Spinal neurons that possess the substance P receptor are required for the development of central sensitization

Abstract

In previous studies, we have shown that loss of spinal neurons that possess the substance P receptor (SPR) attenuated pain and hyperalgesia produced by capsaicin, inflammation, and nerve injury. To determine the role of SPR-expressing neurons in modulating pain and hyperalgesia, responses of superficial and deep lumbar spinal dorsal horn neurons evoked by mechanical and heat stimuli and by capsaicin were made after ablation of SPR-expressing neurons using the selective cytotoxin conjugate substance P-saporin (SP-SAP). Morphological analysis and electrophysiological recordings were made after intrathecal infusion of vehicle, saporin alone, or SP-SAP. SP-SAP, but not vehicle or SAP alone, produced an approximately 62% decrease in SPR-expressing neurons in the dorsal horn. Loss of SPR-expressing neurons diminished the responses of remaining neurons to intraplantar injection of capsaicin. Peak responses to 10 microg of capsaicin were approximately 65% lower in animals pretreated with SP-SAP compared with controls. Additionally, sensitization to mechanical and heat stimuli that normally follows capsaicin was rarely observed. Importantly, responses to mechanical and heat stimuli in the absence of capsaicin were not altered after SP-SAP treatment. In addition, nociceptive neurons did not exhibit windup in the SP-SAP-treated group. These results demonstrate that SPR-expressing neurons located in the dorsal horn are a pivotal component of the spinal circuits involved in triggering central sensitization and hyperalgesia. It appears that this relatively small population of neurons can regulate the physiological properties of other nociceptive neurons and drive central sensitization.

Fig. 1.

Loss of SPR-expressing neurons after intrathecal infusion of SP-SAP. A, Confocal images showing representative examples of SPR-IR in animals pretreated intrathecally with vehicle, SAP alone, or SP-SAP. A dramatic reduction in SPR-IR is evident after SP-SAP. B, Mean ± SEM percentage of neurons that express the SPR after intrathecal vehicle, SAP, or SP-SAP. The number of SPR-expressing cells was obtained from individual animals, and a mean ± SEM was calculated. This mean value was designated as 100%, and the SEM was proportionately adjusted (as a percentage) to provide a measure of variability. Data for SAP- and SP-SAP-treated groups represent the percentage of cells compared with the vehicle-treated group. ∗Significant differences from vehicle.

Fig. 2.

Location of recording sites for all dorsal horn neurons. Neurons studied in rats pretreated with vehicle, SAP, and SP-SAP were distributed in the superficial and deep dorsal horn in all groups.

Fig. 3.

The mean ± SEM number of impulses evoked by heat after pretreatment with vehicle, SAP, and SP-SAP. Responses evoked by heat stimuli of 35–51°C did not differ between groups before capsaicin injection.

Fig. 4.

Responses evoked by 10 μg of capsaicin are diminished in animals pretreated with SP-SAP. A, Responses of single WDR neurons to intradermal injection of capsaicin from vehicle-, SAP-, and SP-SAP-treated rats. Bin size is 500 msec.B, Mean ± SEM number of impulses per 15 sec interval after capsaicin. Arrows indicate the time of injection. ∗Significant difference between vehicle and SP-SAP groups. Responses to capsaicin were weaker in animals that received SP-SAP compared with those that received vehicle or SAP alone.

Fig. 5.

Sensitization of nociceptive neurons to mechanical stimuli (178 mN bending force) after 10 μg of capsaicin does not occur in animals pretreated with SP-SAP. Left panels, Representative examples illustrating responses of WDR neurons to mechanical stimuli before and after capsaicin in vehicle-, SAP-, and SP-SAP-treated groups. RFs are indicated by the stippled area, and test sites for mechanical stimulation are indicted by the dots within the RF. Arrows point to specific test sites at which pairs of responses (before and after capsaicin) were obtained. The capsaicin injection is indicated by the×. Horizontal bars denote time of stimulation (2 sec). Right panels, Mean ± SEM number of impulses evoked by a single mechanical stimulus before and after capsaicin in animals pretreated with vehicle, SAP, or SP-SAP. ∗Significant difference after capsaicin compared with before capsaicin.

Fig. 6.

Sensitization to heat produced by 10 μg of capsaicin in animals pretreated with intrathecal infusion of vehicle.A, Responses of an HT neuron to heat stimuli of 41, 47, and 51°C before and after capsaicin. Also shown is the localization of the recording site for these neurons and its RF (stippled area). The arrow points to location of capsaicin injection. B, Mean ± SEM number of impulses evoked by heat stimuli before and after capsaicin for all neurons. Mean responses to heat increased after capsaicin. C, Mean ± SEM heat threshold for all neurons before and after capsaicin. Response threshold decreased after capsaicin. ∗Significant difference between mean values obtained before and after capsaicin.

Fig. 7.

Sensitization to heat in SAP-treated animals after 10 μg of capsaicin. A, Responses of a WDR neuron to heat stimuli before and after capsaicin. Also shown are the recording site and the RF (stippled area) for this neuron. Thearrow points to location of capsaicin injection.B, Mean ± SEM number of impulses evoked by heat stimuli before and after capsaicin for all neurons. Responses to heat increased after capsaicin. C, Mean ± SEM heat threshold for all neurons before and after capsaicin. Response threshold decreased after capsaicin. ∗Significant difference after capsaicin.

Fig. 8.

Lack of capsaicin-evoked sensitization to heat in animals pretreated with SP-SAP. A, Responses of a WDR neuron to heat stimuli before and after capsaicin. The recording site and the RF (stippled area) are shown for this neuron. The arrow points to location of capsaicin (10 μg) injection. B, Mean ± SEM number of impulses evoked by heat stimuli before and after capsaicin for all neurons. There was a tendency for heat-evoked responses to decrease after capsaicin.C, Mean ± SEM heat threshold for all neurons before and after capsaicin.

Fig. 9.

Neuronal discharges evoked by 100 μg of capsaicin do not differ between groups pretreated with vehicle or with SP-SAP. The mean number of impulses and the temporal profile of the capsaicin-evoked responses were nearly identical for both groups. Responses shown are the mean ± SEM number of impulses evoked during each consecutive 15 sec interval after capsaicin. Thearrow indicates the time of injection.

Fig. 10.

Capsaicin (100 μg) produces sensitization after pretreatment with vehicle, but this sensitization fails to occur after SP-SAP pretreatment. Top row, Data from animals pretreated with vehicle. A, Mean ± SEM number of impulses evoked by a von Frey monofilament (178 mN) before and after capsaicin. B, Mean ± SEM number of impulses evoked by heat stimuli before and after capsaicin. C, Mean ± SEM heat response threshold before and after capsaicin.Bottom row, Responses to mechanical (D) and heat (E,F) stimuli before and after capsaicin in animals pretreated with SP-SAP. ∗Significant difference after capsaicin.

Fig. 11.

Nociceptive spinal neurons in SP-SAP-treated animals do not exhibit windup. A, Responses to electrical stimulation of a WDR neuron recorded from a vehicle- (top) and an SP-SAP (bottom)-pretreated animal. Responses are shown for the 1st, 6th, and 12th electrical stimulus. The time frame of the C-fiber component is within thedashed line. The C-fiber response was facilitated in the vehicle-treated animal on the 6th and 12th trial but not in the SP-SAP-treated animal. Arrows indicate time of electrical stimulation. B, Mean ± SEM normalized C-fiber responses evoked by 12 successive electrical stimuli in rats pretreated with vehicle or SP-SAP. Responses were normalized to the response evoked by the first stimulus.