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GAP-43 analysis in DRG neurons Sections of C5 and C6 DRG (10m m) were double-immunostained for b III tubulin (Promega, 1:1000) to identify all neurons, and for the growth-associated protein GAP-43 (gift from G. Wilkin, 1:2000), using 7-amino-4-methylcoumarin-3-acetic-acid- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibodies. The percentage of GAP-43-positive cells was determined in four sections per animal.
Axon tract tracing in the spinal cord Ascending dorsal column axons were labelled using the CTB tracer29, injected into the left median nerve of anaesthetized rats. Longitudinal sections (20m m) were immunostained for glial brillary acidic protein, to identify the lesion site, and for CTB, to identify the labelled dorsal column axons29. Descending CST axons were labelled with 1m l BDA (Molecular Probes; 10% in saline) injected under anaesthesia at four sites over the left primary motor cortex, 1 mm below the dorsal surface of the brain. BDA-labelled bres were visualized in parasaggittal spinal cord sections (20m m) with extra-avidin conjugated to uorescein isothiocyanate. All BDA-labelled bres observed within a 1-mm square grid were counted at measured intervals from 4 mm above to 5 mm below the lesion site by an experimenter, blinded to treatment. Due to variability in labelling, axon numbers were calculated as a percentage of the bres seen 4 mm above the lesion, where the CST was intact. To conrm a complete CST lesion, transverse lumbar spinal cord sections (20m m) were immunostained with an antibody against theg -subunit of protein kinase C (PKC-g , Santa Cruz, 1:1000), visualized with a TRITC-conjugated secondary antibody.
ElectrophysiologyIn terminal electrophysiological experiments, the sensory motor cortex and cervical spinal cord were exposed in urethane-anaesthetized (1.5 g kg21) rats. Cortical evoked potentials were elicited by electrical stimulation (ve square-wave pulses at 400 Hz, 100m A, 200m s, delivered every 2 s) of the left sensory motor cortex using a 0.5-mm concentric needle electrode lowered 1 mm into the cortex. For each experiment we mapped the optimal stimulation site, located 12 mm lateral and 1 mm rostrocaudal from Bregma. Postsynaptic potentials evoked by the cortical stimuli were recorded with a silver ball electrode placed medially on the contralateral cord surface. At each recording site (1 segment above and 17 mm below the lesion at C4), 64 responses were averaged...