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Articles, Development/Plasticity/Repair

Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy

Xu Maisano, Elizabeth Litvina, Stephanie Tagliatela, Gloster B. Aaron, Laura B. Grabel and Janice R. Naegele
Journal of Neuroscience 4 January 2012, 32 (1) 46-61; https://doi.org/10.1523/JNEUROSCI.2683-11.2012
Xu Maisano
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Elizabeth Litvina
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Stephanie Tagliatela
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Gloster B. Aaron
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Laura B. Grabel
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Janice R. Naegele
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  • Figure 1.
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    Figure 1.

    Time course of neuronal differentiation during derivation of neural progenitors from ES cells. A–F, Neuronal differentiation in vitro of the ES cells viewed with Nomarski optics (A–C) or epifluorescence (D–F). The neural stem cell gene sox1 drives GFP expression in this cell line, indicating commitment to neural lineages by day 9. Distinctive neural rosettes form during the first replating step (arrows, B,E). G–I, Day 14 neural progenitors express nestin (G), MAP2 (H), and neuron-specific β-3 tubulin (I). Cultures also contain mitotically active cells expressing the cell cycle antigen phospho-histone H3 (G). J, Day 14 neural progenitors in the cultures also express the ventral forebrain marker Mash1 (16.5 ± 1.4%, n = 4). K, Neuronal subsets coexpress MAP2 and calbindin (5.0 ± 0.7%, n = 3) and display unipolar, bipolar, or multipolar dendritic morphology (M–O′). L, Additional neurons coexpress MAP2 and calretinin (7.6 ± 0.9%, n = 3) and exhibit a range of bipolar or multipolar dendritic morphologies (P–R′). S, S′, Some calretinin-expressing neurons also exhibit immature dendritic spines. Scale bars: A–F, 100 μm; G–J, 100 μm; K, L, S, S′, 20 μm; M–R′, 10 μm.

  • Figure 2.
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    Figure 2.

    mRNA expression profiles for Sox1-GFP ESNPs. A, The expression of transcription factor mRNA increases from day 2 to day14, except for Oct 4, a transcription factor expressed by pluripotent stem cells, which decreases during this period. B, RT-qPCR analyses of MAP2, Dlx2, GAD2, and Pax6 mRNA expression at different stages of differentiation were compared with expression profiles of pluripotent ES cells on day 2. Map2 mRNA increased by 143-fold by day 14, Pax6 mRNA increased 53-fold, and Dlx2 mRNA increased 103-fold. Strikingly, GAD2 mRNA increased 540-fold. Significance values were calculated by ANOVA; *p < 0.1 between days 2 and 14; **p < 0.05 and a significant difference comparing day 2 with day 14; #p < 0.1, significant difference comparing day 11 with day 14; ##p < 0.05, significant difference in expression between days 11 and 14. White, Day 2; black, day 9; light gray, day 11; dark gray, day 14.

  • Figure 3.
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    Figure 3.

    FISH analyses show that transplanted ESNPs in the dentate gyrus differentiate into GABAergic interneurons. A–J, Approximately 90% of the transplanted cells differentiated into neurons, ascertained by immunofluorescent staining for NeuN (87.4 ± 2.6% NeuN+ cells; n = 6). B–J, Magnified views of boxed regions in A show NeuN-expressing transplanted cells. K–O, GAD1/2 mRNA expression was detected in nearly half of the transplanted cells (48.9 ± 3.0%; n = 6), as assessed by FISH. Arrowheads indicate RFP+ cells expressing GAD1/2 mRNA. Scale bars: A, 50 μm; B–J, L–O, 20 μm; K, 200 μm.

  • Figure 4.
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    Figure 4.

    ESNPs differentiate into neurochemically distinct interneuron types after transplantation into the hilus of the dentate gyrus. A–D, Confocal images of RFP+ ESNP-derived neurons in the dentate gyrus. Approximately 5% of transplanted cells express the calcium-binding protein parvalbumin (4.6 ± 0.7%; n = 4; arrowheads). E–H, Approximately 8% express calbindin indicated by arrowheads (7.5 ± 0.7%; n = 5). Rare cells coexpress calbindin and ctip2 (arrows), suggesting that some ESNPs differentiate into excitatory, glutamatergic neurons. I–K, Nearly 10% (9.3 ± 0.4%, n = 4) of the cells in hilar transplants expressed calretinin (arrows). L, Quantification of the neurochemical phenotypes ascertained for ESNP-derived neurons. Scale bars: A–K, 20 μm.

  • Figure 5.
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    Figure 5.

    Transplanted cells displace mossy fibers in the hilus but fail to suppress MFS in the inner molecular layer. Dual immunohistochemical staining for transplanted ESNP-derived neurons in RFP (red) and mossy fibers expressing ZnT-3 (green) was used to evaluate MFS in the hilus and iml of the dentate gyrus in TLE mice. A, MFS is not observed in the granule cell layer or iml in normal mice without prior seizure experience, B, TLE mice show moderate MFS by 6 weeks after SE after control stereotaxic injections of cell-culture medium devoid of ESNPs. C, More than 3 months after SE, much more extensive MFS in the iml is typical (five of eight TLE cases showed extensive MFS in iml). In the hilus, however, mossy fibers rarely overlapped with ESNP-derived neuronal arbors, and the mossy fiber projections were often displaced in the vicinity of the graft cores (n = 8). D, H, Typically, RFP+ axons from ESNP transplants invaded the iml and overlapped extensively with mossy fibers (n = 5 of 8). E–G, I–K, L, M–O, Only three of eight mice with transplants exhibited reduced MFS (L), despite extensive innervation of the iml by the transplanted neurons in all cases (E–G, I–K, M–O). hi, Hilus; gcl, granule cell layer. E–G, I–K, M–O are magnified views of boxed regions in D, H, and L, respectively. Arrowheads in E–G, I–K, M–O represent the outer limits of ZnT3 staining. Scale bars: A–C, 50 μm; D, H, L, 200 μm; E–G, I–K, M–O, 100 μm.

  • Figure 6.
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    Figure 6.

    Locations of ESNP-derived neurons that were electrophysiologically characterized. Whole-cell current- and voltage-clamp recordings were performed 55–81 d after transplantation. A–C, Transplanted cells were selected in the red fluorescent channel, and the recordings were made under DIC optics. The arrows point to an RFP+ neuron in contact with a patch pipette. C, Overlay of the red fluorescent view and DIC view after obtaining a gigaohm seal of the RFP+ neuron. D, Diagram showing locations of the neurons that were recorded and filled with biocytin. Transplanted neurons were located in the dentate gyrus, with one exception in the stratum oriens of CA1 (cell 19). The morphologies of 15 neurons were examined after biocytin staining. Scale bars: A–C, 50 μm.

  • Figure 7.
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    Figure 7.

    The majority of neurons displayed electrophysiological characteristics of endogenous hippocampal GABAergic interneurons. Top, Neurons are classified based on the presence (types I and II) or absence of Ih current (types III, IV, and V). Types I and II are distinguished by AP drop and AP amplitude. Types III and IV are distinguished by the maximum number of APs in response to depolarizing currents. The type V category contains one cell with a large AP half-width recorded from the granule cell layer. A, Type I cells exhibit Ih currents (arrow) and prominent AP drops. B, Type II cells exhibit Ih currents (arrow) and significantly smaller APs compared with other types. This type also exhibits trains of fast APs. C, Type III neurons resemble type I cells, but without Ih currents. D, Type IV neurons exhibit small AP drops and fast-spiking APs without Ih currents. E, Type V neurons are found in the granule cell layer of the dentate gyrus and resemble granule neurons based on AP half-width and small fast afterhyperpolarizations. F, A GIN neuron recorded from the hilus exhibits firing patterns similar to those from ESNP-derived transplants. Calibration is as indicated.

  • Figure 8.
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    Figure 8.

    Electrophysiology of transplanted neurons into the host brain circuitry. A, The top trace shows a 2 s sample from a voltage-clamp recording at −70 mV demonstrating spontaneous EPSPs in a transplanted neuron. In the bottom traces, for comparison, spontaneous EPSPs were recorded from an endogenous somatostatin-expressing interneuron in a GIN mouse. B, Enlarged views of the individual EPSCs from neurons in A are presented in the highlighted region. Calibration is as indicated.

  • Figure 9.
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    Figure 9.

    Transplanted cells exhibit morphologies and firing patterns resembling endogenous GABAergic interneurons. A–D, An example of one of the transplanted neurons (cell 32) located in the granule cell layer that exhibited typical basket cell morphology (A) and a fast-spiking firing pattern (D). Boxed regions in A are shown in B and C. Primary dendrites emerged from the soma and bifurcated in the hilus before traveling obliquely to the granule cell layer (B). This cell has sparse dendritic spines (C). E, F, Another transplanted neuron (cell 8; E) located near the upper blade of the dentate gyrus has a dendritic morphology resembling HIPP cells. Electrophysiological classification of this cell was type I with Ih current (F). G, I, A neuron (cell 15) located near the upper blade of the granule cell layer forms dendrites running parallel with the dentate gyrus. H, The primary dendrites are aspiny, but dendritic spines are present ∼200 μm from the soma (arrows). This cell is classified electrophysiologically as type IV, with bursting rebound action potentials. Scale bars: A, E, H, 50 μm; B, C, 25 μm; G, 100 μm.

Tables

  • Figures
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    Table 1.

    Primers used for PCR analyses

    GeneSequence
    Oct45′-GGCGTTCTCTTTGGAAAGGTGTTC-3′
    5′-CTCGAACCACATCCTTCTCT-3′
    Nkx2.15′-AACAGCGGCCATGCAGCAGCAC-3′
    5′-CCATGTTCTTGCTCACGTCC-3′
    Mash15′-CTCGTCCTCTCCGGAACTGATG-3′
    5′-CGACAGGACGCCCGCCTGAAA G-3′
    Ngn25′-AGCACCGCCTCCGTGAAGAG-3′
    5′-ACGGTGCAGCGCATCAAGAA-3′
    Pax75′-CCAAGATTCTTTGCCGCTAC-3′
    5′-CAGGATGCCGTCGATGCTGT-3′
    Pax65′-GCAAACACATCTGGATAATGGG-3′
    5′-TGTGAGTAAAATTCTGGGCAGG-3′
    Emx 15′-AGCGACGTTCCCCAGGACGGGCTGC-3′
    5′-CTGAGGTCACTTGGTC-3′
    GAPDH5′-ACCACAGTCCATGCCATCAC-3′
    5′-TCCACCACCCTGTTGCTGTA-3′
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    Table 2.

    Analysis of ESNP transplants

    Case numberSurvival (days)Graft locationCells characterized by electrophysiologyImmunohistochemistry
    SE 011510-0670DG and hilusNot doneYes
    SE 011810-0291DG and OMLNot doneYes
    SE 011810-0491OMLNot doneYes
    SE 112309-0472DG and hilus31 (DG), 32 (DG)Not done
    SE 041410-0181Hilus25, 26, 27 (H), 28Not done
    SE 041410-0655OML1 (H), 2, 3 (OML)Not done
    SE 041410-0776OMLNo accessNot done
    SE 041410-1262CA39, 10Not done
    SE 041910-0257Hilus5 (H), 6, 7 (H), 8 (H)Yes
    SE 041910-0371Hilus and CA118 (H), 19 (CA1), 20, 21Yes
    SE 041910-0675OML22, 23 (OML), 24 (OML)Not done
    SE 041910-0880OML29 (OML), 30Not done
    SE 041410-0956OML4 (OML)Not done
    SE 041910-1067OML11, 12 (OML), 13, 14Yes
    SE 041910-1268Hilus15 (H), 16, 17Yes
    • One mouse (SE 041910-04) was excluded from the study because of a tumor. Animal identification was assigned as the date of pilocarpine-induced status epilepticus. DG, Dentate gyrus granule cell layer; OML, outer molecular layer; H, hilus.

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    Table 3.

    Categorization of transplanted ESNP-derived neurons based on electrophysiological properties

    ParameterType I (10)Type II (4)Type III (11)Type IV (4)Type V (1)GIN (5)p < 0.05
    Membrane potential (mV)−48.50 ± 2.4−52.3 ± 3.1−49.4 ± 2.5−58.0 ± 2.7−68−44.7 ± 2.6
    Delay to spike (ms)19.7 ± 4.215.2 ± 6.118.0 ± 3.912.7 ± 4.344.216.6 ± 4.2
    Spike threshold (mV)−38.7 ± 1.7−39.0 ± 4.0−40.8 ± 2.3−40.8 ± 4.3−22.5−41.8 ± 1.2
    AP amplitude (mV)67.1 ± 2.929 ± 2.663 ± 3.962 ± 6.231.359.5 ± 6.0I–II,
    II–III,
    II–IV, II–GIN
    AP half-width (ms)0.82 ± 0.061.13 ± 0.431.04 ± 0.10.78 ± 0.092.10.96 ± 0.1
    AP drop (mV)6.0 ± 1.4−2.3 ± 0.47.1 ± 1.63.2 ± 1.44.50.8 ± 1.2I–II,
    II–III
    Maximum frequency (Hz)46.5 ± 3.250.7 ± 12.350.4 ± 8.155.2 ± 8.718.469.9 ± 17.6
    Maximum number of APs19.4 ± 2.424.5 ± 6.09.5 ± 1.025.8 ± 5.0820.3 ± 6.7I–III,
    II–III,
    III–IV
    Adaptation2.0 ± 0.22.2–0.32.3 ± 0.32.1 ± 0.22.31.6 ± 0.3
    AHP1 amplitude (mV)−18.1 ± 2.6−7.9 ± 1.6−15.0 ± 2.0−16.6 ± 1.9−9.3−11.6 ± 1.8
    AHP2 amplitude (mV)−4.1 ± 0.9−6.1 ± 2.3−0.7 ± 0.7−3.3 ± 1.20−1.6 ± 1.6II–III
    Voltage sag ratio1.47 ± 0.081.63 ± 0.261.01 ± 0.011.03 ± 0.0311.17 ± 0.07I–IV,
    I–III,
    II–III,
    II–IV, II–GIN
    Input resistance (MΩ)717 ± 98450 ± 51784 ± 107641 ± 92377529 ± 83
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4 Jan 2012
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Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy
Xu Maisano, Elizabeth Litvina, Stephanie Tagliatela, Gloster B. Aaron, Laura B. Grabel, Janice R. Naegele
Journal of Neuroscience 4 January 2012, 32 (1) 46-61; DOI: 10.1523/JNEUROSCI.2683-11.2012

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Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy
Xu Maisano, Elizabeth Litvina, Stephanie Tagliatela, Gloster B. Aaron, Laura B. Grabel, Janice R. Naegele
Journal of Neuroscience 4 January 2012, 32 (1) 46-61; DOI: 10.1523/JNEUROSCI.2683-11.2012
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