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Research ArticleNew Research, Disorders of the Nervous System

Enhanced GABAergic Inputs Contribute to Functional Alterations of Cholinergic Interneurons in the R6/2 Mouse Model of Huntington’s Disease

Sandra M. Holley, Prasad R. Joshi, Anna Parievsky, Laurie Galvan, Jane Y. Chen, Yvette E. Fisher, My N. Huynh, Carlos Cepeda and Michael S. Levine
eNeuro 24 February 2015, 2 (1) ENEURO.0008-14.2015; https://doi.org/10.1523/ENEURO.0008-14.2015
Sandra M. Holley
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Prasad R. Joshi
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Anna Parievsky
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Laurie Galvan
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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  • ORCID record for Laurie Galvan
Jane Y. Chen
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Yvette E. Fisher
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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My N. Huynh
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Carlos Cepeda
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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Michael S. Levine
Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, California 90095
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    Figure 1

    A, LCIs were stained with ChAT antibody in WT and symptomatic R6/2 mice. Less intense staining and reduced cell size were evident. B, Bar graphs show that the mean somatic cross-sectional area was significantly smaller in R6/2 LCIs. In addition, mean striatal area was significantly reduced in hemi-slices from R6/2 mice. Striatal volume assessed stereologically was also significantly reduced. The number of striatal ChAT+ neurons was similar but, because of reduced striatal volume, the mean density of ChAT+ neurons was significantly increased in R6/2 mice. *p < 0.05, **p < 0.01.

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    Figure 2

    A, Infrared differential interference contrast optics was used to visualize LCIs (arrows) in WT and symptomatic R6/2 mice. B-D, Representative traces of tonic firing of LCIs in the cell-attached configuration. In WTs, more LCIs fired action potentials regularly (B). In R6/2s, some LCIs fired regularly (C) but a greater number of irregular and bursting cells (D) were recorded. E, High degree of autocorrelation reflected regular firing patterns of LCIs from WT mice. F, Although firing of some LCIs from R6/2 animals was highly autocorrelated, the autocorrelation coefficient (peak amplitude or coefficient value of the first lag interval) was lower than in LCIs from WTs. G, Low or no autocorrelations were observed in irregular firing and bursting LCIs, particularly from R6/2s. H, Spontaneous firing rates in LCIs from WT and R6/2 mice were almost identical. I, In contrast, the autocorrelation coefficient, a measure of firing regularity, was significantly decreased in LCIs from R6/2 mice. J, Scatter plots showing the correlation between firing frequency and coefficient of variation. There was a very good correlation between both in LCIs from WT mice. In contrast, this correlation was markedly reduced in cells from R6/2 mice. *p < 0.05.

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    Figure 3

    A, B, Representative traces of spontaneous IPSCs in LCIs from R6/2 and WT mice at 65 d using Cs-Meth (A) or CsCl (B) internal solutions. In both cases, spontaneous IPSCs occurred more frequently in LCIs from R6/2 mice. C, D, Amplitude−frequency histograms of sIPSCs using Cs-Meth (C) and CsCl (D) internal solutions. In most amplitude bins, there was an increase in frequency in LCIs from R6/2 mice. The insets show the mean sIPSC frequency. Regardless of the internal solution used, there was a significant increase in LCIs frequency in R6/2s. E, F, Cumulative probability histograms of interevent intervals in WT and R6/2 mice at 65 d using Cs-Meth (E) and CsCl (F) internal solutions. A significant increase in short interevent intervals was observed in LCIs from R6/2 mice. G, H, Average frequency of mIPSCs in LCIs from WT and R6/2 mice was similar using Cs-Meth (G) or CsCl (H) internal solutions. The percent reduction in IPSC frequency after TTX was greater in LCIs from R6/2 mice and this difference was statistically significant for the CsCl internal solution (H, right). *p < 0.05, ***p < 0.001.

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    Figure 4

    A, Representative traces of eIPSCs recorded with Cs-Meth internal solution and holding the membrane at +10 mV in LCIs from WT and symptomatic R6/2 mice. eIPSCs had multiple components and amplitude of the earliest peak was measured (arrows). B, Graph shows the input−output relationship. Current response amplitudes were significantly increased at intensities of 0.04 mA and higher. C, eIPSCs recorded in LCIs at −70 mV with high Cl− internal solution in WT and R6/2 mice at 65 d. D, Similar to recordings with Cs-Meth, there was a significant increase in IPSC amplitudes in cells from R6/2 mice. *p < 0.05, **p < 0.01.

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    Figure 5

    A, Confocal image (z-stack) of a LCI recorded and filled with biocytin (red). The LCI is surrounded by ChR2-EYFP terminals from SOM interneurons (green). Arrows indicate SOM-expressing interneurons. B, IPSCs evoked in LCIs by optogenetic stimulation of SOM-expressing interneurons. Responses were significantly larger in LCIs from R6/2 mice. These responses were completely blocked by BIC. C, Graphs show significant increases in amplitude and charge in LCIs from R6/2 mice compared to WTs. D, LCI surrounded by ChR2-EYFP terminals from PV interneurons. Arrow indicates a PV-expressing interneuron. E, IPSCs evoked in LCIs by optogenetic stimulation of PV-expressing interneurons were smaller than those evoked by SOM terminal stimulation and were not different between WT and R6/2 animals. Responses were completely blocked by BIC. F, Graphs show the lack of significant differences in peak amplitude and charge in LCIs from WT and R6/2 mice. *p < 0.05.

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    Figure 6

    A, Representative traces of eEPSCs in LCIs recorded at −70 mV using Cs-Meth internal solution. The peak amplitude of the response was similar in LCIs from WT and R6/2 mice. B, Graph shows the input−output relationship of eEPSCs. No statistical differences in amplitude were observed. C, Striatal LCI (red) surrounded by EYFP terminals in a R6/2 mouse injected with ChR2 in the Cm/Pf nuclear complex. D, Optogenetic stimulation of thalamic afferents induced similar AMPA (top) and NMDA (bottom) receptor-mediated currents in LCIs from WT and R6/2 mice. E, Graphs show that the amplitude and charge of AMPA (top) and NMDA (bottom) responses were not significantly different.

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    Figure 7

    A, Representative traces of responses induced by bath application of increasing concentrations of GABA (10, 100, and 1000 μM) in large striatal neurons (presumably LCIs) from WT and symptomatic R6/2 mice. B, Graph shows no significant differences in peak current density (peak current divided by cell capacitance) of GABA responses. C, D, AMPA- and NMDA-induced peak current densities also were similar in R6/2 and WT mice.

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    Table 1:

    Passive membrane properties of LCIs in R6/2 mice

    Cm (pF)Rm (MΩ)Tau (ms)
    Presymptomatic
    WT (n = 10)89.5 ± 7151 ± 221.2 ± 0.2
    R6/2 (n = 12)81.4 ± 5177 ± 280.9 ± 0.1
    Symptomatic
    WT (n = 31)89.6 ± 4198 ± 151.3 ± 0.1
    R6/2 (n = 26)80.0 ± 3*335 ± 23***1.2 ± 0.1
    • ↵* p < 0.05,

    • ↵*** p < 0.001.

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Enhanced GABAergic Inputs Contribute to Functional Alterations of Cholinergic Interneurons in the R6/2 Mouse Model of Huntington’s Disease
Sandra M. Holley, Prasad R. Joshi, Anna Parievsky, Laurie Galvan, Jane Y. Chen, Yvette E. Fisher, My N. Huynh, Carlos Cepeda, Michael S. Levine
eNeuro 24 February 2015, 2 (1) ENEURO.0008-14.2015; DOI: 10.1523/ENEURO.0008-14.2015

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Enhanced GABAergic Inputs Contribute to Functional Alterations of Cholinergic Interneurons in the R6/2 Mouse Model of Huntington’s Disease
Sandra M. Holley, Prasad R. Joshi, Anna Parievsky, Laurie Galvan, Jane Y. Chen, Yvette E. Fisher, My N. Huynh, Carlos Cepeda, Michael S. Levine
eNeuro 24 February 2015, 2 (1) ENEURO.0008-14.2015; DOI: 10.1523/ENEURO.0008-14.2015
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Keywords

  • cholinergic interneurons
  • GABA
  • Huntington’s disease
  • optogenetics
  • R6/2 mouse model
  • striatum

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