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Spatial organization of activity evoked by focal stimulation within the rat spinal dorsal horn as visualized by voltage-sensitive dye imaging in the slice - PubMed

  • ️Tue Jan 01 2019

Spatial organization of activity evoked by focal stimulation within the rat spinal dorsal horn as visualized by voltage-sensitive dye imaging in the slice

Masaharu Mizuno et al. J Neurophysiol. 2019.

Abstract

In a prior study using laser scanning photostimulation, we found a pronounced cell type-specific mediolateral asymmetry in the local synaptic connectivity in the superficial laminae of the spinal dorsal horn (Kosugi M, Kato G, Lukashov S, Pendse G, Puskar Z, Kozsurek M, Strassman AM. J Physiol 591: 1935-1949, 2013). To obtain information on dorsal horn organization that might complement findings from microelectrode studies, voltage-sensitive dye imaging was used in the present study to examine patterns of activity evoked by focal electrical stimulation, in the presence and absence of synaptic blocking agents, at different positions in transverse, parasagittal, and horizontal slices of the dorsal horn of 2- to 3-wk -old male rats. A pronounced difference in responsiveness was found between medial and lateral dorsal horn, in that medial sites in the superficial dorsal horn showed much larger synaptic responses to focal stimulation than lateral sites. This difference appeared to be a result of a difference in the intrinsic elements of the dorsal horn, rather than a difference in the inputs from the white matter, because the stimulus intensities were subthreshold for evoking synaptic responses from stimulation at sites in the white matter, although it is also possible that the greater responsiveness is due, at least in part, to activation of Aβ primary afferent fibers that pass through the medial dorsal horn. The results raise the possibility of differences between medial and dorsal horn that need to be taken into account in the interpretation of studies of dorsal horn organization.NEW & NOTEWORTHY We used voltage-sensitive dye imaging to obtain information on spatial aspects of dorsal horn organization that are difficult to examine with single-cell approaches because of the limitations of microelectrode sampling. The most noteworthy finding was a previously unreported, extreme difference between medial and lateral dorsal horn in responsiveness to focal stimulation that appears to result, at least in part, from a greater degree of excitability or local connectivity in medial dorsal horn.

Keywords: nociception; pain; somatosensory; superficial dorsal horn.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.

Comparison of the voltage-sensitive dye (VSD) response evoked by graded intensities of focal electrical stimulation in the white matter (A; ~50 µm dorsal to the border between the white matter and the dorsal horn) vs. the dorsal horn, in the approximate region of the lamina II/III border (B; ~120 µm ventral to the white matter border), in a transverse slice (right side). In most experiments, stimulus threshold for evoking a response was ~8-fold lower at sites in the dorsal horn (lamina II or II/III border region) than in the overlying white matter. In all figures, focal stimulation consisted of a 30-ms train of ten 1-ms anodal pulses, 333 Hz. Time point illustrated is the end of the stimulus train. Circle in each image marks the stimulating electrode position. Images are transverse slice through right dorsal horn (lateral direction is to the right; ×20 objective). VSD images represent the average of 16 stimulus trials delivered with a 16-s intertrial interval. dF/Fmax, %change in fluorescence; Lam, lamina; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide.

Fig. 2.
Fig. 2.

Effect of chemical blockade of synaptic transmission on voltage-sensitive dye (VSD) responses evoked by focal electrical stimulation in the superficial dorsal horn, in a transverse slice (right side; ×10 objective). Time point illustrated is 15 ms after the end of the 30-ms stimulus train. Stimulus amplitude was 3 µA; cross in each image marks the stimulating electrode position. AP5,

d

-2-amino-5-phosphonovalerate (50 µM); Bic, bicuculline (10 µM); dF/Fmax, %change in fluorescence; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (10 µM); Stry, strychnine (300 nM).

Fig. 3.
Fig. 3.

Mediolateral asymmetry of voltage-sensitive dye (VSD) responses evoked by focal electrical stimulation at different positions across the mediolateral axis of the dorsal horn. Inset: a composite infrared-differential interference contrast image of the slice from which VSD images in A–D were obtained. Cross on each image marks the stimulus position. Stimulus intensity is shown at bottom left in A–D (1.5, 1.5, 3, and 24 µA, respectively); intensity was increased for the more lateral positions because of the higher response threshold at these positions. Distribution of activity evoked from stimulation at the most medial site (A) shows a pronounced lateral asymmetry that reverses direction with stimulation at more lateral sites (C and D). Time point illustrated is immediately after the end of the 30-ms stimulus train. Images show transverse slice through right dorsal horn (×10 objective). dF/Fmax, %change in fluorescence.

Fig. 4.
Fig. 4.

Comparison of response amplitudes at stimulation sites in medial vs. lateral dorsal horn. A1: plot of response amplitude (mean and SD) evoked by stimulation at 6 positions (a–f) across the mediolateral axis of the dorsal horn, in the superficial laminae, for one stimulus intensity (6 µA). Estimated laminar position in this stimulus series was the innermost part of lamina II, ~90 µm below the white matter border. Stimulus positions (a–f) are marked on a transverse outline of the right dorsal horn (inset above graph). For the 6 positions, n = 6, 2, 5, 3, 3, and 4 slices, respectively. The data were compiled from 6 slices (6 animals). Two of the slices were stimulated at all 6 positions, and the other 4 slices were each stimulated at 2–3 positions. A2: plots of response amplitude as a function of stimulus intensity for a medial and a lateral position (positions a and f, n = 6 and 4 slices, respectively). Plots are from the same data plotted in A1. B1–B3: plots from a set of experiments in which stimuli were delivered at different depths below the white matter (i.e., distance from white matter border, as measured along an axis perpendicular to white matter border), at either a medial or a lateral position. Medial sites were similar in mediolateral position to a in A1; lateral sites were at the mediolateral position of f or further lateral; n = 8 and 4 slices (animals) for medial and lateral dorsal horn, respectively (separate slices for medial and lateral sites; separate experiments from A1 and A2). Each slice was stimulated at 3–6 depths below the white matter; n = 4, 6, 8, 6, 5, 3, and 4 slices for the 7 sites in B1, and n = 4, 4, 4, and 2 slices for the 4 sites in B2. In each slice, each site was stimulated at 3–5 stimulus intensities (i.e., not all of the plotted intensities in A2 and B3 were tested at each site). Time point used for these measurements was 30 ms after the end of the 30-ms stimulus train. dF/Fmax, %change in fluorescence.

Fig. 5.
Fig. 5.

A: voltage-sensitive dye (VSD) responses evoked by single-shock stimulation (1 ms, 1.4 mA) of the L5 dorsal root, and effect of chemical blockade of synaptic transmission, in a transverse slice (right side; ×10 objective). (Note that all other figures show responses evoked by focal stimulation in the dorsal horn.) Time point illustrated is 140 ms after the stimulus. B: time course of actions of the synaptic inhibitors bicuculline (Bic; 10 µM), strychnine (Stry; 300 nM),

d

-2-amino-5-phosphonovalerate (AP5; 50 µM), and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX; 10 µM), obtained by sequential subtraction of data shown in A. Curve for Bic/Stry was obtained by subtracting the baseline response (first VSD image) from the response with Bic+Stry (second VSD image). AP5 curve was obtained from the difference of the response before and after addition of AP5 (subtraction of third panel from second panel), and similarly for the NBQX curve (subtraction of fourth panel from third panel). Response amplitude was measured from a circular area of ~70-µm diameter within the zone of peak response, in the superficial dorsal horn. dF/Fmax, %change in fluorescence; Stim., stimulus onset.

Fig. 6.
Fig. 6.

A: time series of voltage-sensitive dye (VSD) images during and after focal electrical stimulation in a parasagittal slice, to illustrate the development of the “patchy” or discontinuous distribution of activity along the rostrocaudal axis. Time points shown at left of each image are relative to the onset of the 30-ms stimulus train (Stim.; indicated by the red vertical bar). Note that the time intervals between the images are not equal throughout the series. Circle on each image marks the stimulus position. Dashed lines mark approximate position of the laminae I–II borders. Dorsal direction is at top. B: plot of time course of activity for the same stimulus trial shown in A. Time is represented on the y-axis (the dorsoventral axis is not represented in this plot). Activity is shown for a single rostrocaudally oriented line drawn approximately parallel to the laminar borders through the zone of peak activity (black line in top image). Arrowhead marks the rostrocaudal position of the stimulus. Distance calibration is same as in A. Stimulus intensity is 36 µA, which is considerably higher than the intensities used for the experiments illustrated in transverse slices (Figs. 1–4). All VSD images, ×10 objective.

Fig. 7.
Fig. 7.

Effect of chemical blockade of synaptic transmission on voltage-sensitive dye (VSD) responses evoked by focal electrical stimulation in the superficial dorsal horn, in a parasagittal slice. A: activity during (A1) and 12 ms after (A2) the end of the stimulus train. Circle on each image marks the stimulus position. Dashed lines mark approximate position of the laminae I–II borders. B: plot of time course of activity for the stimulus trial shown in A. Time is represented on the y-axis (the dorsoventral axis is not represented in this plot). Activity is shown for a single rostrocaudally oriented line drawn approximately parallel to the laminar borders through the zone of peak activity (black line in rightmost image of A2 image). Stimulus (Stim.) train is indicated by the red vertical bar. Arrowheads mark the rostrocaudal position of the stimulus. Distance calibration is same as in A. Stimulus intensity is 24 µA. All VSD images, ×10 objective. AP5,

d

-2-amino-5-phosphonovalerate (50 µM); Bic, bicuculline (10 µM); dF/Fmax, %change in fluorescence; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (10 µM); Stry, strychnine (300 nM).

Fig. 8.
Fig. 8.

A time series of voltage-sensitive dye (VSD) images during focal electrical stimulation in a horizontal slice through lamina II. Medial direction is at top and rostral direction is to the left. Time points at top left of each VSD image are relative to the onset of the 30-ms stimulus train. Circle on each image marks the stimulus position. Closed arrow indicates a rostrocaudally oriented patchy band of activity that spreads from the stimulus site, and open arrow indicates a more medial zone of activity that develops somewhat later in the stimulus train. Stimulus intensity is 24 µA. All VSD images, ×10 objective. dF/Fmax, %change in fluorescence.

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