Thalamic degeneration in MPTP-treated Parkinsonian monkeys: impact upon glutamatergic innervation of striatal cholinergic interneurons

Abstract

In both Parkinson's disease (PD) patients and MPTP-treated non-human primates, there is a profound neuronal degeneration of the intralaminar centromedian/parafascicular (CM/Pf) thalamic complex. Although this thalamic pathology has long been established in PD (and other neurodegenerative disorders), the impact of CM/Pf cell loss on the integrity of the thalamo-striatal glutamatergic system and its regulatory functions upon striatal neurons remain unknown. In the striatum, cholinergic interneurons (ChIs) are important constituents of the striatal microcircuitry and represent one of the main targets of CM/Pf-striatal projections. Using light and electron microscopy approaches, we have analyzed the potential impact of CM/Pf neuronal loss on the anatomy of the synaptic connections between thalamic terminals (vGluT2-positive) and ChIs neurons in the striatum of parkinsonian monkeys treated chronically with MPTP. The following conclusions can be drawn from our observations: (1) as reported in PD patients, and in our previous monkey study, CM/Pf neurons undergo profound degeneration in monkeys chronically treated with low doses of MPTP. (2) In the caudate (head and body) nucleus of parkinsonian monkeys, there is an increased density of ChIs. (3) Despite the robust loss of CM/Pf neurons, no significant change was found in the density of thalamostriatal (vGluT2-positive) terminals, and in the prevalence of vGluT2-positive terminals in contact with ChIs in parkinsonian monkeys. These findings provide new information about the state of thalamic innervation of the striatum in parkinsonian monkeys with CM/Pf degeneration, and bring up an additional level of intricacy to the consequences of thalamic pathology upon the functional microcircuitry of the thalamostriatal system in parkinsonism. Future studies are needed to assess the importance of CM/Pf neuronal loss, and its potential consequences on the neuroplastic changes induced in the synaptic organization of the thalamostriatal system, in the development of early cognitive impairments in PD.

Keywords: Non-human primates; Parafascicular; Parkinson’s disease; Striatum; Thalamostriatal; vGluT2.

Fig. 1 :

Cell loss in the intralaminar CM/Pf nuclei of MPTP-treated monkeys. Light microscope images of Nissl-stained coronal sections of the CM in a control (a) and a MPTP-treated monkey (b). Histograms comparing the total number of neurons (c), and the volume of the CM/Pf nuclei (d) between control (N=3) and MPTP-treated (N=2) monkeys. In MPTP-treated parkinsonian monkeys, there is a drastic decrease (45%) in the total neurons number, and in the volume (35%) of the CM/Pf complex compared with controls. Scale bar in a (applies to b) = 50μm

Fig. 2:

Light micrographs of striatal cholinergic interneurons in the caudate (a-d) and putamen (e, f) of control (a,c,e) and MPTP-treated monkeys (b,d,f). In both control and parkinsonian monkeys, these neurons have large cell bodies with different morphologies (ovoid, elongated or triangular) and prominent primary dendrites. The length and ramification of their immunostained dendritic trees vary, but usually the thick primary dendrites branch close to the cell body and give rise to dendritic trees that often extend over long distances in the striatal neuropil. Scale bar in b (applies to a) = 20μm and in f (applies to c-e) = 25μm

Fig. 3:

Stereological analysis of the number and density of cholinergic interneurons (ChAT-IR neurons) in the striatum of control and MPTP-treated parkinsonian monkeys. (a) Histograms comparing the change in the estimated total number (mean±SEM) of ChAT-IR neurons in the head and body of the caudate nucleus, and in the pre- and post-commissural putamen of control (N=3) and MPTP-treated parkinsonian monkeys (N=3). (b) Stereological estimated volume (Cavalieri’s analysis) of the caudate nucleus and putamen in control (N=3) and MPTP- treated (N=3) monkeys. The values in the histograms represent the mean±SEM per group. (c) Density of ChAT-IR neurons in the caudate nucleus and putamen of control (N=3) and MPTP- treated parkinsonian (N=3) monkeys. Note the increased density of cholinergic interneurons in the head and body of the caudate nucleus (*, t-test; head, p=0.043 and body, p=0.045)

Fig. 4:

Electron micrographs of vGluT2-positive (vGluT2+) terminals forming asymmetric synapses with dendrites (d) and dendritic spines (sp) in the caudate (b, c) and putamen (a, d) of control (a, b) and MPTP-treated-parkinsonian monkeys (c, d). In the same field, vGluT2- negative (vGluT2-) terminals form asymmetric synapses with dendritic spines. Scale bar in a (applies to b and c) and in d = 0.5μm

Fig. 5:

Histograms comparing the density of vGluT2-positive terminals in the caudate (a) and putamen (b) of control (N=3) and MPTP-treated (N=3) parkinsonian monkeys. This analysis revealed that the density of vGluT2+ terminals forming synapses onto dendrites and spines in both the head and body of the caudate, and the pre- and post-commissural putamen was not statistically different between control and MPTP-treated monkeys (t-test, p values: Caudate head axo-spine=0.442; axo-dendrite= 0.145. Caudate body axo-spine=0.0961; axo-dendrite=0.0474. Pre-commissural putamen axo-spine=0.066; axo-dendrite=0.035. Postcommissural putamen axo-spine=0.516; axo-dendrite=0.486

Fig. 6 :

Electron micrographs of ChAT-immunostained (peroxidase) dendrites and vGluT2-immunolabeled (silver-intensified gold particles) terminals in the putamen (a, b, d, e) and caudate (c, f) of control (a, b, c) and MPTP-treated monkeys (d, e, f). (a) A large-sized ChAT+ dendrite (diameter >1μm) forms an asymmetric synapse (white arrows) with a vGluT2-negative (vGluT2-) terminal. In the same field, some vGluT2-positive (vGluT2+) terminals form asymmetric synapses (black arrows) with non-labeled dendrites (d) and a dendritic spine (sp). (b) A vGluT2+ terminal forms an asymmetric synapse with a small-sized (diameter <0.5μm) ChAT+ dendrite (double white arrows). (c) Medium-sized (diameter between 0.5–1μm) ChAT-immunostained dendrite receiving an asymmetric synapse from a vGluT2-negative (white arrows). (d-f) Large- (d) and medium-sized (e, f) ChAT+ dendrites form asymmetric synapses with vGluT2- (white arrows in d and f) and vGluT2+ (double white arrows in e) terminals. Scale bars in a = 0.5μm, in b (applies to c) = 0.5 μm and in d = 0.5μm (applies to d and f)

Fig. 7:

Histograms comparing the percentages of ChAT-positive dendrites that form asymmetric synapses with vGluT2-positive terminals in the caudate and putamen of control and MPTP-treated monkeys. In both the caudate and putamen, no significant differences (Chi-square test) between control and parkinsonian monkeys were found in the proportions of ChAT-positive dendrites receiving vGluT2-positive terminals