Loss of Homeostasis in the Direct Pathway in a Mouse Model of Asymptomatic Parkinson's Disease

Abstract

The characteristic slowness of movement in Parkinson's disease relates to an imbalance in the activity of striatal medium spiny neurons (MSNs) of the direct (dMSNs) and indirect (iMSNs) pathways. However, it is still unclear whether this imbalance emerges during the asymptomatic phase of the disease or if it correlates with symptom severity. Here, we have used in vivo juxtacellular recordings and transgenic mice showing MSN-type-specific expression of fluorescent proteins to examine striatal imbalance after lesioning dopaminergic neurons of the substantia nigra. Multivariate clustering analysis of behavioral data discriminated 2 groups of dopamine-lesioned mice: asymptomatic (42 ± 7% dopaminergic neuron loss) and symptomatic (85 ± 5% cell loss). Contrary to the view that both pathways have similar gain in control conditions, dMSNs respond more intensely than iMSNs to cortical inputs in control animals. Importantly, asymptomatic mice show significant functional disconnection of dMSNs from motor cortex without changes in iMSN connectivity. Moreover, not only the gain but also the timing of the pathways is altered in symptomatic parkinsonism, where iMSNs fire significantly more and earlier than dMSNs. Therefore, cortical drive to dMSNs decreases after partial nigrostriatal lesions producing no behavioral impairment, but additional alterations in the gain and timing of iMSNs characterize symptomatic rodent parkinsonism.

Significance statement: Prevailing models of Parkinson's disease state that motor symptoms arise from an imbalance in the activity of medium spiny neurons (MSNs) from the direct (dMSNs) and indirect (iMSNs) pathways. Therefore, it is hypothesized that symptom severity and the magnitude of this imbalanced activity are correlated. Using a mouse model of Parkinson's disease, we found that behaviorally undetectable nigrostriatal lesions induced a significant disconnection of dMSNs from the motor cortex. In contrast, iMSNs show an increased connectivity with the motor cortex, but only after a severe dopaminergic lesion associated with an evident parkinsonian syndrome. Overall, our data suggest that the lack of symptoms after a partial dopaminergic lesion is not due to compensatory mechanisms maintaining the activity of both striatal pathways balanced.

Keywords: 6-OHDA; functional connectivity; in vivo electrophysiology; medium spiny neurons; striatum.

Figure 1.

In vivo juxtacellular recording allows to identify dMSNs and iMSNs in transgenic mice. a, Schematic representation of electrode positioning for stimulation (cortex and thalamus) and recording (striatum). b, Example of cell firing modulation during current injection to label recoded neurons. c, Confocal image from striatum of a double-transgenic mouse showing two neurobiotin-filled neurons (blue). The upper left neuron (1) expressing EGFP but not Tomato was classified as an iMSN. The opposite colocalization pattern in cell 2 led to classification as dMSN. Right, High-magnification confocal images from red (Tomato), green (EGFP), and blue (neurobiotin) channels for each labeled neuron. Scale bars, 10 μm. Cx, Cortex; Th, thalamus; St, striatum; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.

Figure 2.

Characterization of BAC Drd1a-Tomato, Drd2-EGFP, and Drd1a-tomato/Drd2-EGFP mice. a, Top, Confocal image showing DAPI staining (blue channel) of striatum from a Drd1a-tomato/Drd2-EGFP double-transgenic mice. Bottom, Pie chart showing the relative abundance of Tomato⁺ (dMSNs) and EGFP⁺ (iMSNs) cells, expressed as the percentage of total DAPI⁺ neurons (small condensed nuclei from glia were excluded). Data are from three mice. B, Top, Representative examples of high-magnification confocal images from striatum showing no colocalization of striatal interneuron markers with endogenous labeled cells resulting from the Drd1a-tdTomato (top) and Drd2-EGFP (bottom) transgenes. Bottom, Table summarizing quantitative data for each interneurons marker. C, Neurobiotin-filled neurons (green) after juxtacelullar in vivo recording in Drd1a-tdTomato mice. Confocal images from representative Tomato⁺ MSN (left), Tomato⁻ MSN (middle; inset depicts dendritic spines at high magnification), and an aspiny Tomato⁻ interneuron (right). Representative electrophysiological traces correspond to three trials of cortical stimulation (arrows) or spontaneous activity (interneuron).

Figure 3.

Higher corticostriatal gain of the direct pathway in control transgenic mice. a, Threshold current required to induce responses to cortical stimulation in dMSNs (n = 11) and iMSNs (n = 14) recorded from sham Drd1a-tdTomato (red and green circles, respectively) and Drd1a-tdTomato/Drd2-EGFP mice (red and green empty circles, respectively). *p = 0.001, Mann–Whitney test. b, Intensity–response curves to cortical stimulation of dMSNs (n = 11; red) and iMSNs (n = 14; green) in control mice. *p < 0.05, Bonferroni post hoc test after significant interaction, repeated-measures ANOVA, p < 0.0001. Data are mean ± SEM. c, Intensity–response curves to cortical stimulation of MSNs segregated by transgenic mouse line. dMSNs (n = 9) and putative iMSNs (n = 10) recorded from Drd1a-tdTomato mice (red and green squares and lines, respectively) exhibit similar gain to dMSNs (n = 2) and iMSNs (n = 4) recorded from Drd1a-tdTomato/Drd2-EGFP double-transgenic mice (light red lines and green empty squares, respectively). *p < 0.05, Bonferroni post hoc test after significant interaction, repeated-measures ANOVA on data from Drd1a-tdTomato mice, p < 0.0001. Data are mean ± SEM. d, Representative traces showing the responses to cortical stimulation of a dMSN (left) and an iMSN (right) at different phases (peak in gray and trough in black) of LFP slow oscillations. Red bars indicate stimulus onset and artifact. Scale bar, 10 ms. e, Probability of evoking a spike by cortical stimulation does not depend on the phase of the LFP slow oscillation in which the stimulation occurs. This likelihood was measured at current intensities evoking spikes in ∼50% of the trials for dMSNs and iMSNs (nonsignificant main effects and interaction, two-way ANOVA). f, Current intensity evoking spikes in ∼50% of the trials (I_50%) was significantly smaller for dMSNs in control animals (t test, p = 0.021).

Figure 4.

Unbiased separation of asymptomatic from symptomatic 6-OHDA-treated mice. a, Dendrogram obtained by unsupervised multivariate clustering analysis of behavioral parameters shown in b defines two clusters. Sy, Symptomatic; As, asymptomatic; Sh, sham. b, Behavioral alterations in symptomatic mice. Left, Voluntary use of affected forelimb in the cylinder test (p < 0.0001, one-way ANOVA followed by Bonferroni post hoc test, *p < 0.05 vs either sham or asymptomatic mice). Middle, Ipsilateral/total rotations in an open field (p = 0.012, one-way ANOVA followed by Bonferroni post hoc test, *p < 0.05 vs either sham or asymptomatic mice). Right, Incoordination in a motor skill task, the accelerated rotarod (p = 0.03, repeated-measures ANOVA interaction, Bonferroni post hoc test, *p < 0.05). Data are mean ± SEM. c, Confocal reconstructions of substantia nigra illustrating representative lesions of TH⁺ neurons (green) in Drd1a-tdTomato animals (red: dMSNs terminals in substantia nigra reticulata). d, Representative coronal sections of the striatum of sham (left), asymptomatic (middle), and symptomatic (right) mice immunostained with antibodies against TH. White lines show the limits of the quadrants used for regional quantification of TH immunoreactivity. Two-way ANOVA showed a significant effect of group and nonsignificant effects of region and interaction (see text for details). e, Percentage of TH⁺ neuron loss in sham, asymptomatic, and symptomatic mice relative to the nonlesioned side. *p < 0.05 versus sham and symptomatic mice, Tukey post hoc after significant one-way ANOVA: F_{(2, 35)} = 67.21 p < 0.0001). f, Linear regression analysis of forelimb use asymmetry as function of percentage TH⁺ cell loss (p < 0.0006). Each point represents one animal.

Figure 5.

Attenuation of direct pathway functional connectivity after an asymptomatic partial dopaminergic lesion. a, Schematic representation of the position of all recorded MSNs in sham, asymptomatic, and symptomatic mice on a plate of Paxinos and Franklin mouse brain atlas (2001). Numbers of neurons recorded per experimental group were as follows: for dMSNs, n = 9 in sham, 12 in asymptomatic and n = 7 in symptomatic mice; for iMSNs, n = 11 in sham, 8 in asymptomatic, and 20 in symptomatic mice. b, Light microscopic images of coronal sections showing the position of the bipolar electrodes in a representative animal. c, d, Intensity–response curves to motor cortex stimulation for dMSNs (c) and iMSNs (d) recorded from sham (circles), asymptomatic (squares), and symptomatic (triangles) mice. For dMSNs, repeated-measures ANOVA significant interaction, p < 0.0001, followed by Bonferroni post hoc tests, *p < 0.05 asymptomatic versus sham, #p < 0.05 symptomatic versus asymptomatic. For iMSNs, repeated-measures ANOVA significant interaction, p = 0.03, followed by Bonferroni post hoc tests, *p < 0.05 symptomatic versus both sham and asymptomatic. e–g, Threshold (e), gain at 500 μA (f), and latency (g) differences across groups (two-way ANOVA, significant interactions followed by LSD post hoc tests, *p < 0.05 versus all other groups within cell type). Data are mean ± SEM.

Figure 6.

Severe dopaminergic neuron depletion causes thalamostriatal functional disconnection of dMSNs and hyperresponsiveness of iMSNs. a, Representative traces showing responses to thalamic stimulation (arrow) in representative dMSNs (left) and iMSNs (right) from sham (Sh) and symptomatic (Sy) groups. Scale bar, 10 ms. b, Threshold current required to evoke responses to thalamic stimulation in dMSNs and iMSNs from sham, asymptomatic, and symptomatic mice. Data are mean ± SEM (*p < 0.05 vs sham, LSD post hoc tests after significant two-way ANOVA interaction, p < 0.0001).

Figure 7.

Attenuation of dMSN functional connectivity correlates better with dopaminergic cell loss and behavioral impairment than iMSN hyperresponsiveness. Linear regressions of threshold current (a, d), spike response to motor cortex stimulation at 500 μA (b, e), and response peak latency at 500 μA (c, f) on the percentage of remaining TH⁺ cells (a–c) or forelimb use asymmetry index (d–f) for dMSNs (red) and iMSNs (green). Plotted lines correspond to linear regressions remaining significant after Bonferroni correction for multiple comparisons. Each dot corresponds to a recorded neuron.