Early structural and functional plasticity alterations in a susceptibility period of DYT1 dystonia mouse striatum

7 figures, 1 table and 1 additional file

Figures

Electrophysiological and synaptic properties of striatal SPNs.

(A) Superimposed traces showing voltage responses to both depolarizing (+600 pA) and hyperpolarizing (−200 pA) current steps in SPNs recorded from P26 Tor1a+/+ (black) and Tor1a+/Δgag (red) mice. The insets display single action potentials (amplitude: Tor1a+/+69.62 ± 1.14 mV, N = 11, n = 11; Tor1a+/Δgag66.65 ± 1.68 mV, N = 8, n = 11; Student’s t test p>0.05). (B) Summary plot of paired-pulse ratio values showing similar facilitation in both genotypes. Each data point represents mean ± SEM. P26 Tor1a+/+ mice N = 3, 25 ms: 1.24 ± 0.20, n = 5; 50 ms: 1.20 ± 0.12, n = 5, Student’s t test p<0.05; P26 Tor1a+/Δgag mice N = 3, 25 ms: 1.22 ± 0.05, n = 5; 50 ms: 1.19 ± 0.08, n = 5; Student’s t test p<0.05. Insets represent sample traces showing facilitation at ISI = 50 ms in both genotypes. (C) Representative sEPSCs recordings in PTX from SPNs of P26 Tor1a+/+ and Tor1a+/Δgag mice. HP: −70 mV. The summary plots show no significant difference between genotypes in sEPSCs frequency and amplitude (Student’s t test p>0.05). (D) Representative whole-cell recordings in PTX plus TTX of mEPSC from P26 Tor1a+/+ and Tor1a+/Δgag SPNs. HP: −70 mV. Plots show a significant difference in the amplitude of mEPSCs recorded from Tor1a+/Δgag mice compared to wild-types (Tor1a+/+, 7.45 ± 1.09, N = 9, n = 9; Tor1a+/Δgag, 10.11 ± 0.97, N = 8, n = 9; Student’s t test *p<0.05). (E) Representative recordings in MK-801 and CNQX of sIPSCs from P26 Tor1a+/+ and Tor1a+/Δgag SPNs. HP:+10 mV. The summary plots show no significant difference in sIPSC frequency and amplitude (Student’s t test p>0.05). (F) Representative traces of mIPSCs recorded in MK-801, CNQX and TTX. HP:+10 mV. The summary plots show no difference in frequency and amplitude between genotypes (Student’s t test p>0.05). Data are presented as mean ± SEM.

https://doi.org/10.7554/eLife.33331.002
Figure 1—source data 1

Electrophysiological and synaptic properties of striatal SPNs.

https://doi.org/10.7554/eLife.33331.003
Altered developmental profile of corticostriatal long-term synaptic plasticity expression in Tor1a+/Δgag mice.

(A) (Top) Developmental time-course of LTD expression in Tor1a+/+ mice. HFS protocol (arrow) induces LTD in SPNs recorded from Tor1a+/+ mice after P28 (59.63 ± 2.63% of control; N = 8, n = 8; paired Student’s t test p<0.05), but not from P15 to P27 (99.46 ± 4.65, N = 9, n = 10; paired Student’s t test p>0.05). (Bottom) Representative EPSP traces recorded before (pre) and 20 min after (post) HFS protocol delivery. (B) (Top) In Tor1a+/Δgag mice, HFS protocol fails to induce any LTD, irrespective of the postnatal age (P15-27, 96.85 ± 11.35% of control; N = 8, n = 12; P28-35, 100.29 ± 4.16% of control, N = 8, n = 12; paired Student’s t test p>0.05). (Bottom) Representative traces of EPSPs recorded pre- and post-HFS. (C) (Top) Time-course of corticostriatal LTP expression during postnatal development in Tor1a+/+ mice. HFS of corticostriatal afferents (arrow) induces LTP expression in Tor1a+/+ mice after P24 (148.80 ± 15.39% of control; N = 6, n = 10; paired Student’s t test p<0.05), but not at P15-23 (104.68 ± 8.99% of control; N = 6, n = 10; paired Student’s t test p>0.05). (Bottom) Sample EPSPs recorded pre- and post-HFS protocol in Tor1a+/+ mice. (D) (Top) SPNs recorded from Tor1a+/Δgag mice exhibit a premature LTP (P15-23, 174.68 ± 22.59% of control; N = 6, n = 10; P24-35, 172.35 ± 11.06% of control; N = 9, n = 10; paired Student’s t test p<0.05). (Bottom) EPSP traces recorded pre- and post-LTP induction. (E) Mean plot comparing LTD expression at different postnatal days in Tor1a+/+ and Tor1a+/Δgag SPNs. (Inset) Confocal imaging of two SPNs recorded from Tor1a+/Δgag slices filled with biocytin (green) and immunolabelled for ENK (red) and DARPP-32 (cyano), marker of SPNs. Both ENK-positive and ENK-negative biocytin-labeled SPNs showed lack of LTD (scale bar: 10 µm). (F) Mean plot comparing LTP expression at different postnatal days in Tor1a+/+ and Tor1a+/Δgag SPNs. Values are presented as mean ± SEM.

https://doi.org/10.7554/eLife.33331.004
Figure 2—source data 1

Altered developmental profile of corticostriatal long-term synaptic plasticity expression in Tor1a+/Δgag mice.

https://doi.org/10.7554/eLife.33331.005
Electrophysiological characterization of AMPAR and NMDAR currents at corticostriatal synapses of SPNs in both Tor1a+/+ and Tor1a+/Δgag mice.

(A) (Left) Representative EPSCs traces recorded at HP=+40 mV from SPNs of juvenile Tor1a+/+ and Tor1a+/Δgag mice. The NMDAR antagonist MK-801 isolates the AMPAR-mediated EPSC component (black trace), while the NMDAR-EPSC (grey trace) is obtained by digital subtraction of the AMPAR EPSC from the dual-component EPSC (red). (Right) Summary plot of NMDA/AMPA current ratio calculated in SPNs from P26 Tor1a+/+ and Tor1a+/Δgag mice. A significant decrease of NMDA/AMPA ratio was detected in P26 Tor1a+/Δgag mice, compared to Tor1a+/+ (Tor1a+/+, 2.92 ± 0.38, N = 3, n = 8; Tor1a+/Δgag, 1.81 ± 0.25, N = 3, n = 6; Student’s t test, p<0.05). (B) (Left) Representative EPSCs traces recorded at HP =+40 mV from SPNs of adult Tor1a+/+ and Tor1a+/Δgag mice. (Right) Summary plot of NMDA/AMPA current ratio showing no significant difference between genotypes (Tor1a+/+, 1.75 ± 0.15, N = 3, n = 7; Tor1a+/Δgag, 2.01 ± 0.12, N = 3, n = 7; Student’s t test, p>0.05). (C) AMPAR-mediated currents recorded at different HP in P26 Tor1a+/+ and Tor1a+/Δgag SPNs. The IV relationship shows a significant increase in the current recorded at more hyperpolarized range from P26 Tor1a+/Δgag SPNs (HP=−70 mV: two-way ANOVA, *p<0.01). (Left) Summary plot of rectification index values of P26 Tor1a+/+ and Tor1a+/Δgag SPNs (Tor1a+/+, 0.50 ± 0.07, n = 7; Tor1a+/Δgag, 0.43 ± 0.04, n = 8; Student’s t test p>0.05). (D) AMPAR-mediated currents recorded in the presence of the GluA2-lacking AMPAR antagonist NASPM at P26. HP =−70 mV; to-way ANOVA, *p<0.01). (Left) Summary plots of the rectification index measured at P26 (Tor1a+/+, 0.53 ± 0.04, n = 5, N = 6; Tor1a+/Δgag, 0.46 ± 0.03, n = 7; Student’s t test, p>0.05). (E) Normalized IV relationships of NMDAR-mediated currents show no difference between genotypes at P26 (two-way ANOVA, p>0.05). (F) Representative NMDA-mediated EPSCs recorded at HP =+40 mV from P26 SPNs. (G) Summary plots display rise and decay time of NMDA-EPSCs recorded at HP =+40 mV in SPNs from P26 Tor1a+/+ and Tor1a+/Δgag mice (rise time: Tor1a+/+, 7.78 ± 0.42, n = 9; Tor1a+/Δgag, 9.23 ± 1.37, n = 7; Student’s t test p>0.05; decay time: Tor1a+/+, 502.50 ± 20.06, n = 9; Tor1a+/Δgag, 422.10 ± 30.15, n = 7, Student’s t test, *p<0.05). Values are presented as mean ± SEM.

https://doi.org/10.7554/eLife.33331.006
Figure 3—source data 1

Electrophysiological characterization of AMPAR and NMDAR currents at corticostriatal synapses of SPNs in both Tor1a+/+ and Tor1a+/Δgag mice.

https://doi.org/10.7554/eLife.33331.007
Molecular analysis of the SPNs postsynaptic compartment in P26 and P60 Tor1a+/Δgag compared to age-matched wild-type mice.

WB analyses were performed on the post-synaptic TIF fraction in a minimum of three different animals per genotype. (A) WB analysis for GluN2A, GluN2B, PSD-95 and tubulin in P26 (left panel) and P60 (right panel) Tor1a+/Δgag and age-matched Tor1a+/+ mice. (C) WB analysis for GluA1, GluA1p845, GluA2 and tubulin in P26 (left panel) and P60 (right panel) Tor1a+/Δgag and age-matched Tor1a+/+ mice. (B,D) The histogram shows the quantification of protein levels following normalization on tubulin (P26 Tor1a+/Δgag compared to Tor1a+/+, GluA1: 142.8 ± 9.8%, n = 5, p<0.05; GluA1-p845: 200.9 ± 36.6%, n = 5, p<0.05; GluA2: 175.1 ± 16.6%, n = 5, p<0.05; GluN2A: 197.3 ± 34.0%, n = 5, p<0.05; P60 Tor1a+/Δgag GluA1: 90.0 ± 23.4%, n = 5, p>0.05; GluA1-p845: 77.7 ± 14.2%, n = 5, p>0.05; GluA2: 103.2 ± 16.2%, n = 5, p>0.05; GluN2A: 88.8 ± 18.0%, n = 5,p>0.05). All values are mean ± SEM expressed as % of Tor1a+/+ mice.

https://doi.org/10.7554/eLife.33331.008
Analysis of dendritic spines morphology in P26 and P60 Tor1a+/Δgag compared to age-matched Tor1a+/+mice.

(A) Histogram representing dendritic spine density in P26 Tor1a+/Δgag and Tor1a+/+ mice (Tor1a+/+, 10.25 ± 0.75 spines/10 μm, n = 10; Tor1a+/Δgag, 7.89 ± 0.70 spines/10 μm, n = 10; unpaired Student’s t test *p<0.05). (B,C) Histograms showing the quantification of dendritic spine size (B, spine length and head width) and dendritic spine type (C, mushroom, stubby, thin) in P26 Tor1a+/Δgag compared to Tor1a+/+ mice (dendritic spine width Tor1a+/+, 0.51 ± 0.02 μm, n = 10; Tor1a+/ Δgag, 0.64 ± 0.04 μm, n = 10, unpaired Student’s t-test *p<0.05; mushroom-type spines Tor1a+/+, 33.92 ± 2.32%, n = 10; Tor1a+/Δgag, 47.81 ± 5.79%, n = 10, unpaired Student’s t-test *p<0.05). (D) Representative images show dendrites of P26 Tor1a+/Δgag and Tor1a+/+ mice. (E) Histogram representing dendritic spine density in P60 Tor1a+/Δgag and Tor1a+/+ mice (Tor1a+/+, 9.94 ± 0.41 spines/10 μm, n = 10; Tor1a+/ Δgag, 10.76 ± 0.50 spines/10 μm, n = 10; unpaired Student’s t-test p>0.05). (F,G) Histograms showing the quantification of dendritic spine size (F, spine length and head width) and dendritic spine type (G, mushroom, stubby, thin) in P60 Tor1a+/Δgag, compared to Tor1a+/+ mice (spine width Tor1a+/+, 0.600 ± 0.012 μm, n = 10; Tor1a+/Δgag, 0.602 ± 0.027 μm, n = 10; p>0.05; mushroom-type spines Tor1a+/+, 61.40 ± 4.81%, n = 10; Tor1a+/Δgag, 47.92 ± 3.67%, n = 10; *p<0.05; thin spines Tor1a+/+, 19.04 ± 3.85%, n = 10; Tor1a+/ Δgag, 34.64 ± 4.16%, n = 10; *p<0.05; unpaired Student’s t-test). (H) Representative images show dendrites of P60 Tor1a+/Δgag and Tor1a+/+ mice. Data were collected in a minimum of three different animals per genotype.

https://doi.org/10.7554/eLife.33331.009
Figure 5—source data 1

Analysis of dendritic spines morphology in P26 Tor1a+/Δgag compared to age-matched Tor1a+/+ mice.

https://doi.org/10.7554/eLife.33331.010
Figure 5—source data 2

Analysis of dendritic spines morphology in P60 Tor1a+/Δgag compared to age-matched Tor1a+/+ mice.

https://doi.org/10.7554/eLife.33331.011
BDNF protein expression in the striatum of Tor1a+/+and Tor1a+/Δgag mice.

(A, B) Striatal BDNF protein expression in Tor1a+/+ and Tor1a+/Δgag mice at postnatal stages (P15, P26, P60). The graphs show the quantification of BDNF/proBDNF ratio at the various ages. Data are represented as mean ± SEM (Tor1a+/+ P15: 0.67 ± 0.12, N = 4; P26: 0.22 ± 0.08, N = 4; P60: 0.57 ± 0.14, N = 3; Tor1a+/Δgag P15: 0.54 ± 0.08, N = 4; P26: 0.18 ± 0.06, N = 4; P60: 0.32 ± 0.03, N = 4; one-way ANOVA, *p<0.05; **p<0.01). (C) (Left) Representative WB of proBDNF and BDNF protein levels relative to β-actin in striatal extracts (30 μg) derived from P26 Tor1a+/+ and Tor1a+/Δgag mice. (Right) The graphs show the quantitative analysis. The amount of proBDNF and BDNF was quantified relative to β-actin and normalized to wild-type mice. Data are represented as mean ± SEM (proBDNF Tor1a+/+ 1.00 ± 0.12, n = 10; Tor1a+/Δgag1.95 ± 0.29, n = 8; BDNF Tor1a+/+: 1.00 ± 0.28, n = 8, Tor1a+/Δgag2.19 ± 0.50, n = 8, Student’s t test: *p<0.05; **p<0.01). (D) Bdnf mRNA is upregulated in the cortex of Tor1a+/Δgag determined by qRT-PCR. The 2-ΔΔCt method was used to determine the relative expression, and all of the values are expressed relative to the levels of the wild-type mice as mean ± SEM (Tor1a+/+ 1.000 ± 0.084, n = 10; Tor1a+/Δgag1.399 ± 0.163, n = 8; Student’s t test: *p<0.05). (E) (Left) Representative Western blots of proBDNF and BDNF proteins relative to β-actin in striatal extracts (15 μg) derived from Tor1a+/+ and Tor1a+/Δgag adult mice. (Right) The graphs show the quantitative analysis. The amount of proBDNF and BDNF was quantified relative to β-actin and normalized to wild-type mice. Data are represented as mean ± SEM (proBDNF Tor1a+/+ 1.00 ± 0.19, n = 7, Tor1a+/Δgag1.15 ± 0.17, n = 7, p>0.05; BDNF Tor1a+/+: 1.00 ± 0.23 n = 7, Tor1a+/Δgag0.99 ± 0.25, n = 7, Student’s t test: p>0.05).

https://doi.org/10.7554/eLife.33331.012
Figure 6—source data 1

BDNF protein expression in the striatum of Tor1a+/+ and Tor1a+/Δgag mice.

https://doi.org/10.7554/eLife.33331.013
In vivo ANA-12 treatment rescues synaptic plasticity deficits in juvenile Tor1a+/Δgag mice.

(A) Time-course of corticostriatal LTD in juvenile Tor1a+/+ and Tor1a+/Δgag mice (P28-35): after in vivo treatment with the TrkB antagonist ANA-12, the HFS protocol (arrow) induces corticostriatal LTD expression in juvenile Tor1a+/Δgag mice (Tor1a+/+ P28-35, 65.31 ± 1.44% of control; N = 3, n = 12, p<0.05; Tor1a+/Δgag P28-35, 63.41 ± 4.39% of control; N = 3, n = 10; paired Student’s t test p<0.05). (Bottom) Representative EPSPs recorded before (pre) and 20 min after (post) HFS protocol. (B) Time-course of corticostriatal LTP after in vivo ANA-12 treatment: LTP displays a physiological amplitude in SPNs from in P24-35 Tor1a+/Δgag compared to wild-type littermates (Tor1a+/+ P24-35, 144.55 ± 2.67% of control; N = 3, n = 8; Tor1a+/Δgag P24-35, 148.11 ± 10.55% of control; N = 3, n = 9; Tor1a+/Δgag vs. Tor1a+/+ Student’s t test p>0.05). (Bottom) Sample traces recorded pre and post LTP induction. (C) AMPAR-mediated currents recorded from P26 SPNs at HP from −70 mV to + 40 mV after in vivo treatment of Tor1a+/+ and Tor1a+/Δgag mice with ANA-12. The treatment normalizes the current-voltage relationship in Tor1a+/Δgag neurons (HP=−70 mV: 2-way ANOVA p>0.05) and the rectification index (Tor1a+/+, 0.51 ± 0.03, N = 3, n = 3; Tor1a+/Δgag, 0.45 ± 0.04, N = 3, n = 5; Student’s t test p>0.05) (D) In vivo treatment with ANA-12 does not restore corticostriatal LTD in adult (P60) SPNs recorded from Tor1a+/Δgag mice (vehicle: 95.66 ± 9.09% of control, N = 3, n = 8; ANA-12: 98.75 ± 11% of control, N = 3, n = 4; paired Student’s t test p>0.05). (E) Slice pre-treatment with pirenzepine (100 nM) does not rescue LTD expression in P28-35 Tor1a+/Δgag SPNs (vehicle: 101.54 ± 1.07% of control, N = 3, n = 3; pirenzepine: 100.34 ± 8.96% of control; N = 3, n = 3; paired Student’s t test p>0.05). (Bottom) Superimposed traces of EPSPs recorded pre and 20 min post HFS delivery.

https://doi.org/10.7554/eLife.33331.014
Figure 7—source data 1

In vivo ANA-12 treatment rescues synaptic plasticity deficits in juvenile Tor1a+/Δgag mice.

https://doi.org/10.7554/eLife.33331.015

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Tor1aMGI:1353568Gene ID: 30931official full name: torsin family 1, member A (torsin A)
Strain, strain background (M. musculus)C57BL/6J miceCharles Rivercatalog number B6JSIFE10SZ - C57BL/6J SPF/VAF;
RRID:IMSR_JAX:000664
Genetic reagent (M. musculus)heterozygous knock-in Tor1a+/ΔgagGoodchild et al. (2005)-maintained on the C57BL/6J background
Antibodymonoclonal anti-PSD-95Neuromabclone (k28/43) - catalog number 75–028; RRID:AB_2292909dilution 1:2000 in I-Block
Antibodymonoclonal anti-GluN2BNeuromabclone 59/20 - catalog number 75–097; RRID:AB_10673405dilution 1:1000 in I-Block
Antibodypolyclonal anti-GluA1Merck Milliporecatalog number AB1504; RRID:AB_2113602dilution 1:1000 in I-Block
Antibodypolyclonal anti-phospho-GluA1 (Ser845)Merck Milliporecatalog number 04–1073; RRID:AB_1977219dilution 1:1000 in I-Block
Antibodypolyclonal anti-GluN2ASigma-Aldrichcatalog number M264 RRID:AB_260485dilution 1:1000 in I-Block
Antibodymonoclonal anti-GluA2Neuromabclone L21/32 - catalog number 75–002; RRID:AB_2232661dilution 1:1000 in I-Block
Antibodymonoclonal anti-α-tubulinSigma-Aldrichclone DM1A - catalog number T9026; RRID:AB_477593dilution 1:5000 in I-Block
Antibodygoat anti-DARPP-32R and D systemcatalog number AF6259; RRID:AB_10641854dilution 1:500 in I-Block
Antibodymouse anti-EnkephalinMilliporecatalog number MAB350; RRID:AB_2268028dilution 1:1000 in I-Block
Antibodymouse anti-β-actinSigma Aldrichcatalog number A5441; RRID:AB_476744dilution 1:20000 in I-Block
Commercial assay or kitClarity Western ECL SubstrateBioRad-reagent used to visualize protein bands with Chemidoc Imaging System
Commercial assay or kitECL reagentGEHealthcarecatalog number
GERPN2232
reagent used to visualize protein bands with membranes were exposed to film
Commercial assay or kitTRI-reagentSigma Aldrichcatalog number T9424reagent used to RNA extraction
Commercial assay or kitDNAase IInvitrogencatalog number AMPD1-1KTreagent used for elimination of DNA from RNA
Commercial assay or kitTranscriptor First Strand cDNA Synthesis KitRochecatalog number04379012001reagent used to reverse transcribe RNA
Commercial assay or kitExtract-N-Amp Tissue PCR KitSIGMAcatalog number XNAT2genotyping primers UP- AGT CTG TGG CTG GCT CTC C; Low- CCT CAG GCTGCT CAC AAC C
Chemical compound, drugANA-12Sigma-Aldrichcatalog number SML0209in vivo administration
Chemical compound, drugCNQX disodium saltTocriscatalog number 0190/10application in bath during electrophysiology analysis
Chemical compound, drug(+)-MK 801 maleateTocriscatalog number 0924/10application in bath during electrophysiology analysis
Chemical compound, drugTetrodotoxin citrate (TTX)Tocriscatalog number 1069/1application in bath during electrophysiology analysis
Chemical compound, drugPicrotoxinTocriscatalog number 1128/1application in bath during electrophysiology analysis
Chemical compound, drugBiocytinTocriscatalog number 3349/10electrodes filled with biocytin, versatile marker used for neuroanatomical investigations of neuron IHC
Chemical compound, drugNaspm trihydrochlorideTocriscatalog number 2766/10application in bath during electrophysiology analysis
Software, algorithmImageLabBioRad-software used for quantification of protein bands in western blotting experiments
Software, algorithmImageJ softwareNIH;Schneider et al. (2012)RRID:SCR_003070software used for the quantification of protein bands in western blotting and confocal laser scanning microscope
Software, algorithmClampFit 9pClampMolecular Devices; RRID:SCR_011323data analysis
Software, algorithmOrigin 8.0MicrocalRRID:SCR_002815data analysis
Software, algorithmPrism 5.3GraphPadRRID:SCR_002798data analysis

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  1. Marta Maltese
  2. Jennifer Stanic
  3. Annalisa Tassone
  4. Giuseppe Sciamanna
  5. Giulia Ponterio
  6. Valentina Vanni
  7. Giuseppina Martella
  8. Paola Imbriani
  9. Paola Bonsi
  10. Nicola Biagio Mercuri
  11. Fabrizio Gardoni
  12. Antonio Pisani
(2018)
Early structural and functional plasticity alterations in a susceptibility period of DYT1 dystonia mouse striatum
eLife 7:e33331.
https://doi.org/10.7554/eLife.33331