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Research ArticleNew Research, Disorders of the Nervous System

Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin

Corwin R. Butler, Jeffery A. Boychuk and Bret N. Smith
eNeuro 25 September 2017, 4 (5) ENEURO.0134-17.2017; DOI: https://doi.org/10.1523/ENEURO.0134-17.2017
Corwin R. Butler
1Department of Physiology College of Medicine, University of Kentucky, Lexington, KY 40536
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  • ORCID record for Corwin R. Butler
Jeffery A. Boychuk
1Department of Physiology College of Medicine, University of Kentucky, Lexington, KY 40536
2Epilepsy Center, University of Kentucky, Lexington, KY 40536
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Bret N. Smith
1Department of Physiology College of Medicine, University of Kentucky, Lexington, KY 40536
2Epilepsy Center, University of Kentucky, Lexington, KY 40536
3Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY 40536
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  • Figure 1.
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    Figure 1.

    Rapamycin treatment reduces, but does not normalize, the increased activity of hilar inhibitory interneuron 8–12 weeks after CCI injury. A, Representative traces showing spontaneous action potential firing from three different treatment groups: control (i.e., sham and contralateral neurons), ipsilateral to CCI injury + vehicle (CCI Ipsi), and ipsilateral to CCI injury + 3 mg/kg rapamycin (CCI + Rapa Ipsi). B, Mean spontaneous action potential firing in sham, CCI Contra, CCI Ipsi, CCI + Rapa Contra, and CCI + Rapa Ipsi groups. Error bars indicate SEM; *p < 0.05 compared to sham and contralateral hemispheres; #p < 0.05 for CCI Ipsi versus CCI + Rapa Ipsi.

  • Figure 2.
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    Figure 2.

    Rapamycin treatment reduces, but does not normalize, the increase in sEPSC frequency in hilar inhibitory interneurons 8–12 weeks after CCI injury. A, Representative traces showing sEPSCs in eGFP+ neurons from three different treatment groups: control (i.e., sham and contralateral neurons), ipsilateral to CCI injury + vehicle (CCI Ipsi), and ipsilateral to CCI injury + 3 mg/kg rapamycin (CCI + Rapa Ipsi). Expanded sections of the trace under the black line are indicated by arrows. B, Mean sEPSC frequency, amplitude, and whole-cell capacitance in sham, CCI Contra, CCI Ipsi, CCI + Rapa Contra, and CCI + Rapa Ipsi groups. Error bars indicate SEM; *p < 0.05 compared to sham and contralateral hemispheres; #p < 0.05 for CCI Ipsi versus CCI + Rapa Ipsi; †p < 0.05 compared to sham, CCI Contra, and CCI Ipsi.

  • Figure 3.
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    Figure 3.

    Rapamycin treatment abrogates the injury-induced increase in synaptic input from DGCs to hilar inhibitory interneurons 8–12 weeks after CCI injury. A, Representative eEPSC responses in eGFP+ interneurons to glutamate photostimulation applied to DGCs from three different treatment groups: control (i.e., sham and contralateral hemispheres), CCI Ipsi, and CCI + Rapa Ipsi. Bars above traces indicate glutamate photostimulation period. The relative position of the recorded hilar interneuron (green) and numbered stimulation sites in the dentate gyrus are shown on the stereotyped drawing above each set of traces; numbers for traces correspond to stimulation site numbers in the drawing. B, Individual and mean percentage of effective stimulation sites in control, CCI Ipsi, and CCI + Rapa Ipsi groups. C, The synaptic response of a eGFP+ hilar interneuron after photostimulation of DGCs (top trace) is blocked in the presence of TTX (2 µM; bottom trace), additional stimulation sweeps in gray. Error bars indicate SEM; *p < 0.05 compared to control. The number of stimulation sites, cells, and animals from separate treatment groups are presented in Table 3.

  • Figure 4.
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    Figure 4.

    Rapamycin fails to suppress the injury-induced increase in eEPSC responses from CA3 pyramidal cells to eGFP+ hilar interneurons 8–12 weeks after injury. A, Representative traces showing responses in eGFP+ hilar interneurons to glutamate photostimulation applied to CA3 pyramids from three different treatment groups: control, CCI Ipsi, and CCI + Rapa Ipsi. Bars above traces indicate glutamate photostimulation period. The relative position of the recorded hilar interneuron (green) and numbered stimulation sites in the CA3 pyramidal cell layer are shown on the stereotyped drawing above each set of traces. B, Individual and mean percentage of effective stimulation sites in control, CCI Ipsi, and CCI + Rapa Ipsi groups. Error bars indicate SEM; *p < 0.05 compared to control. The number of stimulation sites, cells, and animals from separate treatment groups are presented in Table 3.

  • Figure 5.
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    Figure 5.

    eEPSC responses in DGCs after glutamate photostimulation of CA3 pyramidal cells 8–12 weeks after injury. A, Individual and mean percentage of effective stimulation sites. Responses in DGCs from three different treatment groups: sham, CCI Contra, and CCI Ipsi in normal ACSF. B, Individual and mean percentage of effective stimulation sites for DGCs in the presence of 30 μM bicuculline from three different treatment groups: control (i.e., sham and contralateral hemispheres), CCI Contra and Ipsi, and CCI + Rapa Contra and Ipsi. The number of stimulation sites, cells, and animals from separate treatment groups are presented in Tables 4, 5.

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    Figure 6.

    Diagramatic representation of effects of rapamycin treatment on dentate gyrus circuitry after focal brain injury. A, Normal circuit in dentate gyrus. The projection of a DGC onto a CA3 pyramidal cell is shown (solid black arrow). GABAergic hilar inhibitory interneurons are also present, but are not robustly innervated by DGCs or CA3 pyramidal cells (dashed arrows). B, Functional synaptic input to surviving hilar interneurons arising from activity in both DGCs and CA3 pyramidal cells (red arrows) are increased ipsilateral to CCI injury, as are connections between DGCs (red oval; Hunt et al., 2010, 2011; Butler et al., 2015). C, mTOR inhibition after CCI injury reduces reorganization of functional DGC connections with surviving hilar inhibitory interneurons, but aberrant excitatory connection arising from CA3 pyramidal cell activity is sustained. This cartoon does not discriminate between mono- and polysynaptic connections. Line thickness is used as a surrogate marker for the percentage effective stimulation sites as assessed by glutamate photolysis here and in Hunt et al. (2010).

Tables

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    Table 1.

    Statistical table

    Outcome measureData structureType of testPower
    a. ΔsEPSC frequencyNominal data, non-normal distributionKruskal Wallis0.05
    b. Weight changeNormal distributionOne-way ANOVA0.99
    c. Action potential firing rateNormal distributionOne-way ANOVA0.98
    d. sEPSC frequencyNormal distributionOne-way ANOVA0.8
    e. sEPSC amplitudeNormal distributionOne-way ANOVA0.06
    f. eGFP+ neuron whole-cell capacitanceNormal distributionOne-way ANOVA0.72
    g. DGC RMPNormal distributionOne-way ANOVA0.05
    h. eGFP+ neuron RMPNormal distributionOne-way ANOVA0.06
    i. CA3 neuron RMPNormal distributionOne-way ANOVA0.11
    j. Direct photostimulation evoked AP’s in DGCsNormal distributionOne-way ANOVA0.05
    k. Direct photostimulation evoked AP’s in eGFP+ neuronsNormal distributionOne-way ANOVA0.05
    l. Direct photostimulation evoked AP’s in CA3 neuronsNormal distributionOne-way ANOVA0.05
    m. % effective stimulation sites to DG photostimulation per eGFP+ neuron, controlsNominal data, non-normal distributionKruskal Wallis0.05
    n. % effective stimulation sites to DG photostimulation per eGFP+ neuronNominal data, non-normal distributionKruskal Wallis0.68
    o. % effective stimulation sites to CA3 photostimulation per eGFP+ neuron, controlsNominal data, non-normal distributionKruskal Wallis0.05
    p. % effective stimulation sites to CA3 photostimulation per eGFP+ neuronNominal data, non-normal distributionKruskal Wallis0.4
    q. % effective stimulation sites to CA3 photostimulation per DGC nACSFNominal data, non-normal distributionKruskal Wallis0.05
    r. % responsive sites to CA3 photostimulation per DGC ACSF w/30 μM BicNominal data, non-normal distributionKruskal Wallis0.12
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    Table 2.

    RMP and direct photoactivation measures for DGCs, eGFP hilar neurons, and CA3 pyramidal neurons

    Cell typeGroupNumber of cellsNumber of animalsDirect photoactivation (number of APs)RMP (mV)
    DGCControl66325.31 ± 0.43−67.11 ± 0.97
    DGCCCI ipsi2476.04 ± 0.80−66.73 ± 2.18
    DGCCCI + Rapa ipsi1475.54 ± 0.92−65.63 ± 2.03
    eGFP+ neuronControl20153.88 ± 0.57−54.72 ± 1.95
    eGFP+ neuronCCI ipsi1063.44 ± 0.62−52.33 ± 1.63
    eGFP+ neuronCCI + Rapa ipsi873.78 ± 0.72−60.64 ± 3.46
    CA3 neuronControl16113.79 ± 0.35−55.76 ± 1.78
    CA3 neuronCCI ipsi744.03 ± 0.76−49.53 ± 2.77
    CA3 neuronCCI + Rapa ipsi633.53 ± 0.61−48.39 ± 4.23
    • No significant differences were detected within any cell type.

    • View popup
    Table 3.

    Responses of eGFP+ hilar inhibitory interneurons to photostimulation of DGCs and CA3 neurons

    GroupResponsive DG stimulation sitesResponsive CA3 stimulation sitesNumber of cellsNumber of animalsNet eEPSC frequency DG stimulatio nNet eEPSC frequency CA3 stimulation
    Sham1/340/18950.13 ± 0.080.09 ± 0.10
    CCI contra3/361/14650.31 ± 0.130.28 ± 0.14
    CCI ipsi22/55*10/25*1060.90 ± 0.19*1.31 ± 0.43*
    CCI + Rapa contra1/261/10550.04 ± 0.100.02 ± 0.38
    CCI + Rapa ipsi7/408/18*870.33 ± 0.111.27 ± 0.44*
    • Significant differences from control indicated with an asterisk.

    • View popup
    Table 4.

    Responses of DGCs to photostimulation of CA3 neurons in normal ACSF

    GroupNumber of effective stimulation sitesNumber of cellsNumber of animals
    Sham1/67144
    CCI contra1/46106
    CCI ipsi3/57125
    • View popup
    Table 5.

    Responses of DGCs from control groups to photostimulation of CA3 neurons in ACSF containing 30 μM bicuculline

    GroupNumber of effective stimulation sitesNumber of cellsNumber of animalsNet eEPSC frequency
    Sham2/571140.22 ± 0.06
    CCI contra0/40960.13 ± 0.06
    CCI ipsi3/491260.08 ± 0.04
    CCI + Rapa contra0/491070.05 ± 0.04
    CCI + Rapa ipsi1/781470.01 ± 0.01
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Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin
Corwin R. Butler, Jeffery A. Boychuk, Bret N. Smith
eNeuro 25 September 2017, 4 (5) ENEURO.0134-17.2017; DOI: 10.1523/ENEURO.0134-17.2017

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Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin
Corwin R. Butler, Jeffery A. Boychuk, Bret N. Smith
eNeuro 25 September 2017, 4 (5) ENEURO.0134-17.2017; DOI: 10.1523/ENEURO.0134-17.2017
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Keywords

  • dentate gyrus
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