In vivo measures of nigrostriatal neuronal response to unilateral MPTP treatment

Abstract

A single unilateral intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into non-human primates causes injury to the nigrostriatal pathway including nigral cell bodies, axons and striatal terminal fields. In this model, motor parkinsonism correlates well with the loss of nigral dopaminergic cell bodies but only correlates with in vitro measures of nigrostriatal terminal fields when nigral cell loss does not exceed 50%. The goals of this study are to determine the relationship of motor parkinsonism with the degree of injury to nigrostriatal axons, as reflected by in vitro fiber length density measures, and compare in vivo with in vitro measures of striatal terminal fields. We determined axon integrity by measuring fiber length density with tyrosine hydroxylase (TH) immunohistology and dopamine transporter (DAT) density with DAT immunohistology. We then calculated the terminal arbor size and compared these measures with previously published data of quantified in vivo positron emission tomography (PET) measures of presynaptic dopaminergic neurons, autoradiographic measures of DAT and vesicular monoamine transporter type 2 (VMAT2), striatal dopamine, nigral cell counts, and parkinsonian motor ratings in the same animals. Our data demonstrate that in vivo and in vitro measures of striatal terminal fields correlate with each other regardless of the method of measurement. PET-based in vivo striatal measures accurately reflect in vitro measures of DAT and VMAT2. Terminal arbor size and other terminal field measures correlate with nigral TH immunoreactive (TH-ir) cell counts only when nigral TH-ir cell loss does not exceed 50%. Fiber length density was the only striatal measure that linearly correlated with motor ratings (Spearman: r=-0.81, p<0.001, n=16).

Keywords: MPTP; Nigrostriatal; PET; Striatal terminal field.

Figure 1

In vivo PET data had a tight correlation with corresponding in vitro measures. CFT BP_ND tightly correlated with DAT Bmax (A); striatal DTBZ BP_ND correlated strongly with VMAT2 Bmax (B). The value for each monkey was expressed as the ratio of the injected side to the control side.

Figure 2

Representative coronal sections of post-commissural striatum showing dopamine transporter (DAT) immunoreactivity of the unlesioned side (A) and lesioned side (B) under high power (x100 objective) from a monkey given MPTP 0.25 mg/kg. The arrows indicated the DAT varicosities. Scale bar 100 μm (A and B).

Figure 3

Relationship between DAT density and other striatal terminal field measures including CFT BP_ND, DTBZ BP_ND and FD K_occ, striatal dopamine, DAT Bmax, and VMAT2 Bmax (A-F). Note, that all correlations remained significant when including only a single point in the lower left part of the graphs (Spearman: r = 0.89, p < 0.001; r = 0.91, p < 0.0001; r = 0.94, p < 0.0001; r = 0.92, p < 0.0005; r = 0.89, p < 0.001; r = 0.86, p = 0.002, respectively; n = 10).

Figure 4

Relationship between DAT terminal arbor size and nigral cell count and other striatal terminal field measures including CFT BP_ND, DTBZ BP_ND and FD K_occ, striatal dopamine, DAT Bmax, and VMAT2 Bmax (A–G). In figure A, the ratio of residual TH-ir cells in the substantia nigra pars compacta (SNpc) are labeled as following: control (open triangles), 16–50% (solid triangles), 51–100% (open circles). Note, that all correlations between DAT terminal arbor size and above striatal terminal field measures remained significant when including only a single point in the lower left part of the graphs (Spearman: r = 0.87, p = 0.001; r = 0.89, p = 0.001; r = 0.9, p = 0.001, r = 0.89, p < 0.0001; r = 0.98, p < 0.0001; r = 0.85, p < 0.0001; r = 0.92, p < 0.0005; respectively; n = 10).

Figure 4

Relationship between DAT terminal arbor size and nigral cell count and other striatal terminal field measures including CFT BP_ND, DTBZ BP_ND and FD K_occ, striatal dopamine, DAT Bmax, and VMAT2 Bmax (A–G). In figure A, the ratio of residual TH-ir cells in the substantia nigra pars compacta (SNpc) are labeled as following: control (open triangles), 16–50% (solid triangles), 51–100% (open circles). Note, that all correlations between DAT terminal arbor size and above striatal terminal field measures remained significant when including only a single point in the lower left part of the graphs (Spearman: r = 0.87, p = 0.001; r = 0.89, p = 0.001; r = 0.9, p = 0.001, r = 0.89, p < 0.0001; r = 0.98, p < 0.0001; r = 0.85, p < 0.0001; r = 0.92, p < 0.0005; respectively; n = 10).

Figure 5

Representative coronal sections of post-commissural striatum showing TH immunoreactivity of the unlesioned side (A) and lesioned side (B) under high power (x100 objective) from a monkey given MPTP 0.25 mg/kg. The arrows indicated the TH immunoreactive fibers. Scale bar = 100 μm (A and B).

Figure 6

The fiber length density had a dichotomous relationship with in vitro or in vivo striatal terminal field measures DAT Bmax (A), VMAT2 Bmax (B) and dopamine (C), CFT BP_ND (D), DTBZ BP_ND (E) and FD K_occ (F). Animal 11 is the only statistical outlier identified by SPSS defined as values below or above 1.5* interquartile range. The value for each monkey was expressed as the ratio of the injected side to the control side.

Figure 7

Relationship between fiber length density and DAT density (A) and residual TH-ir neurons in SNpc (B). Fiber length density linearly correlated with motor ratings (C). Note, in figure B, the ratio of residual TH-ir cells in SNpc are labeled as following: control (open triangles), 16–50% (solid triangles), 51–100% (open circles). The value for each monkey was expressed as the ratio of the injected side to the control side.

Figure 8

The structures (regions of interest, ROI) analyzed in the right (R) and left (L) hemispheres are shown: dorsal caudate (LC, RC) and dorsal putamen (LP, RP).