In vivo recording, forepaw denervation, and isolation of slices: Methods for mapping the forepaw/lower jaw border in anesthetized adult rat primary

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1 Supplementary Methods In vivo recording, forepaw denervation, and isolation of slices: Methods for mapping the forepaw/lower jaw border in anesthetized adult rat primary somatosensory cortex (S1), forepaw denervation, re-mapping of the border and isolation of slices were similar to those used previously 1. These methods are briefly summarized here. For all surgical procedures adult Sprague-Dawley rats ( g.) were anesthetized until areflexic with pentobarbital (50 mg/kg, intraperitoneal), and mounted in a stereotaxic frame. Supplemental doses of anesthetic were administered as needed. For all recovery surgeries, aseptic procedures were used. All procedures conformed to NIH guidelines and were approved by Institutional Animal Care and Use Committee at the University of California Riverside. To determine the amount of shift in the forepaw/lower jaw border, the region of S1 around the border was mapped twice: before the denervation and then after the desired duration of denervation. The first map was derived using transdural electrode penetrations. Since the rat dura is relatively transparent, it was possible to see the larger surface blood vessels on the cortex. Carbon-fiber electrodes that had been cut back on the glass supporting the fiber were used to penetrate the dura and cortex. The forepaw or lower jaw was stimulated with a fine probe to elicit multiunit cutaneous responses in S1. Responsiveness to forepaw and/or lower jaw stimulation was determined subjectively by listening to audio monitor output. Penetrations were introduced into the forepaw zone, 1-2 mm rostral to Bregma; subsequent penetrations were introduced more laterally until regions that responded to tactile stimulation of the lower jaw were encountered. Recordings were all at an approximate depth of µm. The location of penetrations was recorded on the computer image of the cortex by using surface vasculature landmarks. Penetrations spaced <50 µm apart were then made to locate the

2 border more exactly. Typically, 3 of these rows of penetrations were made, arranged perpendicular to the forepaw/lower jaw border, which is normally oriented roughly parallel to the midline. Rows were separated by µm. Three or four locations on the forepaw/lower jaw border were then marked with a recording electrode that had been repeatedly dipped in a 1-2% DiI solution (in ethanol) and the DiI crystals allowed to deposit on the electrode 2. The electrode was introduced into the cortex to recording depth (~600 µm), the responses of the penetration confirmed, and the electrode left in the cortex for 3-5 minutes. Reorganization of the forepaw/lower jaw region was then induced by peripheral denervation of the forepaw. The radial and median nerves of the forepaw contralateral to the mapped hemisphere were exposed, a 1-2 mm segment was cut from each nerve, and the remaining cut ends of the nerves tightly ligated with 6-0 suture thread to prevent nerve regeneration. Animals were then allowed to recover for the desired duration of denervation (7, 14 or 28 days) before the second mapping of the forepaw/lower jaw border was performed. After the appropriate duration of denervation, the second (reorganized) map of the forepaw/lower jaw border in S1was derived in a similar manner to the first. The craniotomy was re-exposed, the dura removed, and the exposed cortex covered by silicone oil. Response mapping of the forepaw/lower jaw region was again performed using carbon-fiber electrodes. The novel forepaw/lower jaw border (i.e. the reorganized border) was then marked with DiI, as above. The shift in the border was defined as the distance between the initial and second DiI mark, which had significantly different appearances (Figure 2A). 400 µm thick coronal slices were taken from the marked region and maintained in vitro in standard mammalian bicarbonate buffer (in mm: NaCl, 119; KCl, 2.5; NaH 2 PO 4, 1.25; MgSO 4, 1.3; CaCl 2, 2.5; NaHCO 3, 26.2; glucose, 11; saturated

3 with 95%O 2 /5%CO 2 ) for intracellular recording. Neurons for recording were obtained using blind whole-cell recording. In vitro filling with biocytin: Patch electrodes were pulled on a Flaming/Brown puller to a tip diameter of µm and filled with, in mm: Cs-Gluconate or K-gluconate, 128; CsCl or KCl, 7; EGTA, 1; HEPES, 10; QX-314, 10; Mg-ATP, 2; Na-GTP, 0.2; biocytin %; ph Electrodes had tip resistances of 3-8 M. Only neurons with initial resting potentials of <- 60 mv and stable input resistances of >50 M were used. Generally cells were filled with biocytin for minutes. 31 neurons from 20 animals were analyzed from animals after 7 days of denervation, 28 neurons from 17 animals were analyzed from animals after 14 days of denervation, and 31 neurons from 18 animals were analyzed from animals after 28 days of denervation. Analysis of neuronal morphology: Slices were fixed in 4% paraformaldehyde overnight, rinsed, permeablized with 0.25% Triton X-100 and reacted with ALEXA 488-conjugated streptavidin (0.002%; Molecular Probes, Inc.). Labeled cells were then visualized with a laser scanning confocal microscope (LSM-510, Carl Zeiss, Inc.). The complete, 2-dimensional morphology of labeled processes was obtained for well-filled cells from Z-series taken through the entire depth of the slice, the gain and black level of these images was set to maximize contrast. Images were stored on computer disk, composited, and analyzed using NIH Image, Adobe Photoshop and Canvas 5.0 software. Processes filled were both dendrites and axons. We attempted to restrict analysis to dendrites by excluding the very thin axonal processes. Furthermore, in these thin slices, axons are very truncated. Thus,

4 we are confident that the results reflect effects on dendrites. All analyses were performed blind to the location of the borders and to the duration of denervation. To examine the processes for bias, a modified Scholl procedure 3, using superimposed circles centered on the soma with radii of 20, 40, 60, 80, etc. µm, was used. The neuron s processes were then divided into two regions by a line perpendicular to the cortical surface that bisected the soma (see Figure 2B). The total number of intersections of filled processes with each of these circles was determined for the half circle adjacent to the border (referred to as the border side ) and for the half circle further away from the border (referred to as the nonborder side ). This number of intersections was termed the complexity for that side of the neuron. The bias ratio (i.e. the border/non-border ratio) was defined as the ratio between the complexity on the side toward the border divided by the complexity from the side away from the border. Although these measures were dominated by dendritic process intersections, both dendritic and axonal processes were included, and both the length and number of branches of the arbors contributed to this measure. To further refine the measure of bias, both the number of branch points and the total length of all processes were determined for the two sides of the neuron (i.e., nearer to or farther from the border). Branch points were counted by visual inspection of micrographs; intersections of processes that were clearly due to superimposition of processes in different optical planes were not counted as branch points. Total length of processes was determined using NIH Image software by manually tracing all labeled processes on each side of the neuron and measuring the resulting line segments. These line segments were summed for each side of the neuron. The border/non-border ratio was also calculated for these two parameters. Statistical significance was determined using one-way ANOVA followed by a Fisher s PLSD for individual comparisons, or using planned unpaired Student s t-tests. A P value of <0.5 was taken to be significant.

5 References: 1. Hickmott, P. W. & Merzenich, M. M. J. Neurophysiol. 88, (2002). 2. DiCarlo, J. J., Lane, J. W., Hsiao, S. S. & Johnson, K. O. J. Neurosci. Meth. 64, (1996) 3. Hickmott, P. W. & Merzenich, M. M. J. Comp. Neurol. 409, (1999).