Timeline and scheme of in vivo experiment.

<p>A) Rats were pre-trained on the Montoya staircase test and the horizontal ladder for three weeks. One week before the SCI (pyramidotomy) animals were injected with AAVs overexpressing FGFR1 or mCherry into the sensorimotor cortex innervating the less preferred paw. During spinal cord surgeries the medullary pyramids were cut unilaterally in the brainstem innervating the preferred paw of all animals. Three days post-surgery the animals were assessed behaviourally and thereafter every week for ten weeks. Four weeks prior to the end of the study all animals were injected with BDA on the same side as to the vector injection. B) Scheme of the different surgical procedures. 1) Injection of AAVs expressing either CMV-FGFR1-2A-EGFP or CMV-mCherry-2A-EGFP, 2) unilateral pyramidotomy, 3) BDA injection for axon tracing, and 4) counting fibres that sprouted over the cervical midline.</p

Tracing of intact corticospinal axons.

<p>A) A representative picture of BDA traced C7 spinal cord Dotted line shows outline of the grey matter and dorsal column. Scale bar, 100μm. B) Crossing of intact corticospinal fibres was assessed by counting injecting BDA in the motor cortex innervating the unlesioned forepaw. Fibres that crossed over were counted at C7 at the midline, two different distances from the midline (D1 and D2). Sprouting of the non-decussated fibres was counted on the line called “Ipsi”. C) Number of fibres that crossed a given line in C7 for control and FGFR1 overexpressing animals.</p

Wild type and mutant FGFR1 reduced neurite length on growth permissive PLL and growth inhibitory CSPGs when compared to control cells.

<p>A) Schematic diagram of the pMaxGFP vector highlighting distinct features. CMV: cytomegalovirus. B) Representative pictures of cerebellar granule neurons grown on CSPG. C,D) Cells were transfected with 1μg pMaxGFP and 4μg of either wild type or mutant FGFR1 by electroporation and then cultured on PLL (C) or CSPGs (D) for 48 hours. Control cells were transfected with 1μg pMaxGFP. Cells were stained for βIII tubulin and DAPI and analysed for neurite outgrowth by fluorescence microscopy. Data are mean ± SEM (n = 8) and significance is shown for Dunnett’s post hoc test values when groups were compared to cells transfected with pMaxGFP alone. *P < 0.05; **P < 0.01; ***P < 0.001; ns–not significant.</p

Schematic diagram of the CMV- FGFR1-2A-eGFP vector.

<p>The two genes are separated by a 2A sequence to achieve expression of two separate proteins from the single cytomegalovirus (CMV) promoter (19). ITR: Inverted Terminal Repeat Sequences; CMV: cytomegalovirus; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; AmpR: ampicillin resistance. Created using Serial Cloner 2.6.</p

FGFR1 overexpression did not increase dexterity in the Montoya Staircase or reduce errors on the horizontal ladder.

<p>A) Cortical expression of FGFR1 transcript in mCherry and FGFR1 injected animals. A separate cohort of rats was used for this experiment. qRTPCR was performed with RNA extracted 1 to 2.5 weeks after injection of AAV. The expression values of FGFR1 expression was normalised to GAPDH expression. Graph shows means ± SEM, p = 0.02. B) A representative picture of a coronal section of the spinal cord at the level of C2 stained using antibodies against GFP, 10 weeks after pyramidotomy. GFP positive fibres were found unilaterally in the dorsal columns, confirming expression in CST axons. Dotted line shows outline of dorsal columns on the side. Scale bar, 200μm. C) All animals were tested on the horizontal ladder before surgery (baseline) and after 10 weeks. Depicted is the percentage of errors made when crossing the ladder. Graph shows means ± SEM. D) All animals were tested on the Montoya staircase test three days after surgery and thereafter weekly for ten weeks. BL = base line. Graph shows means ± SEM.</p