Cell type-specific plasticity of striatal projection neurons in parkinsonism and L-DOPA-induced dyskinesia

Abstract

The striatum is widely viewed as the fulcrum of pathophysiology in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). In these disease states, the balance in activity of striatal direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) is disrupted, leading to aberrant action selection. However, it is unclear whether countervailing mechanisms are engaged in these states. Here we report that iSPN intrinsic excitability and excitatory corticostriatal synaptic connectivity were lower in PD models than normal; L-DOPA treatment restored these properties. Conversely, dSPN intrinsic excitability was elevated in tissue from PD models and suppressed in LID models. Although the synaptic connectivity of dSPNs did not change in PD models, it fell with L-DOPA treatment. In neither case, however, was the strength of corticostriatal connections globally scaled. Thus, SPNs manifested homeostatic adaptations in intrinsic excitability and in the number but not strength of excitatory corticostriatal synapses.

Figure 1. iSPN intrinsic and dendritic excitability was reduced in iSPNs from parkinsonian mice and restored by high-dose L-DOPA

Intrinsic excitability was assessed by somatic current injection. (a) Sample traces are provided with vertical and horizontal scale bars denoting 25mV and 200msec and (b) current-response curves (shaded regions illustrate S.E.M.). Intrinsic excitability was decreased in iSPNs from parkinsonian mice and restored by high-dose L-DOPA (control n=13; parkinsonian n=13; dyskinetic n=12; two-way ANOVA). Dendritic excitability was assessed by somatically driven theta burst stimulation.(c) Cartoon (left) and iSPN 2PLSM image provided (top right) with a high magnification 2PLSM image depicting dendritic segment with a line representing the region assessed for Ca²⁺ fluorescence (bottom); scale bars denote 10μm with an eccentricity denoting 80μm from the soma in the low magnification image.(d) Sample fluorescent traces provided with vertical and horizontal scale bars denoting 0.5 ΔF/F₀ and 200msec, respectively. (e) Dendritic excitability was decreased in shaft (top; control median: 1.54 n=17; parkinsonian median: 1.25 n=19; dsykinetic median: 1.28 n=24) and spine heads (bottom; control median: 1.27 n=14; parkinsonian median: 1.05 n=23;dyskinetic median: 1.04 n=26) of parkinsonian mice and restored by high-dose L-DOPA; Kruskal-Wallis. Sample traces provided below 2PLSM images; *p<0.05; response areas refer to (S*ΔF/F₀).

Figure 2. dSPN intrinsic excitability was increased in parkinsonian mice and partially restored by high-dose L-DOPA with a decrease in dendritic excitability

(a) Sample traces are provided with vertical and horizontal scale bars denoting 25 mV and 200 msec and (b) current-response curves (shaded regions illustrate S.E.M.). Intrinsic excitability was increased in dSPNs from parkinsonian mice and partially restored by high-dose L-DOPA (control n=24; parkinsonian n=20; dyskinetic n=21; two-way ANOVA). Dendritic excitability was assessed by somatically driven theta burst stimulation. (c) Cartoon (left) and iSPN 2PLSM image provided (top right) with a high magnification 2PLSM image depicting dendritic segment with a line representing the region assessed for Ca²⁺ fluorescence (bottom); scale bars denote 10μm with an eccentricity denoting 80μm from the soma in the low magnification image. (d) Sample fluorescent traces provided with vertical and horizontal scale bars denoting 0.5 ΔF/F₀ and 200msec, respectively.(e) dSPN dendritic excitability was decreased in the dendritic shaft (top; control median: 1.52 n=26; parkinsonian median: 1.01 n=22; dsykinetic median: 0.94 n=16) and spine heads(bottom; control median: 1.06 n=21; parkinsonian median: 1.35 n=24; dyskinetic median: 0.98 n=17) of high dose L-DOPA treated mice; Kruskal-Wallis. Sample traces provided below 2PLSM images; *p<0.05; response areas refer to (S*ΔF/F₀).

Figure 3. iSPN dendritic arborization is reduced in parkinsonian mice and is not restored by high-dose L-DOPA

iSPN neurons were patched and filled with Alexa 568, 2PLSM images obtained, and reconstructed in 3D. (a) Representative reconstructions of iSPNs from control (black), parkinsonian (blue), and dyskinetic (red)mice with eccentricities every 10μm from the soma. (b) Sholl analysis of reconstructed iSPNs; solid lines represent the mean with shaded regions illustrating S.E.M. (c) Total dendritic length was reduced in both parkinsonian (median: 1,627μm) and dyskinetic mice(median: 1,622μm) compared to control(median: 2,333μm). (d) Number of branch points was unchanged by treatment conditions (controlmedian: 12.5, parkinsonian median: 13.0, dyskineticmedian: 16.0). (e) Number of primary dendrites was reduced in both parkinsonian (median: 5.0) and dyskinetic (median: 4.0) mice(controlmedian: 6.0). Control n=14, parkinsonian n=11, and dyskineticn=12; *p<0.05; Kruskal-Wallis.

Figure 4. dSPN dendritic arborization is reduced in parkinsonian mice and is not restored by high-dose L-DOPA

dSPN neurons were patched and filled with Alexa 568, 2PLSM images obtained, and reconstructed in 3D. (a) Representative reconstructions of iSPNs from control (black), parkinsonian (blue), and dyskinetic (red) mice with eccentricities every 10μm from the soma. (b) Sholl analysis of reconstructed dSPNs; solid lines represent the mean with shaded regions illustrating S.E.M. (c) Total dendritic length was reduced in parkinsonian (median: 3,388μm) and dyskinetic mice(median: 2,774μm) compared to control(median: 4,464μm). (d) Number of branch points was decreased in both parkinsonian (median: 25) and dyskinetic (median: 22.5) mice compared to control (median: 36). (e) Number of primary dendrites was decreased in parkinsonian (median: 6) and dyskinetic mice (median: 6) compared to control (median: 8). Control n=13, parkinsonian n=13, and dyskinetic n=11; *p<0.05; Kruskal-Wallis.

Figure 5. iSPN axospinous synapses were reduced in parkinsonian mice and restored by high-dose L-DOPA

(a) 2PLSM image of an iSPN (left) from a control mouse with circles delineating minimal proximal (40μm) and distal (80μm) distances from the soma. (b) Representative 2PLSM images of proximal dendritic segments from control (black), parkinsonian (blue), and dyskinetic (red)mice. (c) iSPN proximal spine density was decreased in parkinsonian mice (median: 0.83 spines/μm) and restored in dyskinetic mice (median: 1.11 spines/μm; controlmedian: 1.30 spines/μm). (d) iSPN distal spine density was also decreased in parkinsonian mic e(median: 0.64 spines/μm) and restored in dyskinetic mice (median 1.07 spines/μm; controlmedian: 1.22 spines/μm). (e) Total number of spines were estimated based on spine density and dendritic anatomy data (Fig. 3); despite a restoration in spine density with dyskinesia, total number of spines remain below controllevels due to reduced dendritic surface area. Total spine estimates for controlmedian: 2,701 spines, parkinsonian median: 1,171 spines, and dyskineticmedian: 1,622 spines; control n=16, parkinsonian n=17, dyskineticn=15; *p<0.05; scale bar denotes 10μm; Kruskal-Wallis.

Figure 6. dSPN axospinous synapses were reduced in L-DOPA treated mice

(a) 2PLSM image of a dSPN (left) from a control mouse with circles delineating minimal proximal (40μm) and distal (80μm) distances from the soma. (b) Representative 2PLSM images of proximal dendritic segments from control, parkinsonian, and dyskinetic mice. dSPN proximal (c) and distal (d) spine density was decreased in L-DOPA treated mice compared to controls. (e) Total number of spines were estimated based on spine density and dendritic anatomy data (Fig. 4); dyskinetic mice (median: 2,096) had fewer spines than control (median: 4,715) and parkinsonian mice (median: 3,426); control n=16, parkinsonian n=17, dyskinetic n=15; *p<0.05;scale bar denotes 10μm; Kruskal-Wallis.

Figure 7. Cortical axospinous synapses in iSPNs were lost in parkinsonian mice and rewired with high-dose L-DOPA

sCRACm was used to assess corticostriatal circuit function in iSPNs from control (black), parkinsonian (blue), and dyskinetic (red)mice. (a) Sample 2PLSM image depicting a patched iSPN (left) and dendritic region (right) assessed for functional corticostriatal circuitry; scale bars denote 10μm. Blue circles depict region stimulated by focal blue laser excitation of ChR2. (b) The cartoon segment of dendrite shows both cortical and thalamic input onto spines and shaft. Only axospinous synapses were tested for cortical circuitry; to the right of the experimental depiction are sample traces showing responses from a corticostriatal synapse, a presumed thalamostriatal synapse, and a second corticostriatal synapse; vertical scale bar denotes 5pA and horizontal scale bar 50msec. Both proximal (c) and distal (d) corticostriatal axospinous synapses were lost in iSPNs from parkinsonian mice and restored by dykinesiogenic L-DOPA (Proximal medians: control 76.92% n=15, parkinsonian 50.00% n=15, dyskinetic73.33% n=15; distal medians: control 76.19% n=15, parkinsonian 47.37% n=13, dyskinetic 71.43% n=15). (e) Individual corticostriatal optogenetically evoked EPSCs (oEPSC) were measured, representative traces are shown to the right of experimental diagram; vertical scale bar denotes 5pA and horizontal scale bar 50msec. oEPSC amplitude was increased in iSPNs from parkinsonian mice and restored in dyskinetic mice. Full-field stimulation using a blue LED was used to assess overall circuit strength. (f) Overall circuit strength was unchanged despite losing axospinous synapses parkinsonian mice sample traces provided (g) with vertical scale and horizontal scale bars denoting 100pA and 100msec, respectively. (h) Input-output curve demonstrating sublinearity of recorded data (solid line) and estimated linear summation (dashed line) from control cells. Box and whisker plots of minimal LED stimulation (i) (control median: 86.67pA n=15, parkinsonian median: 237.7pA n=15, dyskinetic median: 170.3pA n=15) and maximal stimulation (j) (control median: 508.0pA n=15, parkinsonian median: 660.6pA n=15, dyskinetic median: 508.4pA n=15) demonstrate the lack of change in overall corticostriatal circuit strength.*p<0.05; Kruskal-Wallis.

Figure 8. Cortical axospinous synapses in dSPNs were lost in dyskinetic mice

sCRACm was used to assess corticostriatal circuit function in iSPNs from control (black), parkinsonian (blue), and dyskinetic (red) mice. (a) Sample 2PLSM image depicting a patched dSPN (left) and dendritic region (right) assessed for functional corticostriatal circuitry; scale bars denote 10μm. Blue circles depict region stimulated by focal blue laser excitation of ChR2. (b) The cartoon segment of dendrite shows both cortical and thalamic input onto spines and shaft. Only axospinous synapses were tested for cortical circuitry; to the right of the experimental depiction are sample traces showing responses from a corticostriatal synapse, a presumed thalamostriatal synapse, and a second corticostriatal synapse; vertical scale bar denotes 5pA and horizontal scale bar 50msec. Both proximal (c) and distal (d) corticostriatal axospinous synapses were lost in dSPNs from dyskinetic mice (Proximal medians: control 75.00% n=13, parkinsonian 71.82% n=14, and dyskinetic 48.53% n=18; distal medians: control 76.47% n=13, parkinsonian 66.97% n=14, and dyskinetic 51.47% n=14). (e) Individual corticostriatal optogenetically evoked EPSCs (oEPSC) were measured, representative traces are shown to the right of experimental diagram; vertical scale bar denotes 5pA and horizontal scale bar 50msec. EPSC amplitude data was separated into low and high EPSC amplitude groups by the median; in parkinsonian and dyskinetic mice there were fewer high amplitude synapses than in controls. Full-field stimulation using a blue LED was used to assess overall circuit strength. (f) Overall circuit strength was reduced in parkinsonian and dyskinetic mice; sample traces provided (g) with vertical scale and horizontal scale bars denoting 100pA and 100msec, respectively. (h) Input-output curve demonstrating sublinearity of recorded data (solid line) and estimated linear summation (dashed line) from control cells. Box and whisker plots of minimal LED stimulation (i) (control median: 885.4pA n=13, parkinsonian median: 435.8pA n=15, and dyskinetic median: 505.6pA n=11) and maximal stimulation (j) (control median: 1198.0pA n=13, parkinsonian median: 857.8pA n=15, and dyskinetic median: 817.5pA n=11) demonstrate the decreased corticostriatal circuit strength in parkinsonian and dyskinetic mice.*p<0.05; Kruskal-Wallis and Mann-Whitney tests.

Figure 9. Extrasynaptic NMDA/AMPA ratios in iSPNs were increased in parkinsonian mice

(a) 2PLSM image depicting an iSPN (left) with circles delineating proximal (40μm from soma) and distal (80μm from soma) dendritic segments and a high magnification image of a dendritic segment (middle) with yellow asterisks indicating regions targeted with uncaging laser; scale bars denote 10μm. To the right is a cartoon depicting regions exposed to glutamate due to uncaging (yellow circles). (b) Sample traces depicting uncaged glutamate-induced responses used to assess NMDA/AMPA ratios (middle) and data from control (black), parkinsonian (blue), and dyskinetic mice (red); vertical and horizontal scale bars denote 10pA and 100msec, respectively. (c) NMDA/AMPA ratios in iSPNs were increased in parkinsonian mice (control median: 0.96 n=58 spines; parkinsonian median: 1.67 n=44 spines; dyskinetic median: 1.07 n=43 spines). (d) Cartoon depicting full-field stimulation of an iSPN exciting all cortical inputs. (e) Synaptic NMDA/AMPA ratios (determined using cortical optogenetic stimulation)with sample traces on the left and data showing no change on the right; vertical and horizontal scale bars denote 100pA and 100msec, respectively (control median: 0.37 n=16; parkinsonian median: 0.54 n=17; dyskinetic median: 0.54 n=16). (f) To verify findings with uncaged glutamate, full-field optogenetic stimulation was again tested in the presence of TBOA to engage both synaptic and extrasynaptic receptors. Sample traces provided on the left; again, NMDA/AMPA ratio was increased in parkinsonian mice, verifying that changes were due to extrasynaptic receptors (control median: 0.35 n=13; parkinsonian median: 1.14 n=16). *p<0.05; Kruskal-Wallis and Mann-Whitney tests.

Figure 10. Extrasynaptic NMDA/AMPA ratios in dSPNs were increased in dyskinetic mice

(a) 2PLSM image depicting a dSPN (left) with circles delineating proximal (40μm from soma) and distal (80μm from soma) dendritic segments and a high magnification image of a dendritic segment (middle) with yellow asterisks indicating regions targeted with uncaging laser; scale bars denote 10μm. To the right is a cartoon depicting regions exposed to glutamate due to uncaging (yellow circles). (b) Sample traces depicting uncaged glutamate-induced responses used to assess NMDA/AMPA ratios (middle) and data from control (black), parkinsonian (blue), and dyskinetic mice (red); vertical and horizontal scale bars denote 10pA and 100msec. (c) NMDA/AMPA ratios in dSPNs were increased in dyskinetic mice (control median: 0.81 n=61 spines; parkinsonian median: 0.58 n=46 spines; dyskinetic median: 1.04 n=55 spines). (d) Cartoon depicting full-field stimulation with a dSPN exciting all cortical inputs. (e) Synaptic NMDA/AMPA ratios (determined using cortical optogenetic stimulation) with sample traces on the left and data showing no change on the right; vertical and horizontal scale bars denote 100pA and 100msec, respectively (control median: 0.43 n=13; parkinsonian median: 0.61 n=13; dyskinetic median: 0.52 n=14). (f) To verify findings with uncaged glutamate, full-field optogenetic stimulation was again tested in the presence of TBOA to engage both synaptic and extrasynaptic receptors. Sample traces provided on the left; again, NMDA/AMPA ratio was increased in parkinsonian mice, verifying that changes were due to extra synaptic receptors (control median: 0.37 n=17; parkinsonian median: 1.16 n=15). *p<0.05; Kruskal-Wallis and Mann-Whitney tests.