All experiments involving animals were carried out according to the law of animal experimentation issued by the German parliament (“Tierschutzgesetz”) and to the European Communities Council Directive. The present study was approved on May 16 2012, by the Regierungspräsidum Tübingen (Baden-Württemberg, Germany) according to § 4, Abs.3 of the law of animal experimentation.
For all experiments, we used standard protocols established and routinely applied in our labs. All cultures were immunocytochemically characterized using established markers.
NPC were prepared from ED 16 Wistar rat brains. Shortly, brains were dissected from the embryos and collected in Hibernate-E Medium (life technologies, Darmstadt, Germany) supplemented with B27 supplement and GlutaMAX (life technologies). Brains were mechanically dissociated by passing them through a nylon cloth of 110 μm mesh size. After centrifugation at 400 g and 4°C for 10 min, the cell pellet was resuspended in Neurobasal Medium (life technologies) supplemented as above. The cell suspension was passed through a second nylon cloth of 25 μm mesh size, appropriately diluted and seeded at a density of 1–3 million cells/ 21 cm2 surface area in 5 ml medium on p-D-lysine-coated coverslips. Cells were cultured at 37°C and 5% CO2 in a humidified atmosphere. For all experiments, 4–7 DIV cultures were used. Astroglial primary cultures (APC) were prepared from newborn Wistar rat brains as described . For isolation of RNA and preparation of homogenates for Western blotting, 10 DIV cultures were used.
Studies on GP had primarily been carried out with rat tissues and primary cultures derived from rat brain, but studies carried out with tissues and cultures from other species confirmed the results [17, 18]. We therefore used mouse spinal motoneuron and trigeminal neuron cultures for FISH analysis in motoneurons and trigeminal neurons. MNC were prepared from the lumbar spinal cords of ED 13.5 Bl/6 mice as described  and used for experiments after 5 DIV. TGC were prepared from the trigeminal ganglia of PD 0–5 or ED 16.5 CD1 mice as described  and used for experiments after 3–7 DIV.
Tissue sections and immunohistochemistry (IHC)
Tissues were freshly dissected from adult Wistar rats of both sexes (4 animals for spinal cord sections, 3 animals for trigeminal ganglion sections, 2 animals for ventral spinal nerve and trigeminal nerve sections, respectively) and fixed in 4% paraformaldehyde/phosphate-buffered saline (PFA/PBS) at 4°C for 24 h. After cryoprotection, the tissue was frozen by immersion in liquid nitrogen. Sections were cut at 10 μm and stored at -80°C until processed.
Total RNA was isolated from whole rat brain using TRI Reagent Solution (Ambion, Life Technologies) according to the manufacturer’s protocol. About 1 μg of total RNA was reversely transcribed using Oligo(dT) primers and 2 μl of the resulting cDNA solution were used as a template for the PCR amplification of nucleotide sequences from the coding regions of rat glycogen synthase 1 (accession number BC 131849) and rat brain glycogen phosphorylase (accession number NM 0131188). The following primers were used: GS, forward primer 5′-AGCCATCTTTGCGACTCAGC-3′, reverse primer 5′-TGGTAGGACTCAGGGGCTCA-3′; GP, forward primer 5′-TCCCAGACAAGGTAGCCATC-3′, reverse primer 5′-AAGGCCTCATCATCAACCAG-3′. The PCR products were diluted 1:10 and used as templates for a second PCR amplification applying primers including sequences of the T7 and SP6 RNA polymerase promoters, respectively. Primers were the following: GS, forward primer SP6, 5′-ATTTAGGTGACACTATAGACACCCTCACTGTCTCGACAC-3′, reverse primer T7 5′- TAATACGACTCACTATAGGGTGTACTGAGTGAGCTGGAGG-3′; GP, forward primer SP6 5′-ATTTAGGTGACACTATAGATCCCAGACAAGGTA-3′, reverse primer, T7 TAATACGACTCACTATAGGAAGGCCTCATCATCACC-3′. The products from 5 PCRs were pooled and purified. For GS DNA, electrophoresis in a 1% agarose preparative gel with subsequent extraction using a gel extraction kit (QIAGEN, Stockach, Germany) according to the manufacturer’s protocol was applied; GP DNA was purified with a PCR purification kit (QIAGEN) according to the manufacturer’s protocol.
Digoxigenin(DIG)-labeled RNA probes were prepared by in-vitro transcription with T7 RNA polymerase (antisense probe) or SP6 RNA polymerase (sense probe), respectively, using a DIG RNA Labeling Kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. The length of the transcripts was checked by agarose gel electrophoresis, subsequent capillary transfer to a nylon membrane and visualization of the bands with an alkaline phosphatase-coupled DIG antibody and nitroblue tetrazolium chloride/5-bromo-4-chloro-indolylphosphate substrate reaction. The lengths of the riboprobes were approximately 250 nt for GS and 600 nt for GP. Dot blots were used for semi-quantitative analysis of the probe concentrations.
After defined periods of time in culture, cells were fixed in 4% PFA/PBS at 4°C for 10 min, then washed with PBS and subsequently permeabilized with PBS/0.3% Triton X-100 at RT for 10 min. After a washing step in PBS, cells were pre-hybridized at 55°C for 0.5 – 2 h in the following hybridization buffer: 4x saline-sodium citrate (SSC), 4x Denhardt’s, 10% dextrane sulfate, 500 μg/ml salmon sperm DNA, 250 μg yeast tRNA and 50% formamide. Probes were denatured at 90°C for 5 min. Hybridization was performed in hybridization buffer at 55°C overnight with a probe concentration of 5 ng/μl for MNC and 0.1 ng/μl for the other cultures. Stringency washes were performed with 0.2x SSC at 55°C for MNC and 72°C for other culture types for 1 h and then with 0.2x SSC at room temperature (RT) for 5 min. After a blocking step in 0.5% Blocking Reagent (Roche) at RT for 30 min, cells were incubated with sheep anti-DIG peroxidase (POD) conjugate (Fab-fragments, Roche) diluted 1:500 in blocking reagent at RT for 3 h. After two washing steps in 0.1 M Tris/HCl, 0.15 M NaCl pH 7.5, 0,05% Tween 20 cells were incubated with the TSA-Plus Fluorescein reagent (Perkin Elmer, Rodgau-Jügersheim, Germany), diluted 1:50 (MNC) or 1:100 (other cell types) for 10 min (MNC) or 30 min (other cell types). After two washing steps, cells were rinsed with water and embedded in Immumount. Negative controls included replacement of antisense probe by sense probe, omitting of probes and omitting of anti-DIG antibody. In experiments combining FISH with immunocytochemistry (ICC), the relevant primary antibody was incubated together with the anti-DIG/POD conjugate. After the washing steps, cells were incubated with the secondary antibody diluted 1: 1000 at RT for 1 h. For FISH on tissue slices, the protocol was essentially the same with the following exceptions: Sections were postfixed in ice-cold 4% PFA/PBS for 5 min, and after stringency washes, endogenous peroxidase was blocked by incubation in 2% H2O2/1x SSC at RT for 15 min. Images were acquired with a Zeiss Cell Observer microscope using a 40x oil immersion objective and AxioVision software. Immunostaining on tissue sections and NPC that was not combined with FISH was performed as described , with the exception that secondary antibodies were conjugated to Alexa Dye 488 or 568 and diluted 1:1000. For TGC, the staining protocol was essentially identical. In brief, unspecific binding was blocked by incubation in 5% donkey serum/PBS for 30 min prior to incubation with antisera. All antibodies were diluted in 1% donkey serum; GP immunostaining was combined with staining for β-tubulin (1:2000) at RT for 2 h. Secondary antibodies were conjugated to Alexa dye 488 (anti-rabbit) or Cy5 (anti-mouse), diluted 1:500 and applied at RT for 30 min. Nuclei were stained with 4′,6-diamidino-2-indole (DAPI) diluted 1:10 000 in PBS .
Total RNA was isolated from NPC, APC, rat cortex and cerebellum using TRI-Reagent (Ambion) according to the manufacturer’s protocol. After DNase treatment (RQ1 DNase, Promega, Mannheim, Germany), 250 ng of RNA were reversely transcribed applying the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies). For PCR reactions, 3.1 ng of cDNA were used. Reactions were prepared with Fast SYBR Mix (Applied Biosystems) and the following primers: GP, forward primer 5′- TCAGGGATGTAGCCAAGGTC-3′, reverse primer 5′-TCGGTTGTACAGGGTGATGA-3′; GS, forward primer 5′-CCCCCAATGGACTAAATGTG-3′, reverse primer 5′-AGCGTCCAGCGATAAAGAAA-3′; GFAP, forward primer 5′- GAAGAAAACCGCATCACCAT-3′, reverse primer 5′-TCCTTAATGACCTCGCCATC-3′; GAPDH, forward primer 5′-CTCATGACCACAGTCCATGC-3′, reverse primer 5′-TTCAGCTCTGGGATGACCTT-3′; β-actin, forward primer 5′-AGCCATGTACGTAGCCATCC-3′, reverse primer 5′-ACCCTCATAGATGGGCACAG-3′; 18S rRNA, forward primer 5′-GCAATTATTCCCCATGAACG-3′, reverse primer 5′-GGCCTCACTAAACCATCCAA-3′. All PCR reactions were performed in triplicate and repeated at least 6 times. Each experiment included a no-reverse transcription control and a no-template control. Amplification efficiencies were tested for each gene and reached optimal values (>1.8). Ct values were normalized to GAPDH, β-actin and 18S rRNA. Expression rates were expressed as n-fold of the expression rate of the relevant gene in APC.
Western blot analysis
Western blotting was performed as described  with the following exceptions: proteins were transferred to a PVDF membrane using a semi-dry blotting system (BioRad, München, Germany). After a blocking step, the membrane was incubated with goat anti-rabbit IgG/POD conjugate (diluted 1:10 000) at RT for 2 h. The blot was developed applying a chemiluminescence detection system (ECL Western Blotting Substrate (Pierce, Rockford, IL, USA).
Rabbit antiserum against rat brain glycogen phosphorylase isozyme was generated as published . Anti-MAP2 monoclonal antibody was purchased from Millipore (Darmstadt, Germany). Rabbit antiserum against tau and monoclonal antibodies against GFAP and GAP-43 were from Sigma/Aldrich (St. Louis, MO, USA). Donkey antiserum against β-tubulin was purchased from Covance (Princeton, NJ, USA). Fluorescent secondary antibodies were from Molecular probes (Invitrogen), goat anti-rabbit POD conjugate was from Jackson Immuno Research (Dianova, Hamburg, Germany).