Neural precursor integration in organotypic slice cultures

Organotypic slice cultures provide an opportunity to explore migration and incorporation of donor cells under controlled conditions in a real-time setting (Figures 5 and 6). In our 'in vitro transplantation' system, the donor cells are deposited at the surface of cultured brain slices from where they migrate and incorporate into the host tissue (Protocol 6). When combined with EGFP genetic-labelling of the engrafted cells, this system permits direct experimental access to migration, differentiation, and electrophysiological activity at the single-cell level. Indeed, hippocampal slice cultures have been successfully

Organotypic Slice Culture Brain

Figure 4 (see Plate 6) Antigenic profile of mES cell-derived oligodendrocytes generated by CNP-bgeo-based lineage selection. CNP-figeo transfected mES cells were first subjected to controlled differentiation into glial precursors. Subsequent induction and stabilization of a CNP-positive fate permitted the isolation of mES cell-derived oligodendrocyte progenitors. Quantitative marker expression and representative immunofluorescence images of the selected cells are shown in (A) and (B-G), respectively. More than 96% of the cells coexpress CNP and p-gal (A-B). In addition, the majority of cells express nestin (C), PSA (D), A2B5 (E), and 04 (F). The selected population also includes GalC- and NG2-positive cells (G). Nuclei of cells are stained with DAPI (blue). Scale bar = 50mm. For details see Glaser et al. (29).

Figure 4 (see Plate 6) Antigenic profile of mES cell-derived oligodendrocytes generated by CNP-bgeo-based lineage selection. CNP-figeo transfected mES cells were first subjected to controlled differentiation into glial precursors. Subsequent induction and stabilization of a CNP-positive fate permitted the isolation of mES cell-derived oligodendrocyte progenitors. Quantitative marker expression and representative immunofluorescence images of the selected cells are shown in (A) and (B-G), respectively. More than 96% of the cells coexpress CNP and p-gal (A-B). In addition, the majority of cells express nestin (C), PSA (D), A2B5 (E), and 04 (F). The selected population also includes GalC- and NG2-positive cells (G). Nuclei of cells are stained with DAPI (blue). Scale bar = 50mm. For details see Glaser et al. (29).

Figure 5 (see Plate 7) Morphological and functional Integrity of hlppocampal slice cultures used as recipient tissue for'in vitro transplantation' of mES cell-derived neural precursors. (A) A 400mm-thick slice 1d after explantation. Dentate gyrus (DG), pyramidal-cell layer (CA3-CA1), entorhinal cortex (EC), and adjacent regions of the temporal cortex (TC) are well delineated. SC, Schaffer collaterals; MF, mossy fibres; PP, perforant path. (B) Cryostat section (10mm) of a slice preparation maintained in culture for 31 d. Note the morphological preservation of the key anatomic structures (haematoxylin and eosin stain). The inset shows typical field potentialsfollowing orthodromic stimulation of the perforant path (PP-DG) and the Schaffer collaterals (SC-CA1) at the end of the culture period. (C) Anterograde axonal tracing with rhodamine-conjugated dextran (Micro-Ruby®) confirms the integrity of the perforant path at 33d in culture. (D) TIMM stain demonstrating the histological preservation of the mossy fibre system of a slice culture maintained for 33d (20mm cryostat section). Scale bars = 1mm. For details see Scheffler et al. (37).

Figure 5 (see Plate 7) Morphological and functional Integrity of hlppocampal slice cultures used as recipient tissue for'in vitro transplantation' of mES cell-derived neural precursors. (A) A 400mm-thick slice 1d after explantation. Dentate gyrus (DG), pyramidal-cell layer (CA3-CA1), entorhinal cortex (EC), and adjacent regions of the temporal cortex (TC) are well delineated. SC, Schaffer collaterals; MF, mossy fibres; PP, perforant path. (B) Cryostat section (10mm) of a slice preparation maintained in culture for 31 d. Note the morphological preservation of the key anatomic structures (haematoxylin and eosin stain). The inset shows typical field potentialsfollowing orthodromic stimulation of the perforant path (PP-DG) and the Schaffer collaterals (SC-CA1) at the end of the culture period. (C) Anterograde axonal tracing with rhodamine-conjugated dextran (Micro-Ruby®) confirms the integrity of the perforant path at 33d in culture. (D) TIMM stain demonstrating the histological preservation of the mossy fibre system of a slice culture maintained for 33d (20mm cryostat section). Scale bars = 1mm. For details see Scheffler et al. (37).

used to demonstrate network integration of mES cell-derived neurons and glia (37, 38).

Candidate regions for the preparation of slice cultures include the cerebellum and the hippocampus; and the latter offers the possibility to study donor cell integration in the context of ongoing neurogenesis in the dental granule cell layer (37). Typically, organotypic slices are propagated as interface cultures on polyester membranes.

According to our method (Protocol 6), hippocampal slice cultures are prepared from 9-day-old Wistar rats. Using a vibroslicer, 400 mm-thick slices encompassing the dentate gyrus, hippocampus, and entorhinal/temporal cortex (Figure 5A) are propagated on a porous (0.4 mm pore size) polyester membrane in a humidified incubator gassed with 5% CO2 in atmospheric air, and set at 35 °C (39). Cultures are initiated in a horse serum-containing medium, which is gradually replaced over 5 d in culture with a serum-free, defined medium based on DMEM/F12 and including the N2 and B27 supplements. Under these conditions, cultured slices can be maintained for a period of up to 35 d ((37); and see Figure 5).

Figure 6 (see Plate 8) Functional Integration of mES cell-derived neurons Into hippocampal slice cultures. (A) Schematic representation of the implantation site: CA1, CA1 subfield; CA3, CA3 subfield of the hippocampus; DG, dentate gyrus. (B) Confocal image of two EGFP-positive neurons 3 weeks after engraftment (three-dimensional reconstruction of 16 individual planes taken from a fixed slice). Scale bar = 10 mm. (C1-C3) Infrared DIC image (C1) and fluorescence image (C2) of an EGFP-positive donor neuron after formation of a gigaseal. Diffusion of EGFP into the pipette serves as confirmation of the donor cell identity (C3). (D) Functional maturation of engrafted mES cell-derived neurons. Current-clamp recordings during prolonged (D1) and brief (D2) current injections at different time points after transplantation (12-14d, and 19-21 d, as indicated at the left). Top traces represent voltage recordings, whereas bottom traces indicate current injections. Note the progressive development of repetitive discharge properties and action potential morphology with time in culture. For details see Benninger et al. (38). Copyright, the Society for Neuroscience.

Figure 6 (see Plate 8) Functional Integration of mES cell-derived neurons Into hippocampal slice cultures. (A) Schematic representation of the implantation site: CA1, CA1 subfield; CA3, CA3 subfield of the hippocampus; DG, dentate gyrus. (B) Confocal image of two EGFP-positive neurons 3 weeks after engraftment (three-dimensional reconstruction of 16 individual planes taken from a fixed slice). Scale bar = 10 mm. (C1-C3) Infrared DIC image (C1) and fluorescence image (C2) of an EGFP-positive donor neuron after formation of a gigaseal. Diffusion of EGFP into the pipette serves as confirmation of the donor cell identity (C3). (D) Functional maturation of engrafted mES cell-derived neurons. Current-clamp recordings during prolonged (D1) and brief (D2) current injections at different time points after transplantation (12-14d, and 19-21 d, as indicated at the left). Top traces represent voltage recordings, whereas bottom traces indicate current injections. Note the progressive development of repetitive discharge properties and action potential morphology with time in culture. For details see Benninger et al. (38). Copyright, the Society for Neuroscience.

4.1.1 Functional validation of hippocampal slice cultures

The integrity of slice cultures may be assessed using the following techniques:

(a) Recording of field excitatory postsynaptic potentials. Field potential recordings can be used to validate the synaptic connectivity between perforant path and dentate gyrus, as well as between Schaffer collaterals and CA1 pyramidal neurons ((40); and see Figure 5B, inset).

(b) Anterograde axonal tracing. Anterograde tracing of perforant path axons is performed by depositing rhodamine-conjugated dextran (e.g. Micro-Ruby®, Molecular Probes) on top of the entorhinal cortex, and confirming anterograde labelling of the perforant path ((41); and see Figure 5C).

(c) Specialized histological staining. TIMM staining can be used to delineate the mossy fibre system ((42); and see Figure 5D).

For a more detailed description of these methods see Scheffler et al. (37).

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