Peripheral inflammation facilitates Abeta fiber-mediated synaptic input to the substantia gelatinosa of the adult rat spinal cord

Abstract

Whole-cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons in thick adult rat transverse spinal cord slices with attached dorsal roots to study changes in fast synaptic transmission induced by peripheral inflammation. In slices from naive rats, primary afferent stimulation at Abeta fiber intensity elicited polysynaptic EPSCs in only 14 of 57 (25%) SG neurons. In contrast, Abeta fiber stimulation evoked polysynaptic EPSCs in 39 of 62 (63%) SG neurons recorded in slices from rats inflamed by an intraplantar injection of complete Freund's adjuvant (CFA) 48 hr earlier (p < 0.001). Although the peripheral inflammation had no significant effect on the threshold and conduction velocities of Abeta, Adelta, and C fibers recorded in dorsal roots, the mean threshold intensity for eliciting EPSCs was significantly lower in cells recorded from rats with inflammation (naive: 33.2 +/- 15.1 microA, n = 57; inflamed: 22.8 +/- 11.3 microA, n = 62, p < 0.001), and the mean latency of EPSCs elicited by Abeta fiber stimulation in CFA-treated rats was significantly shorter than that recorded from naive rats (3.3 +/- 1.8 msec, n = 36 vs 6.0 +/- 3.5 msec, n = 12; p = 0.010). Abeta fiber stimulation evoked polysynaptic IPSCs in 4 of 25 (16%) cells recorded from naive rat preparations and 14 of 26 (54%) SG neurons from CFA-treated rats (p < 0.001). The mean threshold intensity for IPSCs was also significantly lower in CFA-treated rats (naive: 32.5 +/- 15.7 microA, n = 25; inflamed: 21. 9 +/- 9.9 microA, n = 26, p = 0.013). The facilitation of Abeta fiber-mediated input into the substantia gelatinosa after peripheral inflammation may contribute to altered sensory processing.

Fig. 1.

A, Schematic diagram of the experimental setup. Extracellular recordings were made from dorsal roots, and whole-cell patch-clamp recordings were made from SG neurons in adult rat spinal cord transverse slices with a long attached dorsal root. B, Representative extracellular recording of compound action potentials evoked at graded stimulus intensities (top, 12–50 μA; bottom, 300 μA). The threshold intensities for Aβ, Aδ, and C fibers were 12, 23, and 230 μA, respectively. The stimulus duration for Aβ and Aδ was 0.05 msec and for C fibers was 0.5 msec. Calculated conduction velocities for Aβ, Aδ, and C fibers were 27.3, 8.5, and 0.8 m/sec, respectively. C, The stimulus–response relationship of Aβ and Aδ compound action potentials (n = 5).

Fig. 2.

Identification of SG and SG neurons in the transverse spinal cord slices. A, Photomicrograph of the slice preparation from naive rat showing that the SG can be identified as a translucent pale area in the superficial dorsal horn (dotted area) enabling targeting of the recording electrode to the this region. Scale bar, 600 μm. B, A representative SG neuron injected with Neurobiotin. Scale bar, 20 μm. C, A low-power photomicrograph of a slice from naive rat showing SG neurons filled with Neurobiotin. Note all neurons lie within the middle third of the dorsoventral plane of SG and have the features typical of stalk cells. The dendrites of some cells extend ventrally into deeper laminae as indicated by thearrow. Scale bar, 150 μm.

Fig. 3.

A, The top,middle, and bottom show respectively, EPSCs evoked by Aβ (14–20 μA, 0.05 msec), Aδ (32–50 μA, 0.05 msec), and C (200–500 μA, 0.5 msec) fiber intensities. Four to five traces are superimposed in each panel. Top, Aβ fiber-evoked polysynaptic EPSCs. Middle, Aδ fiber-evoked monosynaptic EPSCs. Bottom, Aδ and C fibers evoked monosynaptic EPSCs. Note that the latencies were constant for the monosynaptic EPSCs and variable in the polysynaptic responses. The above records were obtained from a single neuron. B, Polysynaptic IPSCs evoked by graded stimulation. As the intensity was increased from 15 to 40 μA, 0.05 msec, the latency of the IPSC shortened. C, The effects of strychnine (2 μm) and bicuculline (20 μm) on IPSCs. Strychnine eliminated the short-latency component of the IPSC, whereas bicuculline reduced the longer latency component.

Fig. 4.

Distribution of the minimum stimulus threshold intensities necessary for eliciting EPSCs and IPSCs in cells recorded in the SG in slices from naive and CFA-treated rats. The mean stimulus threshold intensity required to evoke EPSCs in naive and CFA-treated rats was 33.2 ± 15.1 μA (n = 57) for naive and 22.8 ± 11.3 μA (n = 62) for CFA-treated rats (p < 0.001; nested ANOVA). The mean stimulus threshold intensity required to evoke IPSCs in naive and rats with an inflamed hindpaw was 32.5 ± 15.7 μA (n = 25) for naive and 21.9 ± 9.9 μA (n = 26) for the CFA-treated rats (p = 0.013; nested ANOVA). Thearrow indicates the stimulus intensity at which an Aδ volley begins to be detected, all responses below this value are exclusively Aβ. The arrowhead represents the stimulus value at which a maximal Aβ volley is elicited. All responses elicited above this intensity are exclusively Aδ-evoked. For values between the arrow and the arrowhead, the responses evoked may be Aβ- and/or Aδ-evoked.

Fig. 5.

Distribution of the latencies of EPSCs in cells recorded in the SG in slices from naive and CFA-treated rats.A, The EPSC latencies evoked by Aβ fiber intensity (20 μA, 0.05 msec, below Aδ fiber threshold) were significantly shortened in the rats with an inflamed hindpaw; 3.3 ± 1.8 msec versus 6.0 ± 3.5 msec (p = 0.010; nested ANOVA; n = 12 in naive andn = 36 in inflamed rats). Aβ fiber-mediated EPSCs with short latencies (<2.0 msec) were only observed in the preparations from rats with an inflamed hindpaw. B, Distribution of the latencies of EPSCs evoked by supramaximal Aβ fiber stimulation intensity. Mean latency of EPSCs in CFA-treated rats was 2.6 ± 1.0 msec (n = 60), which was significantly shorter than that recorded in naive rats (3.1 ± 1.1 msec; n = 53; nested ANOVA; p< 0.05).

Fig. 6.

Aβ fiber-evoked polysynaptic EPSCs with short and variable latencies recorded in an SG cell from a slice from a rat with an inflamed hindpaw. A, The effect of stimulus intensity. As the intensity was increased (12–25 μA; 0.05 msec), the latency of the Aβ fiber-evoked EPSC shortened. The shortest latency was 1.6 msec at an intensity of 22–25 μA. Thearrowhead identifies the EPSC evoked by the lowest, and the arrow identifies the EPSC evoked by the highest stimulus strengths. B, A shift in latency of the Aβ-evoked EPSCs was observed with 20 Hz repetitive stimulation at 25 μA, indicating a polysynaptic synaptic response. Thearrow identifies the first EPSC, and thearrowhead identifies the last EPSC in the train.C, Stimulation of the dorsal root by a peripheral suction electrode and the entry zone with a focal electrode were performed to calculate the conduction velocity of fibers responsible for the evoked EPSC. Conduction velocity calculated by the difference of latencies was 32.5 m/sec (length of dorsal root, 19.5 mm).