2008), these data suggest that vGluT1 is a reliable marker of cortico-subthalamic terminals in monkeys.

Changes in vGluT1-immunopositive innervation of the dorsolateral STN in MPTP-treated monkeys

Light microscopic observations

At the light microscope level, the STN in normal and MPTP-treated monkeys contained a moderate level of vGluT1 immunoreactivity (Fig. 2A) which, at higher power, was found to be confined to pleomorphic varicose processes of different sizes (Fig. 2B). Our quantitative assessment of the number of labelled varicosities showed that the dorsolateral ‘motor’ territory of the STN contained 3.85 ± 0.15 million vGluT1-immunopositive varicosities per mm3, while this density was 26.1% lower (2.84 ± 0.18 million/mm3) in parkinsonian animals (Fig. 2D). The difference between normal and parkinsonian animals was significant (P = 0.012).

Electron microscopy observations

Density of vGluT1-positive terminals

Because light microscopy observations do not allow us to ascertain whether a vGluT1-immunolabelled element is a vGluT1-containing terminal, we analysed the tissue at the electron microscopy level to ultrastructurally identify and quantify vGluT1-immunopositive terminals in normal and MPTP-treated monkeys (Fig. 3A). Under the electron microscope, vGluT1-containing terminals were easily distinguishable from unlabelled neuropil elements by their association with the dense amorphous peroxidase reaction product. As shown in Fig. 3D, these studies showed that the density of vGluT1-positive terminals was 55.1% lower in the parkinsonian monkeys than in the control animals (14 814 ± 1846 vGluT1-containing terminals per mm2 in normal monkeys; 6649 ± 1178 terminals per mm2 in parkinsonian animals; P = 0.02).

Loss of terminals forming asymmetric synapses

Because it is possible that the parkinsonism-related reduction of the density of vGluT1-immunopositive terminals in the STN of parkinsonian monkeys resulted from a decreased expression of vGluT1 immunoreactivity in (otherwise normal) cortico-subthalamic boutons, we quantified the density of terminals forming asymmetric synapses (with or without vGluT1 immunoreactivity) in the dorsolateral STN of normal and parkinsonian animals (Fig. 3B and C). As shown in Fig. 3E, the density of terminals forming asymmetric synapses was 27.9% lower in parkinsonian monkeys than in controls (13 323 ± 446 terminals forming asymmetric synapses per mm2 in normal monkeys, 9613 ± 1012 per mm2 in parkinsonian monkeys; P = 0.029).

To ensure that these density values were collected from STNs of comparable sizes, the unbiased Cavalieri method was used to measure the volume of the STN in three control and three MPTP-treated monkeys. No significant difference in STN volume was found between normal and MPTP-treated monkeys (P = 0.884; Fig. 2C).

Pattern of innervation of STN neurons by vGluT1-positive terminals

To determine if the remaining vGluT1-positive terminals in the STN of parkinsonian monkeys showed changes in their overall pattern of synaptic connectivity, we compared the distribution of postsynaptic targets in contact with vGluT1-positive terminals (n = 50 terminals per animal) in the dorsolateral STN between normal and MPTP-treated monkeys. In both groups of animals, vGluT1-immunopositive terminals formed asymmetric synapses, primarily with dendritic shafts of STN neurons (70 ± 4% of all synapses of vGluT1-containing terminals in normal animals, and 76 ± 5% in parkinsonian animals) and, to a lesser extent, dendritic spines (30 ± 4% in normal animals, and 24 ± 5% in parkinsonian animals; Fig. 4A–C). There was no significant difference between normal and MPTP-treated monkeys (two-way ANOVA; P = 0.223; Fig. 4C).

The dendritic shafts contacted by vGluT1-positive terminals were further categorized as large ( > 1.0 µm), medium (0.5–1.0 µm) or small ( < 0.5 µm), based on their cross-sectional diameter (Fig. 4A). In normal monkeys, 21.8 ± 7.0%, 47.9 ± 6.5% and 30.3 ± 1.7% of axo-dendritic synapses formed by vGluT1-immunoreactive terminals targeted large, medium and small dendrites, respectively. This pattern was not significantly different (two-way ANOVA; P = 0.173) in the parkinsonian monkeys (27.6 ± 3.3%, 51.7 ± 1.1% and 20.6 ± 3.7%, respectively; Fig. 4C).

Physiological impact of cortico-subthalamic activation on pallidal neurons in normal and parkinsonian monkeys

The decreased number of cortico-subthalamic terminals in parkinsonian monkeys shown in our anatomical studies could result in functional changes of the cortico-subthalamic system. A direct approach to identifying functional alterations of the cortico-subthalamic projection would be to record the activity of single STN neurons in response to cortical stimulation. However, it is difficult to conduct high quality STN neuron recordings for sufficient periods of time (Fernandez et al., 2007) and the presence of large stimulation artefacts often obscures short-latency responses of STN neurons after stimulation. We therefore decided to record stimulation-evoked short latency responses of pallidal neurons (downstream from the STN) instead, as an indirect measure of STN reactivity to cortico-subthalamic inputs. Because previous studies have shown that electrical stimulation of cortico-subthalamic fibres generates short-latency excitatory responses in the pallidum of normal monkeys (Nambu et al., 2000), we hypothesized that these early responses should be affected by changes in the integrity of the cortico-subthalamic projection in MPTP-treated monkeys.

A convenient and efficient method to activate the bulk of the cortico-subthalamic projection is through stimulation of the internal capsule. We therefore stimulated the internal capsule before and after induction of parkinsonism (Fig. 5), and sampled pallidal neurons from equivalent portions of the ventrolateral (‘sensorimotor’) globus pallidus. Short-latency excitatory responses (i.e. with a latency < 10 ms; Fig. 6) were observed in 30.9 ± 4.4% of pallidal neurons (15/44 GPe neurons, 6/24 GPi neurons) under normal conditions (Fig. 7A). In the parkinsonian state, a significantly lower proportion (13.4 ± 5.7%; Student’s t-test, P = 0.046) of pallidal neurons (5/33 GPe neurons, 1/14 GPi neurons) showed early excitation responses (Fig. 7A). Because the number of responding units in the GPi was very low, and because functional changes of the cortico-subthalamic system would be expected to affect similarly both pallidal segments (Nambu et al., 2000) as STN efferents to GPe and GPi are collaterals originating from the same neurons (Shink et al., 1996; Smith et al., 1998), we pooled the data collected from both the GPe and GPi for the subsequent analyses. Long latency responses, likely induced by the recruitment of complex multisynaptic networks, were not considered in the present analysis.

We found that the size of the early excitation responses in responding neurons (measured by calculating the average Z-score of peri-stimulus histogram bins within excitation responses) did not differ between data from the normal and parkinsonian state in either monkey (Fig. 7B). Thus, while the proportion of neurons that responded to internal capsule stimulation was lower in the parkinsonian state, the average amplitude of the responses remained the same between normal and parkinsonian animals. The latencies of the early excitation responses, as well as the ‘failure rate’ also remained unchanged by the induction of parkinsonism (Fig. 7C and D).

Discussion

Our results provide evidence for morphological and functional changes of the glutamatergic cortico-subthalamic projection in parkinsonian monkeys. The decrease in the density of vGluT1-containing cortical terminals and of terminals forming asymmetric synapses, combined with the significant reduction in the proportion of pallidal neurons responding to cortico-subthalamic activation in parkinsonian monkeys shown in this study, suggests that complex pathophysiological changes may affect the hyperdirect cortico-subthalamic system in Parkinson’s disease. Together with evidence for significant synaptic remodelling and altered glutamatergic transmission of the cortico-striatal system in Parkinson’s disease (Raju et al., 2008; Villalba and Smith, 2013), these findings suggest significant changes in the integration, processing and transmission of extrinsic cortical information to the basal ganglia in Parkinson’s disease.

Loss of cortico-subthalamic terminals in parkinsonian monkeys

The possibility that the decrease in vGluT1-positive terminals shown in the STN of parkinsonian monkeys reflects a reduction in the expression level of the vGluT1 protein rather than an actual loss of terminals of the cortico-subthalamic projection cannot be completely ruled out. However, the fact that the number of terminals forming asymmetric synapses (putatively glutamatergic) was also reduced adds credence to a possible terminal loss, but does not rule out the possibility that some of these terminals may have originated from vGluT2-containing subcortical sources. A detailed analysis of changes in the vGluT2-containing afferent system to the STN in parkinsonian monkeys is currently in progress to address this issue.

It is important to consider that the estimates of density reduction of vGluT1-positive ‘terminals’ in the STN of parkinsonian monkeys differed between electron microscopy (55.1%) and light microscopy (26.1%) observations. A contributing factor to these numeric differences could be the fact that light microscopy and electron microscopy data were collected from different monkeys. However, because the extent of striatal tyrosine hydroxylase denervation and the parkinsonian motor scores were comparable between all animals used in the light microscopy and electron microscopy studies, it is unlikely that interindividual differences in the state of parkinsonism accounted for these variations. Another possible contributing factor could be that counts at the electron microscopy level involved only well-defined vesicle-filled terminal-like structures, while the light microscopy-based counts may have included immunoreactive axonal profiles and terminals. Despite these differences, it is noteworthy that both light microscopy and electron microscopy observations strongly suggest a significant reduction in the number of glutamatergic terminals from the sensorimotor hyperdirect cortico-subthalamic projection in MPTP-treated monkeys. Because all animals used in this study displayed comparable parkinsonian motor signs and had reached > 80% nigrostriatal dopaminergic loss, it was not possible to correlate the extent of STN pathology with the severity of nigrostriatal damage. Future studies of the cortico-subthalamic system in animals with different degrees of dopaminergic depletion and a variable range of parkinsonism motor scores should be considered to address this issue.

It is noteworthy that both the anatomical and electrophysiological results reported in this study were gathered from the motor-related regions of the STN and the globus pallidus. Because of the tight topography of the connections between the STN and the globus pallidus (Smith et al., 1998), it was important to relate findings collected from functionally related territories of both structures. The dorsolateral (i.e. motor) region of the STN was specifically examined in the present study because of its relevance in motor processing, and the fact that it represents the main target for deep brain stimulation in Parkinson’s disease (Wichmann et al., 2011). However, preliminary anatomical evidence suggests that the reduced density of vGluT1-positive terminals in the STN of MPTP-treated monkeys also affect the ventromedial (i.e. associative) region of the nucleus (Mathai et al., 2011), raising the potential for a widespread pathology of the hyperdirect cortico-subthalamic system in Parkinson’s disease.

The cortico-striatal system also undergoes complex morphological and functional changes in animal models of Parkinson’s disease (Calabresi et al., 1993, 2007; Onn et al., 2000; Villalba et al., 2009; Villalba and Smith, 2011). In rodent studies, some of the cortico-subthalamic inputs are collaterals of corticofugal systems innervating the striatum and other targets along their course (Kita and Kita, 2012), so that structural changes at the level of the striatum and the STN may reflect changes within the same group of neurons. However, there is no clear evidence that single cortical projection neurons innervate both the striatum and the STN in primates (Parent and Parent, 2006; Smith et al., 2013). Furthermore, the morphological changes affecting the cortico-striatal projection in parkinsonian monkeys differ from those described here. Even though there is a marked reduction in the spine density on striatal medium spiny neurons, the density of vGluT1-immunoreactive cortical terminals is slightly increased or unchanged in the striatum of MPTP-treated parkinsonian monkeys (Raju et al., 2008; Villalba et al., 2013a), and the amount of striatal vGluT1 protein is significantly increased in post-mortem brains of patients with Parkinson’s disease (Kashani et al., 2007). Consistent with these changes at the terminal level, the amount of glutamate is increased, and there is strengthening of cortical synapses in the striatum of rodent models of Parkinson’s disease (Lindefors and Ungerstedt, 1990; Calabresi et al., 1993; Meshul et al., 2000).

In contrast to the findings pertaining to the cortico-striatal projection, our data suggest a significant loss of cortical terminals in the sensorimotor territory of the STN in parkinsonian monkeys. However, this finding does not necessarily translate into reduced glutamatergic transmission in the STN of parkinsonian animals. As described in the striatum of rodent and monkey models of Parkinson’s disease (Ingham et al., 1989; Meshul et al., 2000; Villalba and Smith, 2011, 2013), compensatory structural and functional changes of remaining cortico-subthalamic synapses (or other glutamatergic inputs) may overcome the predicted decrease in glutamatergic synaptic drive of STN neurons in the parkinsonian state, so that our findings are still incompatible with the known increase in the baseline activity of STN neurons (Bergman et al., 1994), and their greater degree of synchrony with cortical activity in Parkinson’s disease (Williams et al., 2002, 2003, 2005; Moran et al., 2008; Moshel et al., 2013; Shimamoto et al., 2013; Devergnas et al., 2014).

Combined with recent data showing morphological and functional changes in GABAergic pallidal terminals in a rodent model of Parkinson’s disease (Bevan et al., 2006, 2007; Fan et al., 2012), our findings provide additional evidence for changes in the synaptic microcircuitry of the STN in parkinsonian animals. The functional consequences of these alterations at the cellular level, and their impact upon the integration, processing and transmission of information through the basal ganglia circuitry await further studies. Whether these plastic changes result from local dopamine denervation of STN neurons and/or functional alterations of multi-synaptic basal ganglia networks also remain to be established (Campbell et al., 1985; Hassani and Feger, 1999; Shen and Johnson, 2000; Ni et al., 2001; Zhu et al., 2002; Cragg et al., 2004; Baufreton and Bevan, 2008; Rommelfanger and Wichmann, 2010). Although unlikely, the possibility that cortical neuronal loss underlies some of these changes should also be considered (MacDonald and Halliday, 2002).

Parkinsonism-related changes in the proportion of pallidal neurons activated by stimulation of the cortico-subthalamic pathway

Our electrophysiology data suggest that a lower proportion of pallidal neurons respond to internal capsule stimulation in MPTP-treated parkinsonian monkeys than in controls. It is possible that the decreased responsiveness of pallidal neurons to the stimulation is due to parkinsonism-related changes affecting transmission along the subthalamo-pallidal projection. However, considered jointly with our anatomical findings, the most likely interpretation is that the decreased responsiveness of pallidal neurons is caused by the partial degeneration of the cortico-subthalamic system in MPTP-induced parkinsonism. We found that the average amplitude, latency and failure rate of the remaining early excitatory responses induced by internal capsule stimulation remained unchanged by the induction of parkinsonism. One possibility to explain the lack of change in the remaining responses is the strict topography and specificity of cortical relationships with individual subthalamo-pallidal neurons so that the cortical deafferentation may render some STN neurons ineffective in transmitting cortical information to the globus pallidus, while leaving intact the transmission through other subthalamo-pallidal neurons. Future studies are needed to directly address this issue.

The possibility must be considered that the internal capsule stimulation used to evoke cortical responses in the STN recruited non-cortico-subthalamic projections that may then have affected pallidal activity. However, the placement of the stimulation electrodes (Fig. 5), the general trajectory of non-cortico-subthalamic projections and, most importantly, the criteria used for identifying early excitation events (i.e. latency <10 ms) render this possibility less likely. Antidromic activation of pallidal neurons is also unlikely because the earliest excitatory responses occurred with a latency of >3 ms, and the responses showed a high ‘failure rate’ within the early excitation response period (Fig. 7D).

The stimulation and recording sites used in the normal and parkinsonian states were reasonably well matched in this study. While the stimulation electrode placement in the posterior limb of the internal capsule was not identical across monkeys (Fig. 5) and stimulation sites, the strength of stimulation was in all cases adequate to stimulate large and overlapping portions of the internal capsule (Ranck, 1975). We also carefully mapped the borders and extent of GPe and GPi in each case to ensure that comparable recording sites in the ventrolateral part of the pallidal complex were sampled in each animal under normal and parkinsonian conditions. The ventrolateral ‘sensorimotor’ GPe/GPi region was chosen as the recording site because it is the main target of inputs from the dorsolateral STN (the site of the pathological changes described in this study) (Smith et al., 1990; Shink et al., 1996), and because it is located far enough from the stimulation site in the internal capsule so that the recordings were not affected by direct current spread.

Degeneration of cortico-subthalamic fibres: potential impact on basal ganglia functions

The temporal interplay of the cortico-subthalamic and cortico-striatal pathways is a critical element in ‘action selection’ models of basal ganglia function (Nambu et al., 2002) and in models that ascribe response inhibition to the hyperdirect pathway (Jahfari et al., 2011; Schmidt et al., 2013; Udupa and Fox, 2013). Central to arguments in favour of these models is the apparent restriction of action selection in patients with Parkinson’s disease (Mink and Thach, 1993; Nambu et al., 2000, 2002; Helmich et al., 2009; Wylie et al., 2010), a deficit possibly related to increased functional connectivity between the motor cortex and the STN (Baudrexel et al., 2011), and a greater entrainment of STN neurons to cortical activity (Mallet et al., 2008; Shimamoto et al., 2013). As mentioned above, if similar changes in the functional connectivity of the cortico-subthalamic system also occur in MPTP-treated monkeys, it is possible that the degeneration of the cortico-subthalamic system in these monkeys is accompanied with compensatory changes at the synaptic level that affect both glutamatergic and non-glutamatergic STN afferents (Fan et al., 2012).

Taking into consideration that some of the antiparkinsonian effects induced by STN deep brain stimulation in Parkinson’s disease are due in part to the antidromic activation of the cortico-subthalamic system (Li et al., 2007; Gradinaru et al., 2009), an important therapeutic implication of our findings may be that the documented loss of cortico-subthalamic terminals limits the overall effectiveness of deep brain stimulation of the STN in Parkinson’s disease.

Funding

This work was supported by the following grants by the National Institutes of Health: P50 NS071669 (Udall Centre grant, T.W., Y.S.), R01 NS037948 (Y.S.), and P51 OD011132 (infrastructure grant to the Yerkes National Primate Research Centre).

Acknowledgements

We thank Ms Susan Jenkins and Mr Damien Pittard for providing expert technical assistance during the anatomical and electrophysiologic components of this study, respectively.

Glossary

Abbreviations

GPe

external globus pallidus

GPi

internal globus pallidus

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

STN

subthalamic nucleus

vGluT1

vesicular glutamate transporter 1

References

  1. Baudrexel S, Witte T, Seifried C, von Wegner F, Beissner F, Klein JC, et al. Resting state fMRI reveals increased subthalamic nucleus-motor cortex connectivity in Parkinson's disease. Neuroimage. 2011;55:1728–38. doi: 10.1016/j.neuroimage.2011.01.017. [DOI] [PubMed] [Google Scholar]
  2. Baufreton J, Atherton JF, Surmeier DJ, Bevan MD. Enhancement of excitatory synaptic integration by GABAergic inhibition in the subthalamic nucleus. J Neurosci. 2005;25:8505–17. doi: 10.1523/JNEUROSCI.1163-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baufreton J, Bevan MD. D2-like dopamine receptor-mediated modulation of activity-dependent plasticity at GABAergic synapses in the subthalamic nucleus. J Physiol. 2008;586:2121–42. doi: 10.1113/jphysiol.2008.151118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergman H, Wichmann T, Karmon B, DeLong MR. The primate subthalamic nucleus. II. Neuronal activity in the MPTP model of parkinsonism. J Neurophysiol. 1994;72:507–20. doi: 10.1152/jn.1994.72.2.507. [DOI] [PubMed] [Google Scholar]
  5. Bevan MD, Francis CM, Bolam JP. The glutamate-enriched cortical and thalamic input to neurons in the subthalamic nucleus of the rat: convergence with GABA-positive terminals. J Comp Neurol. 1995;361:491–511. doi: 10.1002/cne.903610312. [DOI] [PubMed] [Google Scholar]
  6. Bevan MD, Magill PJ, Terman D, Bolam JP, Wilson CJ. Move to the rhythm: oscillations in the subthalamic nucleus-external globus pallidus network. Trends Neurosci. 2002;25:525–531. doi: 10.1016/s0166-2236(02)02235-x. [DOI] [PubMed] [Google Scholar]
  7. Bevan MD, Atherton JF, Baufreton J. Cellular principles underlying normal and pathological activity in the subthalamic nucleus. Curr Opin Neurobiol. 2006;16:621–28. doi: 10.1016/j.conb.2006.10.003. [DOI] [PubMed] [Google Scholar]
  8. Bogenpohl J, Galvan A, Hu X, Wichmann T, Smith Y. Metabotropic glutamate receptor 4 in the basal ganglia of parkinsonian monkeys: ultrastructural localization and electrophysiological effects of activation in the striatopallidal complex. Neuropharmacology. 2013;66:242–52. doi: 10.1016/j.neuropharm.2012.05.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Calabresi P, Mercuri NB, Sancesario G, Bernardi G. Electrophysiology of dopamine-denervated striatal neurons. Implications for Parkinson's disease. Brain. 1993;116(Pt 2):433–52. [PubMed] [Google Scholar]
  10. Calabresi P, Picconi B, Tozzi A, Di Filippo M. Dopamine-mediated regulation of corticostriatal synaptic plasticity. Trends Neurosci. 2007;30:211–19. doi: 10.1016/j.tins.2007.03.001. [DOI] [PubMed] [Google Scholar]
  11. Campbell GA, Eckardt MJ, Weight FF. Dopaminergic mechanisms in subthalamic nucleus of rat: analysis using horseradish peroxidase and microiontophoresis. Brain Res. 1985;333:261–70. doi: 10.1016/0006-8993(85)91580-x. [DOI] [PubMed] [Google Scholar]
  12. Cragg SJ, Baufreton J, Xue Y, Bolam JP, Bevan MD. Synaptic release of dopamine in the subthalamic nucleus. Eur J Neurosci. 2004;20:1788–802. doi: 10.1111/j.1460-9568.2004.03629.x. [DOI] [PubMed] [Google Scholar]
  13. Devergnas A, Pittard D, Bliwise D, Wichmann T. Relationships between oscillatory activity in the cortico-basal ganglia network and parkinsonism in MPTP-treated monkeys. Neurobiol Dis. 2014;68:156–66. doi: 10.1016/j.nbd.2014.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fan KY, Baufreton J, Surmeier DJ, Chan CS, Bevan MD. Proliferation of external globus pallidus-subthalamic nucleus synapses following degeneration of midbrain dopamine neurons. J Neurosci. 2012;32:13718–28. doi: 10.1523/JNEUROSCI.5750-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fernandez HH, Rodriquez RL, Skidmore FM, Okun MS. A Practical Approach to Movement Disorders. 1 edn. New York: Demos Medical Publishing; 2007. Key concepts for surgical therapy for Parkinson's disease and movement disorders. [Google Scholar]
  16. Fremeau RT, Jr, Voglmaier S, Seal RP, Edwards RH. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 2004;27:98–103. doi: 10.1016/j.tins.2003.11.005. [DOI] [PubMed] [Google Scholar]
  17. Fremeau RT, Jr, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron. 2001;31:247–60. doi: 10.1016/s0896-6273(01)00344-0. [DOI] [PubMed] [Google Scholar]
  18. Fujiyama F, Unzai T, Nakamura K, Nomura S, Kaneko T. Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum. Eur J Neurosci. 2006;24:2813–24. doi: 10.1111/j.1460-9568.2006.05177.x. [DOI] [PubMed] [Google Scholar]
  19. Fujiyama F, Kuramoto E, Okamoto K, Hioki H, Furuta T, Zhou L, et al. Presynaptic localization of an AMPA-type glutamate receptor in corticostriatal and thalamostriatal axon terminals. Eur J Neurosci. 2004;20:3322–30. doi: 10.1111/j.1460-9568.2004.03807.x. [DOI] [PubMed] [Google Scholar]
  20. Galvan A, Hu X, Smith Y, Wichmann T. Localization and function of GABA transporters in the globus pallidus of parkinsonian monkeys. Exp Neurol. 2010;223:505–15. doi: 10.1016/j.expneurol.2010.01.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Galvan A, Hu X, Smith Y, Wichmann T. Localization and pharmacological modulation of GABA-B receptors in the globus pallidus of parkinsonian monkeys. Exp Neurol. 2011;229:429–39. doi: 10.1016/j.expneurol.2011.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Garber JC, Barbee RW, Bielitzki JT, Clayton LA, Donovan JC, Hendriksen CFM, et al. Guide for the Care and Use of Laboratory Animals. 8 edn. Washington, DC: The National Academies Press; 2010. [Google Scholar]
  23. Gonzales KK, Pare JF, Wichmann T, Smith Y. GABAergic inputs from direct and indirect striatal projection neurons onto cholinergic interneurons in the primate putamen. J Comp Neurol. 2013;521:2502–22. doi: 10.1002/cne.23295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gradinaru V, Mogri M, Thompson KR, Henderson JM, Deisseroth K. Optical deconstruction of parkinsonian neural circuitry. Science. 2009;324:354–59. doi: 10.1126/science.1167093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gundersen HJ, Osterby R. Optimizing sampling efficiency of stereological studies in biology: or ‘do more less well. J Microsc. 1981;121:65–73. doi: 10.1111/j.1365-2818.1981.tb01199.x. [DOI] [PubMed] [Google Scholar]
  26. Hadipour-Niktarash A, Rommelfanger KS, Masilamoni GJ, Smith Y, Wichmann T. Extrastriatal D2-like receptors modulate basal ganglia pathways in normal and Parkinsonian monkeys. J Neurophysiol. 2012;107:1500–12. doi: 10.1152/jn.00348.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hassani OK, Feger J. Effects of intrasubthalamic injection of dopamine receptor agonists on subthalamic neurons in normal and 6-hydroxydopamine-lesioned rats: an electrophysiological and c-Fos study. Neurosci. 1999;92:533–43. doi: 10.1016/s0306-4522(98)00765-9. [DOI] [PubMed] [Google Scholar]
  28. Haynes WI, Haber SN. The organization of prefrontal-subthalamic inputs in primates provides an anatomical substrate for both functional specificity and integration: implications for Basal Ganglia models and deep brain stimulation. J Neurosci. 2013;33:4804–814. doi: 10.1523/JNEUROSCI.4674-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Helmich RC, Aarts E, de Lange FP, Bloem BR, Toni I. Increased dependence of action selection on recent motor history in Parkinson's disease. J Neurosci. 2009;29:6105–113. doi: 10.1523/JNEUROSCI.0704-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hur EE, Zaborszky L. Vglut2 afferents to the medial prefrontal and primary somatosensory cortices: a combined retrograde tracing in situ hybridization study [corrected] J Comp Neurol. 2005;483:351–73. doi: 10.1002/cne.20444. [DOI] [PubMed] [Google Scholar]
  31. Ingham CA, Hood SH, Arbuthnott GW. Spine density on neostriatal neurones changes with 6-hydroxydopamine lesions and with age. Brain Res. 1989;503:334–38. doi: 10.1016/0006-8993(89)91686-7. [DOI] [PubMed] [Google Scholar]
  32. Jahfari S, Waldorp L, van den Wildenberg WP, Scholte HS, Ridderinkhof KR, Forstmann BU. Effective connectivity reveals important roles for both the hyperdirect (fronto-subthalamic) and the indirect (fronto-striatal-pallidal) fronto-basal ganglia pathways during response inhibition. J Neurosci. 2011;31:6891–99. doi: 10.1523/JNEUROSCI.5253-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kaneko T, Fujiyama F. Complementary distribution of vesicular glutamate transporters in the central nervous system. Neurosci Res. 2002;42:243–50. doi: 10.1016/s0168-0102(02)00009-3. [DOI] [PubMed] [Google Scholar]
  34. Kashani A, Betancur C, Giros B, Hirsch E, El Mestikawy S. Altered expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in Parkinson disease. Neurobiol Aging. 2007;28:568–78. doi: 10.1016/j.neurobiolaging.2006.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kita H, Chang HT, Kitai ST. The morphology of intracellularly labeled rat subthalamic neurons: a light microscopic analysis. J Comp Neurol. 1983;215:245–57. doi: 10.1002/cne.902150302. [DOI] [PubMed] [Google Scholar]
  36. Kita T, Kita H. The subthalamic nucleus is one of multiple innervation sites for long-range corticofugal axons: a single-axon tracing study in the rat. J Neurosci. 2012;32:5990–9. doi: 10.1523/JNEUROSCI.5717-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kliem MA, Pare JF, Khan ZU, Wichmann T, Smith Y. Ultrastructural localization and function of dopamine D1-like receptors in the substantia nigra pars reticulata and the internal segment of the globus pallidus of parkinsonian monkeys. Eur J Neurosci. 2010;31:836–51. doi: 10.1111/j.1460-9568.2010.07109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kubota Y, Hatada S, Kondo S, Karube F, Kawaguchi Y. Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents. J Neurosci. 2007;27:1139–50. doi: 10.1523/JNEUROSCI.3846-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lavoie B, Parent A. Pedunculopontine nucleus in the squirrel monkey: projections to the basal ganglia as revealed by anterograde tract-tracing methods. J Comp Neurol. 1994;344:210–31. doi: 10.1002/cne.903440204. [DOI] [PubMed] [Google Scholar]
  40. Li S, Arbuthnott GW, Jutras MJ, Goldberg JA, Jaeger D. Resonant antidromic cortical circuit activation as a consequence of high-frequency subthalamic deep-brain stimulation. J Neurophysiol. 2007;98:3525–37. doi: 10.1152/jn.00808.2007. [DOI] [PubMed] [Google Scholar]
  41. Liguz-Lecznar M, Skangiel-Kramska J. Vesicular glutamate transporters (VGLUTs): the three musketeers of glutamatergic system. Acta Neurobiol Exp (Wars) 2007;67:207–18. doi: 10.55782/ane-2007-1649. [DOI] [PubMed] [Google Scholar]
  42. Lindefors N, Ungerstedt U. Bilateral regulation of glutamate tissue and extracellular levels in caudate-putamen by midbrain dopamine neurons. Neurosci Lett. 1990;115:248–52. doi: 10.1016/0304-3940(90)90463-j. [DOI] [PubMed] [Google Scholar]
  43. MacDonald V, Halliday GM. SElective loss of pyramidal neurons in the pre-supplementary motior cortex in Parkinson's disease. Mov. Disorders. 2002;17:1166–73. doi: 10.1002/mds.10258. [DOI] [PubMed] [Google Scholar]
  44. Mallet N, Pogosyan A, Sharott A, Csicsvari J, Bolam JP, Brown P, et al. Disrupted dopamine transmission and the emergence of exaggerated beta oscillations in subthalamic nucleus and cerebral cortex. J Neurosci. 2008;28:4795–806. doi: 10.1523/JNEUROSCI.0123-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Masilamoni GJ, Bogenpohl JW, Alagille D, Delevich K, Tamagnan G, Votaw JR, et al. Metabotropic glutamate receptor 5 antagonist protects dopaminergic and noradrenergic neurons from degeneration in MPTP-treated monkeys. Brain. 2011;134:2057–2073. doi: 10.1093/brain/awr137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mathai A, Smith Y. The corticostriatal and corticosubthalamic pathways: two entries, one target. So what? Front Syst Neurosci. 2011;5:64. doi: 10.3389/fnsys.2011.00064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Mathai A, Pare JF, Jenkins S, Smith Y. Glutamatergic inputs to the subthalamic nucleus: a quantitative analysis of the synaptic microcircuitry of vGluT1- and vGluT2-containing terminals in normal and Parkinsonian nonhuman primates. In: Xth Triennial Meeting of the International Basal Ganglia Society. Long Branch, NJ. 2010 [Google Scholar]
  48. Mathai A, Ma Y, Wichmann T, Smith Y. 41st Annual Meeting of the Society for Neuroscience. Washington, DC: 2011. Glutamatergic inputs to the subthalamic nucleus degenerate in experimental parkinsonism. abstract. [Google Scholar]
  49. McMillan J, Galvan A, Wichmann T, Bloomsmith M. The use of positive reinforcement during pole and collar training of rhesus macaques (Macaca mulatta) J Am Assoc Lab Anim Sci. 2010;49:705–6. [PMC free article] [PubMed] [Google Scholar]
  50. Meshul CK, Cogen JP, Cheng HW, Moore C, Krentz L, McNeill TH. Alterations in rat striatal glutamate synapses following a lesion of the cortico- and/or nigrostriatal pathway. Exp Neurol. 2000;165:191–206. doi: 10.1006/exnr.2000.7467. [DOI] [PubMed] [Google Scholar]
  51. Mink JW, Thach WT. Basal ganglia intrinsic circuits and their role in behavior. Curr Opin Neurobiol. 1993;3:950–7. doi: 10.1016/0959-4388(93)90167-w. [DOI] [PubMed] [Google Scholar]
  52. Mitrano DA, Pare JF, Smith Y. Ultrastructural relationships between cortical, thalamic, and amygdala glutamatergic inputs and group I metebotropic glutamate receptors in the rat accumbens. J Comp Neurol. 2010;518:1315–29. doi: 10.1002/cne.22277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Monakow KH, Akert K, Kunzle H. Projections of the precentral motor cortex and other cortical areas of the frontal lobe to the subthalamic nucleus in the monkey. Exp Brain Res. 1978;33:395–403. doi: 10.1007/BF00235561. [DOI] [PubMed] [Google Scholar]
  54. Moran A, Bergman H, Israel Z, Bar-Gad I. Subthalamic nucleus organization revealed by parkinsonian neuronal oscillations and synchrony. Brain. 2008;131:3395–409. doi: 10.1093/brain/awn270. [DOI] [PubMed] [Google Scholar]
  55. Moshel S, Shamir RR, Raz A, de Noriega FR, Eitan R, Bergman H, et al. Subthalamic nucleus long-range synchronization-an independent hallmark of human Parkinson's disease. Front Syst Neurosci. 2013;7:79. doi: 10.3389/fnsys.2013.00079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Nambu A, Tokuno H, Takada M. Functional significance of the cortico-subthalamo-pallidal ‘hyperdirect' pathway. Neurosci Res. 2002;43:111–17. doi: 10.1016/s0168-0102(02)00027-5. [DOI] [PubMed] [Google Scholar]
  57. Nambu A, Takada M, Inase M, Tokuno H. Dual somatotopical representations in the primate subthalamic nucleus: evidence for ordered but reversed body-map transformations from the primary motor cortex and the supplementary motor area. J Neurosci. 1996;16:2671–83. doi: 10.1523/JNEUROSCI.16-08-02671.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Nambu A, Tokuno H, Hamada I, Kita H, Imanishi M, Akazawa T, et al. Excitatory cortical inputs to pallidal neurons via the subthalamic nucleus in the monkey. J Neurophysiol. 2000;84:289–300. doi: 10.1152/jn.2000.84.1.289. [DOI] [PubMed] [Google Scholar]
  59. Ni Z, Gao D, Bouali-Benazzouz R, Benabid AL, Benazzouz A. Effect of microiontophoretic application of dopamine on subthalamic nucleus neuronal activity in normal rats and in rats with unilateral lesion of the nigrostriatal pathway. Eur J Neurosci. 2001;14:373–81. doi: 10.1046/j.0953-816x.2001.01644.x. [DOI] [PubMed] [Google Scholar]
  60. Onn SP, West AR, Grace AA. Dopamine-mediated regulation of striatal neuronal and network interactions. Trends Neurosci. 2000;23:S48–56. doi: 10.1016/s1471-1931(00)00020-3. [DOI] [PubMed] [Google Scholar]
  61. Orieux G, Francois C, Feger J, Hirsch EC. Consequences of dopaminergic denervation on the metabolic activity of the cortical neurons projecting to the subthalamic nucleus in the rat. J Neurosci. 2002;22:8762–70. doi: 10.1523/JNEUROSCI.22-19-08762.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Parent A, Hazrati LN. Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res Brain Res Rev. 1995;20:91–127. doi: 10.1016/0165-0173(94)00007-c. [DOI] [PubMed] [Google Scholar]
  63. Parent M, Parent A. Single-axon tracing study of corticostriatal projections arising from primate motor cortex in primates. J Comp Neurol. 2006;496:202–13. doi: 10.1002/cne.20925. [DOI] [PubMed] [Google Scholar]
  64. Paxinos G, Huang X-F, Toga AW. The Rhesus Monkey Brain in Stereotaxic Coordinates. 1st edn. San Diego, CA: Academic Press; 1999. [Google Scholar]
  65. Peters A, Palay SL, Webster HD. The Fine Structure of the Nervous System: Neurons and their Supporting Cells. 3rd edn. New York: Oxford University Press; 1991. [Google Scholar]
  66. Raju DV, Shah DJ, Wright TM, Hall RA, Smith Y. Differential synaptology of vGluT2-containing thalamostriatal afferents between the patch and matrix compartments in rats. J Comp Neurol. 2006;499:231–43. doi: 10.1002/cne.21099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Raju DV, Ahern TH, Shah DJ, Wright TM, Standaert DG, Hall RA, et al. Differential synaptic plasticity of the corticostriatal and thalamostriatal systems in an MPTP-treated monkey model of parkinsonism. Eur J Neurosci. 2008;27:1647–58. doi: 10.1111/j.1460-9568.2008.06136.x. [DOI] [PubMed] [Google Scholar]
  68. Ranck JB., Jr Which elements are excited in electrical stimulation of mammalian central nervous system: a review. Brain Res. 1975;98:417–40. doi: 10.1016/0006-8993(75)90364-9. [DOI] [PubMed] [Google Scholar]
  69. Rommelfanger KS, Wichmann T. Extrastriatal dopaminergic circuits of the Basal Ganglia. Front Neuroanat. 2010;4:139. doi: 10.3389/fnana.2010.00139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Sadikot AF, Parent A, Francois C. Efferent connections of the centromedian and parafascicular thalamic nuclei in the squirrel monkey: a PHA-L study of subcortical projections. J Comp Neurol. 1992;315:137–59. doi: 10.1002/cne.903150203. [DOI] [PubMed] [Google Scholar]
  71. Schmitz C, Hof PR. Design-based stereology in neuroscience. Neuroscience. 2005;130:813–31. doi: 10.1016/j.neuroscience.2004.08.050. [DOI] [PubMed] [Google Scholar]
  72. Schmidt R, Leventhal DK, Mallet N, Chen F, Berke JD. Canceling actions involves a race between basal ganglia pathways. Nat Neurosci. 2013;16:1118–24. doi: 10.1038/nn.3456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Shen KZ, Johnson SW. Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro. J Physiol. 2000;525(Pt 2):331–41. doi: 10.1111/j.1469-7793.2000.00331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Shimamoto SA, Ryapolova-Webb ES, Ostrem JL, Galifianakis NB, Miller KJ, Starr PA. Subthalamic nucleus neurons are synchronized to primary motor cortex local field potentials in Parkinson's disease. J Neurosci. 2013;33:7220–33. doi: 10.1523/JNEUROSCI.4676-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Shink E, Bevan MD, Bolam JP, Smith Y. The subthalamic nucleus and the external pallidum: two tightly interconnected structures that control the output of the basal ganglia in the monkey. Neurosci. 1996;73:335–357. doi: 10.1016/0306-4522(96)00022-x. [DOI] [PubMed] [Google Scholar]
  76. Smith Y, Hazrati LN, Parent A. Efferent projections of the subthalamic nucleus in the squirrel monkey as studied by the PHA-L anterograde tracing method. J Comp Neurol. 1990;294:306–23. doi: 10.1002/cne.902940213. [DOI] [PubMed] [Google Scholar]
  77. Smith Y, Wichmann T, Delong MR. Corticostriatal and mesocortical dopamine systems: do species differences matter? Nat Rev Neurosci. 2013;15:63. doi: 10.1038/nrn3469-c1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Udupa K, Fox SH. Subthalamic nucleus and striatum: the red and green signals to regulate the traffic of basal ganglia circuitry. Mov Disorders. 2013;28:1802. doi: 10.1002/mds.25694. [DOI] [PubMed] [Google Scholar]
  79. Unal G, Pare JF, Smith Y, Pare D. Cortical inputs innervate calbindin-immunoreactive interneurons in the rat basolateral amygdaloid complex. J Comp Neurol. 2014;522:1915–28. doi: 10.1002/cne.23511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Villalba R, Wichmann T, Smith Y. Preferential loss of thalamostriatal over corticostriatal glutamatergic synapses in parkinsonian monkeys In: 43rd Annual Meeting of the Society for Neuroscience. San Diego, CA; 2013a; abstr. [Google Scholar]
  81. Villalba RM, Smith Y. Differential structural plasticity of corticostriatal and thalamostriatal axo-spinous synapses in MPTP-treated Parkinsonian monkeys. J Comp Neurol. 2011;519:989–1005. doi: 10.1002/cne.22563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Villalba RM, Smith Y. Differential striatal spine pathology in Parkinson's disease and cocaine addiction: a key role of dopamine? Neurosci. 2013;251:2–20. doi: 10.1016/j.neuroscience.2013.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Villalba RM, Lee H, Smith Y. Dopaminergic denervation and spine loss in the striatum of MPTP-treated monkeys. Exp Neurol. 2009;215:220–27. doi: 10.1016/j.expneurol.2008.09.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Villalba RM, Wichmann T, Smith Y. Neuronal loss in the caudal intralaminar thalamic nuclei in a primate model of Parkinson's disease. Brain Struct Funct. 2013b;219:381–94. doi: 10.1007/s00429-013-0507-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. West MJ, Slomianka L, Gundersen HJ. Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec. 1991;231:482–97. doi: 10.1002/ar.1092310411. [DOI] [PubMed] [Google Scholar]
  86. West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci. 1999;22:51–61. doi: 10.1016/s0166-2236(98)01362-9. [DOI] [PubMed] [Google Scholar]
  87. Wichmann T, DeLong MR. Pathophysiology of Parkinson's disease: the MPTP primate model of the human disorder. Ann N Y Acad Sci. 2003;991:199–213. doi: 10.1111/j.1749-6632.2003.tb07477.x. [DOI] [PubMed] [Google Scholar]
  88. Wichmann T, Dostrovsky JO. Pathological basal ganglia activity in movement disorders. Neuroscience. 2011;198:232–44. doi: 10.1016/j.neuroscience.2011.06.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Wichmann T, Kliem MA, DeLong MR. Antiparkinsonian and behavioral effects of inactivation of the substantia nigra pars reticulata in hemiparkinsonian primates. Exp Neurol. 2001;167:410–24. doi: 10.1006/exnr.2000.7572. [DOI] [PubMed] [Google Scholar]
  90. Wichmann T, Delong MR, Guridi J, Obeso JA. Milestones in research on the pathophysiology of Parkinson's disease. Mov Disord. 2011;26:1032–41. doi: 10.1002/mds.23695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Williams D, Tijssen M, Van Bruggen G, Bosch A, Insola A, Di Lazzaro V, et al. Dopamine-dependent changes in the functional connectivity between basal ganglia and cerebral cortex in humans. Brain. 2002;125:1558–69. doi: 10.1093/brain/awf156. [DOI] [PubMed] [Google Scholar]
  92. Williams D, Kuhn A, Kupsch A, Tijssen M, Van Bruggen G, Speelman H, et al. Behavioural cues are associated with modulations of synchronous oscillations in human subthalamic nucleus. Brain. 2003;126:1975–85. doi: 10.1093/brain/awg194. [DOI] [PubMed] [Google Scholar]
  93. Williams D, Kuhn A, Kupsch A, Tijssen M, Van Bruggen G, Speelman H, et al. The relationship between oscillatory activity and motor reaction time in the parkinsonian subthalamic nucleus. Eur J Neurosci. 2005;21:249–58. doi: 10.1111/j.1460-9568.2004.03817.x. [DOI] [PubMed] [Google Scholar]
  94. Wylie SA, Ridderinkhof KR, Bashore TR, van den Wildenberg WP. The effect of Parkinson's disease on the dynamics of on-line and proactive cognitive control during action selection. J Cogn Neurosci. 2010;22:2058–73. doi: 10.1162/jocn.2009.21326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Zhu ZT, Shen KZ, Johnson SW. Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro. Neuropharmacol. 2002;42:772–81. doi: 10.1016/s0028-3908(02)00035-7. [DOI] [PubMed] [Google Scholar]

Articles from Brain are provided here courtesy of Oxford University Press

RESOURCES