PMC

Dorsal ramus stimulation increases excitability in large and medium diameter cells only after SNL. “Stim”, dorsal ramus stimulation applied periodically throughout experiment. *, p<0.05; **, p<0.01; ***; p<0.001. SA, spontaneous activity. N = 191 – 261 large cells and 45 – 82 medium cells per group, 5 animals per group.

Effects of the various forms of sympathetic blockade in large cells (left) and medium cells (right), in the absence of dorsal ramus stimulation. See text for explanation of experimental groups. Normal, SNL groups replotted from . N = 261 – 336 large cells and 66 – 111 medium cells per group; 5 – 6 animals per group.

Several forms of sympathetic blockade reduce effects of dorsal ramus stimulation in large diameter cells (left) and medium diameter cells (right). See text for explanation of the different experimental groups. Values of the variables after SNL without stimulation, or in control cells (dashed lines) are taken from ; values of SNL + Stim group are replotted from . N = 191 – 249 large cells and 78 – 104 medium cells per group; 5 animals per group.

Effects of dorsal ramus stimulation are relatively slow and long lasting. Example of a cell recorded before any dorsal ramus stimulation, and during and after two rounds of dorsal ramus stimulation. Top: initially, 0.2 nA current injection could evoke an action potential, but there was no spontaneous activity (second trace shows membrane potential on a slower time base; V = −64 mV). Stimulation of the dorsal ramus (third trace) shows stimulation artifacts but does not evoke an action potential or spontaneous activity (V = −63 mV). Membrane potential oscillations and spontaneous firing appeared during a second round of dorsal ramus stimulation, and spontaneous activity continued after the end of the stimulation protocol.

Sprouting early after SNL is reduced by cutting either the grey ramus or the dorsal ramus: A. Whole mount DRG 3 days after ventral ramus ligation and 24 days after sectioning of the grey ramus to that spinal nerve, showing the great reduction in TH-positive sprouting on the DRG surface (compare to ). The arrow indicates the dorsal ramus. Scale bar 500 μm. B and C. Higher magnification view (scale bar 100 μm applies to both panels) of whole mount DRGs in the region near where the spinal nerve meets the DRG. B is another example of a DRG 3 days after ventral ramus ligation, showing TH-positive fiber sprouting over this region of the DRG. Panel C shows an analogous region of a DRG on POD3 after both ventral and dorsal rami were ligated. Note the great reduction in TH-positive fibers over the surface of the DRG if the dorsal ramus is also sectioned.

Histogram of membrane capacitance values for the cells in . All non-spontaneously active (quiescent) cells have been combined into a single distribution (black). Spontaneously active cells after SNL (blue), or after SNL plus dorsal ramus stimulation (red), had a narrower distribution; in general the largest cells were not spontaneously active in any condition. Bins with capacitance values from 250 to 350 are not shown (total frequency of 0.005, non SA cells only). For clarity, SA cells from normal or normal plus stim groups are not shown; the number of cells in these groups was small. Bin size 12.5 pF. See also .

Anatomy in region of normal L4 and L5 DRGs. . Top panel: Schematic diagram showing the normal DRG anatomy (not to scale). See text for explanation. Sensory neurons (blue); Sympathetic postganglionic neurons (red); motor neuron (green). The connections between sympathetic ganglia and the various pathways of preganglionic fibers from more rostral regions of the spinal cord are not shown; however, at this level no white rami are found in rats[]. SNL refers to ligation and transection of the ventral ramus. Diagram modified from ref: []. Bottom: composite low power image of a normal DRG. Neurons are stained with NeuN (green), and tyrosine hydroxylase in red. Note the high proportion of TH-positive fibers in the dorsal ramus, and the much larger diameter of the ventral ramus compared to the dorsal ramus at this spinal level. The dorsal ramus has been folded back from the angle it would have in vivo, which would be coming up from the plane of the figure. Scale bar: 500 μm

Effects of cutting grey rami to the DRG on mechanical pain behavior. Mechanical withdrawal threshold was determined with the von Frey method (see Methods). Ligation of the ventral ramus of the L5 spinal nerve (SNL) was performed on day 0. Baseline behavior plotted on day -1 is average of measurements on three separate preceding days. Average ipsilateral baseline is indicated by the dashed line. Top: cutting the grey ramus to the L5 DRG, 3 weeks before the SNL surgery (n = 8 animals), significantly reduced the mechanical pain sensitivity induced by SNL (n = 13) on the ipsilateral side (left panels) but had no effect on the contralateral side (right panels). Differences between the two ipsilateral groups were significant at all points except baseline and POD 14. Middle: cutting both the L4 and L5 grey rami 3 weeks before the SNL surgery gave a similar effect (n = 15). SNL data is replotted from top figure. Bottom: cutting the grey ramus to the L5 DRG at the time of surgery also reduces mechanical pain behavior, with a delay (n = 5). SNL data is replotted from top figure.

The dorsal ramus is a primary source of sympathetic sprouts in the DRG early after SNL. A: Composite whole mount DRG picture 3 days after (ventral ramus) spinal nerve ligation: showing extensive TH-fiber sprouting onto the surface of the DRG. Many of the sprouting fibers originate in the dorsal ramus onto the dorsal surface of the DRG. Scale bar 500 μm. B. Magnification of the boxed region in A, showing TH-positive fibers coursing from the dorsal ramus towards the DRG. Scale bar 100 μm. C. Whole mount image from another DRG on POD3, in the region on the edge of the DRG near the spinal nerve. TH-positive fibers are shown on the surface of the DRG originating primarily from the dorsal ramus. Scale bar 100 μm. At higher magnification (panel D; scale bar 20 μm), fibers surrounding individual neurons can be observed (* symbols), as previously described in sectioned DRG. Arrows indicate varicosities and growth cone-like structures. E. Image from more distal region of the same DRG on POD3, showing TH-positive fibers in the region of the dorsal ramus. Scale bar 100 μm. At higher magnification (F; scale bar 20 μm) examples of bifurcating or branching fibers can be observed (arrows). G. Schematic showing the proposed origin of sympathetic sprouts in the dorsal ramus after ventral ramus ligation (SNL). Neuron color scheme is as in . The dotted line represents a newly sprouted sympathetic fiber entering the DRG as a collateral from an intact sympathetic axon in the dorsal ramus. Wavy lines indicate fibers in the ventral ramus that have been severed by the SNL.