Nitric Oxide-Mediated Spinal Disinhibition Contributes to the Sensitization of Primate Spinothalamic Tract Neurons

Qing Lin, Jing Wu, Yuan Bo Peng, Minglei Cui, and William D. Willis

Department of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lin, Qing, Jing Wu, Yuan Bo Peng, Minglei Cui, and William D. Willis. Nitric oxide-mediated spinal disinhibition contributes to the sensitization of primate spinothalamic tract neurons. This study concentrated on whether an increase in spinal nitric oxide (NO) diminishes inhibition of spinothalamic tract (STT) cells induced by activating the periaqueductal gray (PAG) or spinal glycinergic and GABAergic receptors, thus contributing to the sensitization of STT neurons. A reduction in inhibition of the responses to cutaneous mechanical stimuli induced by PAG stimulation was seen in wide dynamic range (WDR) STT cells located in the deep layers of the dorsal horn when these neurons were sensitized during administration of a NO donor, 3-morpholinosydnonimine (SIN-1), into the dorsal horn by microdialysis. In contrast, PAG-induced inhibition of the responses of high-threshold (HT) and superficial WDR STT cells was not significantly changed by spinal infusion of SIN-1. A reduction in PAG inhibition when STT cells were sensitized after intradermal injection of capsaicin could be nearly completely blocked by pretreatment of the dorsal horn with a NO synthase inhibitor, 7-nitroindazole. Moreover, spinal inhibition of nociceptive activity of deep WDR STT neurons elicited by iontophoretic release of glycine and GABA agonists was attenuated by administration of SIN-1. This change paralleled the change in PAG-induced inhibition. However, the inhibition of HT and superficial WDR cells induced by glycine and GABA release did not show a significant change when SIN-1 was administered spinally. Combined with our recent results, these data show that the effectiveness of spinal inhibition can be reduced by the NO/cGMP pathway. Thus disinhibition may constitute one mechanism underlying central sensitization.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The preceding paper (Lin et al. 1999) reported that nitric oxide (NO) sensitizes a population of wide dynamic range (WDR) primate spinothalamic tract (STT) neurons in the deep dorsal horn to cutaneous mechanical stimulation of the skin. Endogenous NO is released within the dorsal horn during sensitization of STT cells induced by intradermal injection of capsaicin (CAP), and this sensitization is dependent on the production of NO.

NO formation by neurons is triggered by activation of excitatory amino acid (EAA) receptors (Garthwaite and Balazs 1978; Garthwaite et al. 1988, 1989), which results in an increased activity of nitric oxide synthase (NOS) mainly through Ca2+-dependent mechanisms (Garthwaite et al. 1988; MacDermott et al. 1986; Womack et al. 1988). An increased release of EAAs and neuropeptides takes place within the spinal cord during peripheral tissue injury or inflammation, such as intradermal CAP injection (Gamse et al. 1979; Sorkin and McAdoo 1993). As a result, the responses of STT cells to peripheral stimuli and to iontophoretically applied EAAs are greatly enhanced, apparently due to a cooperative action of EAAs and neuropeptides released by the afferent barrage and acting on N-methyl-D-aspartate (NMDA) and neurokinin receptors (Dougherty et al. 1992-1995; Dougherty and Willis 1991a,b, 1992).

On the other hand, a functional change in inhibitory amino acid (IAA) receptors could also be hypothesized to influence central sensitization. Second-messenger systems, including the NO/cGMP system, may regulate the function of glycine and GABA receptors by phosphorylation of ion channel proteins (Leidenheimer et al. 1992; Porter et al. 1990; Vaello et al. 1994; Zarri et al. 1994). We have found that descending inhibition of STT cells by stimulation in the periaqueductal gray (PAG) is profoundly reduced during sensitization of STT cells following intradermal injection of CAP or after intraspinal administration of phorbol ester, a protein kinase C (PKC) activator (Lin et al. 1996a). Furthermore, we have observed that the inhibition of STT cells produced by iontophoretic release of glycine and GABA receptor agonists was also attenuated following CAP injection or activation of PKC (Lin et al. 1996b).

The present experiments were conducted to determine whether NO attenuates spinal inhibition during central sensitization of STT cells. This was done by 1) determining if 3-morpholinosydnonimine (SIN-1) administered into the spinal cord lessens the inhibition of STT cells produced by PAG stimulation; 2) investigating whether the attenuation of PAG inhibition of STT cells induced by intradermal injection of CAP (Lin et al. 1996a, 1997a) is blocked when NOS is blocked; and 3) examining whether NO decreases the inhibition of STT cells resulting from activation of glycine and GABA receptors.

Preliminary data from this work have been reported previously (Lin et al. 1996d, 1997b).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Data were collected from 24 adult monkeys (Macaca fascicularis, 1.9-2.8 kg). Animal preparation, maintenance and the experimental approaches, such as placement of microdialysis fibers for spinal drug delivery and extracellular recordings from STT cells, were described in the preceding paper (Lin et al. 1999).

PAG stimulation

We employed electrical stimulation in the PAG, as in previous experiments (Gerhart et al. 1984; Lin et al. 1994; Zhang et al. 1991). Stimulation sites were located by introducing a monopolar steel electrode stereotaxically 3.5-4.5 mm caudal to the electrode in the ventral posterolateral nucleus, 0.5-0.75 mm lateral to the midline, and 22-25 mm deep from the surface of the cerebral cortex (Yezierski et al. 1982; Zhang et al. 1991). The electrode tip was placed at a site from which an obvious inhibition of both spontaneous and evoked STT cell activity was obtained when the PAG was stimulated electrically with a 1-s train of square pulses (333 Hz, 0.2 ms) repeated at 2-s intervals at intensities of 100-400 µA (2 times threshold for inhibition). The stimulation sites were mostly distributed in the lateral or ventrolateral parts of PAG at the level of the oculomotor or trochlear nuclei (Lin et al. 1994).

Administration of drugs

Drugs were administered in the dorsal horn in two ways. 1) SIN-1 or 7-nitroindazole (7-NINA) was delivered by microdialysis in the same doses as in the preceding paper. 2) IAA agonists were released by iontophoresis. A carbon filament (3-4 MOmega ) in the central barrel of a seven-barrel micropipette assembly was used to record extracellular activity of STT cells, and up to five other barrels were used to apply drugs iontophoretically. IAA agonists used in this study included GABA (gamma -amino-n-butyric acid; Sigma, 0.5 M, pH 5.0), muscimol (muscimol HBr; RBI, 2 mM, pH 5.0), and glycine (sodium glycinate; Sigma, 0.5 M, pH 8.0). For some cells, SIN-1 was applied by iontophoresis; the concentration in the drug barrel was 40 mM (pH 5.0). One barrel of the electrode array was filled with 0.5 M NaCl (pH 5.0) for current balance. A retaining current (10-15 nA) sufficient to prevent drug leakage was used between drug injections. IAA agonists were delivered by three graded 10-s-long current pulses to produce a graded inhibition of the activity of STT cells evoked by a sustained cutaneous Pinch stimulus during drug administration.

Experimental design

Recordings of the responses of STT neurons to cutaneous mechanical stimuli were used to classify STT neurons and to determine the responsiveness of the cells.

In one group of STT neurons, responses to mechanical stimuli applied to the cutaneous receptive field and inhibition of these responses produced by PAG stimulation were assessed when SIN-1 (10 mM) was administered by microdialysis into the spinal cord dorsal horn. SIN-1 was infused for 30-60 min, and its effects on these responses and PAG-induced inhibition were then compared with baseline. The perfusion fluid was switched back to normal artificial cerebrospinal fluid (ACSF) or saline for drug wash out after testing was completed. The responses of STT cells to mechanical stimuli and PAG inhibition were recorded every 30 min after SIN-1 infusion had been stopped to test for recovery. Some of the same STT cells were used to examine the effects of microdialysis administration of SIN-1 on the inhibition induced by iontophoresis of IAA agonists. This inhibition was evoked after the dorsal horn was treated with the same dose of SIN-1 for 30-60 min, and compared with the baseline inhibition observed before SIN-1 administration. ACSF or normal saline was then used to wash out SIN-1 from the spinal cord, and the STT cells were tested periodically until the inhibition showed partial recovery.

In some STT cells separate from those used for the above experiments, SIN-1 was applied by iontophoresis to examine its action on the inhibition induced by iontophoresis of IAAs. After control responses were recorded, SIN-1 was iontophoresed continuously (70-100 nA) during the period when a glycine or GABA agonist was delivered iontophoretically using three graded current pulses.

The third group of STT cells was used to examine the effects of a NOS inhibitor on the increased responses to mechanical stimuli and the reduction in PAG-induced inhibition that took place during central sensitization following intradermal injection of CAP. Two successive injections of CAP were given 2-3 h apart while recording from the same cell. One of the CAP injections was given during 7-NINA infusion. The experimental sequence was the same as used in the preceding paper. Briefly, 7-NINA (1 mM) was infused by microdialysis before the first CAP injection. PAG-induced inhibition was tested 15 min after the first CAP injection. 7-NINA was then washed out for 2-2.5 h, during which time PAG-induced inhibition was tested every 30 min to demonstrate recovery of the inhibition. A second CAP injection was given after the residue of 7-NINA was believed to be nearly washed out. Tests for PAG inhibition were again performed and compared with the baseline values.

Data analysis

Responses to mechanical stimuli were analyzed in the same way as in the preceding paper. Inhibition was evaluated by calculating the percentage of inhibition of evoked activity. The average control evoked response during application of the cutaneous stimuli was determined immediately preceding the onset of each stimulus train or current pulse. The percentage of inhibition was then calculated by averaging the differences between evoked activity before and during each episode of PAG stimulation or drug ejection and expressed as percentage of inhibition. A repeated measures ANOVA tested responses in each group. If significance was obtained, post hoc testing with paired t-tests assessed differences from the baseline levels. A value of P < 0.05 was considered significant. All values are given as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Observations were made on a total of 46 STT neurons, including 43 WDR cells and 3 high-threshold (HT) cells. The recording sites were located from 944 to 1,945 µm below the surface of the spinal cord, corresponding to laminae I-V (see Lin et al. 1999). STT neurons were divided into three groups. The first group of neurons consisted of 20 WDR and 2 HT cells that were used to examine the effects of microdialysis infusion of SIN-1 on PAG- and IAA-induced inhibition. Of these, 17 WDR and 2 HT STT cells had been used for presenting the effects of SIN-1 on responses to cutaneous stimuli in the preceding paper. Seven WDR neurons in the second group were used to examine the effects of a NOS inhibitor on the reduction in PAG inhibition when STT cells were sensitized after intradermal injection of CAP. The same neurons were used in the preceding paper to describe the effects of 7-NINA on CAP-induced sensitization. The remaining 17 neurons (16 WDR and 1 HT cells) were used in experiments in which SIN-1 was administered iontophoretically while IAA agonists were released iontophoretically to test whether iontophoresis of SIN-1 could affect the IAA-induced inhibition of STT cells. These STT cells were not reported in the preceding paper.

Effects of SIN-1 on PAG-induced inhibition

As described in the previous paper (Lin et al. 1999), microdialysis administration of SIN-1 into the dorsal horn of the spinal cord consistently produced a sensitization of deep WDR STT cells to both weak and strong cutaneous mechanical stimuli. In the present experiments, we observed that the PAG-induced inhibition of responses to both weak and strong mechanical stimuli was reduced in a majority of the cells examined when they were sensitized by SIN-1. Figure 1A shows the results from a deep STT neuron that was also used in the preceding paper to show the enhanced responses to cutaneous mechanical stimuli due to SIN-1. Responses to mechanical stimuli increased during and 0.5 h after SIN-1 infusion. At the same time, PAG-induced inhibition of the responses of this neuron was profoundly reduced (Fig. 1A, 2nd row). The reduction in PAG-induced inhibition outlasted the drug infusion period (Fig. 1A, 3rd row), and this long-lasting effect had the same time course as the increase in the responses to mechanical stimuli (cf., Fig. 2A in the preceding paper, Lin et al. 1999). Responses of all 13 deep WDR cells to mechanical stimuli were enhanced, and an attenuation of the inhibition of responses to Brush, Press, and Pinch stimuli was observed in 12, 8, and 8 cells, respectively. A summary of the results from these deep (1,308-1,945 µm) WDR STT cells (Fig. 1B) shows that the average baseline inhibition of the responses to Brush, Press, and Pinch stimuli was -62 ± 4% (mean ± SE), -80 ± 3%, and -80 ± 3%, respectively. The PAG-induced inhibition of these three graded mechanical-evoked responses was reduced to -31 ± 5%, -49 ± 11%, and -59 ± 7%, respectively, during SIN-1 infusion, and a significant reduction continued even when SIN-1 infusion had been stopped for 0.5 h.



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Fig. 1. A: rate histograms from a representative deep wide dynamic range (WDR) spinothalamic tract (STT) neuron (1,400 µm) showing the changes in periaqueductal gray (PAG)-induced inhibition of responses to Brush, Press, and Pinch stimuli when the spinal dorsal horn was perfused with 3-morpholinosydnonimine (SIN-1) by microdialysis. Responses to mechanical stimuli were increased, and PAG-induced inhibition was reduced during SIN-1 infusion (2nd row). Both effects outlasted the drug infusion even when the drug was washed out (3rd row). The upward marks in the lower part of each histogram indicate the timing of PAG stimulation with 1-s trains. B: bar graph summarizing the grouped data from deep WDR STT neurons (n = 13) for inhibition of responses to mechanical stimuli elicited by stimulating the PAG when the spinal dorsal horn was perfused SIN-1 by microdialysis. *P < 0.05; **P < 0.01, ***P < 0.001, compared with the baseline level.



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Fig. 2. A and B: rate histograms and bar graphs from a representative deep WDR STT neuron (1,456 µm) showing the changes in PAG-induced inhibition of responses to Brush, Press, and Pinch stimuli (A) and in responses to mechanical stimuli (B) produced by capsaicin (CAP) injection and the effects of spinal infusion of 7-nitroindazole (7-NINA) by microdialysis. The upward marks in the lower part of each histogram indicate the timing of PAG stimulation with 1-s trains. The spinal cord was pretreated with 7-NINA for 30-60 min after baseline recordings. Row and bars labeled "15 min after 1st CAP" show the effects of CAP injection during 7-NINA infusion. After that, 7-NINA was washed out, and recordings were made 2 h after 7-NINA. Row and bars labeled "15 min after 2nd CAP" show the effects of CAP injection without treatment with 7-NINA. C: bar graphs summarizing the grouped data (n = 7) for the effects of 7-NINA infusion on the responses in PAG inhibition to CAP injection. The responses to CAP injection while the spinal cord was pretreated with 7-NINA are shown by the left set of bars of each group [1st CAP (7-NINA)], and the responses to CAP injection in which 7-NINA within the spinal cord was washed out for 1.5-2 h are shown by the right set of bars of each group [2nd CAP (ACSF)]. **P < 0.01; ***P < 0.001, compared with the pre-CAP value (Baseline).

In contrast, no significant change in PAG-induced inhibition of WDR STT neurons that were located in superficial layers of the dorsal horn and of HT neurons was seen when SIN-1 was administered into the spinal cord by microdialysis. We reported in the preceding paper that SIN-1 administration produced inconsistent effects on the responses of these STT cells to mechanical stimuli. Seven superficial WDR (depths of 980-1,224 µm) and 2 HT cells were tested for the effects of SIN-1 on PAG-induced inhibition of their responses to mechanical stimuli. An attenuation of the inhibition of responses to Brush, Press, and Pinch stimuli was observed in only three, one, and one cell, respectively.

Effects of a NOS inhibitor on the reduction in PAG-induced inhibition following intradermal injection of capsaicin

PAG-induced inhibition of the responses of STT cells to peripheral stimulation has been shown to be partially blocked when STT cells are sensitized by intradermal CAP injection (Lin et al. 1996a, 1997a). Figure 2A shows rate histograms for a representative deep WDR STT cell that show the effects of intradermal CAP injections on PAG-induced inhibition with and without intraspinal infusion of 7-NINA. Figure 2B shows bar graphs depicting the changes in the total evoked responses of the cell to mechanical stimuli following two successive injections of CAP. Neither the responses of the cell nor PAG-induced inhibition were obviously changed by a 30-min infusion of 7-NINA (2nd row of Fig. 2A and 2nd column of Fig. 2B). The cell was not sensitized and PAG inhibition was unchanged following the first CAP injection (3rd row of Fig. 2A and 3rd column of Fig. 2B). A second CAP injection was given after 7-NINA was washed out for 2 h. Responses to Brush and Press stimuli were enhanced and the inhibitory effects of stimulation in the PAG were reduced (bottom row of Fig. 2A and last column of Fig. 2B). The grouped results from seven deep WDR STT cells are summarized in Fig. 2C. The absence of PAG-induced inhibition when the spinal cord was pretreated with 7-NINA is shown by the left sets of bars [1st CAP (7-NINA)], and the PAG inhibition without 7-NINA pretreatment is shown in the right sets of bars [2nd CAP (ACSF)].

Effects of spinal administration of SIN-1 on the inhibition of STT neurons induced by iontophoretic release of glycine and GABA agonists

We also examined whether the inhibition of STT cells mediated by spinal glycine and GABA receptors was affected when the NO level within the dorsal horn was elevated. Observations were made in the same experiments in which effects of SIN-1 on PAG-induced inhibition were examined. In some of these STT cells, inhibition was evoked by iontophoretic release of glycine and GABA agonists onto the STT cells during microdialysis of SIN-1. Figure 3 is an example showing the inhibition of a deep WDR STT cell produced by glycine and GABA agonists (this cell was also used in Fig. 1 to show the effects of SIN-1 on PAG-induced inhibition). When this cell was sensitized by infusion of SIN-1, IAA-induced inhibition of activity evoked by a prolonged noxious Pinch stimulus was profoundly reduced (2nd row). The time course of this reduction of IAA responses paralleled that of the reduction in PAG-induced inhibition. Recovery was seen ~2 h after the SIN-1 infusion was stopped. Seven deep WDR cells (1,308-1,644 µm) were tested and the attenuation of glycine-induced inhibition was found in all seven cells and of GABA- and muscimol-induced inhibition in six cells. All of these changes were significantly different from the baseline levels (Fig. 4A).



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Fig. 3. Rate histograms from the same deep WDR cell as shown in Fig. 1A for PAG inhibition. Changes in the inhibition of Pinch-evoked activity produced by iontophoretic release of glycine, GABA, and muscimol when the spinal dorsal horn was perfused with SIN-1 by microdialysis are shown. Top row: baseline responses. Second row: responses recorded during SIN-1 infusion. Third and bottom rows: responses obtained 0.5 and 2 h after the end of drug infusion, respectively. Graded current pulses used for delivering drugs iontophoretically are indicated by upward-or downward-going square waves below each histogram. A Pinch stimulus was applied to the receptive field while agonists were released iontophoretically.



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Fig. 4. Bar graphs summarizing the grouped data from deep WDR STT neurons for the effects of SIN-1 administration on the inhibition of Pinch responses elicited by iontophoretic release of glycine and GABA agonists. A: changes in inhibitory amino acid (IAA)-induced inhibition when SIN-1 was infused into the spinal dorsal horn by microdialysis (n = 7). B: changes in IAA-induced inhibition when SIN-1 was applied onto STT cells iontophoretically (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001, compared with the baseline level.

In contrast, Fig. 5 shows recordings of the effects of SIN-1 on IAA-induced inhibition of a superficial WDR cell (980 µm). The IAA-induced inhibition of this cell was not reduced during infusion of SIN-1. In fact, the inhibitions elicited by glyicne and GABA were potentiated. Two superficial WDR cells and two HT cells were tested with SIN-1. Among these, the IAA-induced inhibition was not obviously reduced, except for glycine in one cell. There were no statistically significant changes in inhibition for the grouped data (not shown).



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Fig. 5. Rate histograms from a superficial WDR cell (980 µm) demonstrate the lack of obvious changes in the inhibition of Pinch-evoked activity produced by iontophoretic release of glycine, GABA, and muscimol when the spinal dorsal horn was perfused with SIN-1 by microdialysis. Top row: baseline responses. Second row: responses recorded during SIN-1 infusion. Bottom row: responses obtained 0.5 h after the end of drug infusion. Graded current pulses used for delivering drugs iontophoretically are indicated by upward- or downward-going square waves below each histogram. A Pinch stimulus was applied to the receptive field while agonists were released iontophoretically.

The effects of NO on IAA-induced inhibition were also tested by applying SIN-1 iontophoretically onto STT cells. In a group of cells, including 16 WDR and 1 HT neurons (944-1,700 µm), SIN-1 was iontophoresed continuously (70-100 nA) while a glycine or GABA agonist was delivered iontophoretically by three graded current pulses. A reduction in IAA-induced inhibition was seen in most of the deep WDR cells (1,336-1,700 µm). However, SIN-1 applied by iontophoresis did not produce as long an effect as that produced by microdialysis. As shown in Fig. 6, the inhibition of the Pinch-evoked responses of a deep WDR cell (1,628 µm) produced by IAAs was reduced during continuous iontophoresis of SIN-1 (70 nA), but the inhibition recovered within 0.5 h. An attenuation of inhibition induced by glycine was observed on all cells tested (9/9). A reduction in GABA- and muscimol-induced inhibition was seen on eight (8/10) and six (6/7) cells, respectively. These changes were significantly different from the baseline levels (Fig. 4B).



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Fig. 6. Rate histograms represent changes in the inhibition of Pinch-evoked activity of a deep WDR cell produced by iontophoretic release of glycine, GABA, and muscimol when SIN-1 was administered (70 nA) iontophoretically. Top row: baseline inhibitory responses. Second row: inhibitory responses recorded during iontophoresis of SIN-1. Third row: inhibitory responses obtained 0.5 h after cessation of drug ejection. Graded current pulses used for delivering drugs iontophoretically are indicated by upward- or downward-going square waves below each histogram. A Pinch stimulus was applied to the receptive field while agonists were released iontophoretically.

The effects of iontophoresis of SIN-1 on GABA-induced inhibition were also tested on six superficial WDR cells (944-1,234 µm) and one HT cell (1,600 µm), but a reduction in inhibition was seen in only two superficial WDR cells. Tests for the effects of iontophoresis of SIN-1 on glycine- and muscimol-induced inhibition were completed in six and five cells, respectively. A reduction in inhibition produced by glycine and muscimol was seen only in two neurons (1 superficial WDR and 1 HT cell), respectively. No statistically significant change in inhibition in this group of cells was observed during SIN-1 application.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In a recent study (Lin et al. 1997a), we found that a long-lasting enhancement of the responses of STT neurons to peripheral mechanical stimuli was accompanied by a profound attenuation of PAG-induced inhibition when the spinal dorsal horn was perfused with the cGMP analogue, 8-bromo-cGMP. The present experiments show that the central sensitization of STT cells induced by exogenous administration of a NO donor, SIN-1, is also associated with a reduction in PAG-induced inhibition. Parallel with results using 8-bromo-cGMP, sensitization of responses to mechanical stimuli and attenuation of PAG-induced inhibition of these responses were consistently seen only in WDR STT neurons located in the deep layers of the dorsal horn and not in superficially located WDR STT cells or HT STT cells. The reduction in PAG-induced inhibition during the sensitization of these deep WDR cells to mechanical stimuli following intradermal injection of CAP (Lin et al. 1996a) could be prevented by pretreatment with an nNOS inhibitor, 7-NINA.

Moreover, in this study we have found that spinal inhibition of nociceptive activity of deep WDR STT neurons mediated by glycine and GABA receptors became less effective during administration of SIN-1. This paralleled the change in inhibition resulting from PAG stimulation, indicating that changes in the effectiveness of spinal inhibition during central sensitization could be due to an action of second-messenger pathways on IAA receptors (Lin et al. 1996b). Combined with our observations on the effects of PKC and cGMP on the inhibition induced by PAG stimulation and by IAA receptor activation (Lin et al. 1996a,b, 1997a), our current data strengthen the evidence that secondary hyperalgesia and allodynia may reflect in part a reduction in the effectiveness of spinal inhibitory mechanisms by certain second-messenger systems, such as the PKC and NO/cGMP cascades.

Glycine and GABA are well established as inhibitory transmitters in the spinal dorsal horn. Immunocytochemistry demonstrates numerous glycine and GABA immunoreactive neurons in laminae I-IV of the dorsal horn (Mitchell et al. 1993; Powell and Todd 1992; Proudlock et al. 1993; Todd 1990; Todd and Sullivan 1990). Glycine and GABA immunoreactive terminals are found to connect with other dorsal horn neurons, including STT cells, in the formation of local circuits that exert an inhibitory modulation of spinal nociceptive transmission (Carlton et al. 1992; Mitchell et al. 1993; Powell and Todd 1992; Todd 1990). Our previous work suggested that glycine and GABA produce a tonic inhibition of STT cells by a postsynaptic action mediated by IAA receptors on STT cells and, in the case of GABA, by presynaptically depressing glutamate release from primary afferents (Lin et al. 1994, 1996c; Willcockson et al. 1984). Glycine and GABA release were shown to contribute to the inhibition of nociceptive responses by pathway descending from the brain (Lin et al. 1994; McGowan and Hammond 1993; Sorkin et al. 1993). It is thus plausible that a reduced effectiveness of spinal inhibition that takes place during central sensitization would exacerbate the central sensitization.

nNOS immunoreactivity has been verified in the dorsal root ganglia (Ruda et al. 1994; Zhang et al. 1993) and in afferent terminals of the dorsal horn, as well as in neurons in laminae I/II and X (Dun et al. 1993; Saito et al. 1994; Traub et al. 1994; Valtschanoff et al. 1992). Experiments have indicated that NO production increases when peripheral tissue is damaged or inflamed (Lam et al. 1996; Wu et al. 1998a,b) and that the subsequent elevation in intracellular cGMP levels may be a primary mode of action for NO on spinal nociceptive transmission (Meller et al. 1992a,b; Meller and Gebhart 1993). Because NO is a diffusible molecule, acting in a nonsynaptic manner, it can diffuse a considerable distance to reach its target (Baringa 1991), where it acts to change the excitability of neurons by modulating the release of neurotransmitters and synaptic efficacy (Boulton et al. 1994; Montague et al. 1994; Sorkin 1993; Zhuo et al. 1993, 1994). In addition, it is also possible that NO may act on the neuron in which it is produced, or it may diffuse from its site of production to act on adjacent neurons (Bult et al. 1990; Meller and Gebhart 1993). Primary afferent terminals in laminae I and II synapse on NOS-positive dendrites (Bernardi et al. 1995). These dendrites belong to NOS-positive interneurons in laminae II and III (Valtschanoff et al. 1992). A finding that is especially pertinent to the present study is that an increase in NO production can reduce GABA-gated currents in cerebellar granule cells and in retinal amacrine cells (Wexler et al. 1998; Zarri et al. 1994). Our experiments show that elevation of NO by SIN-1 administration within the spinal dorsal horn can also reduce the inhibitory actions mediated by glycine and GABA receptors on deep WDR STT cells. Superficial WDR and HT STT cells are unaffected. The effectiveness of the inhibition produced by stimulation in the PAG is also reduced when STT cells are sensitized by spinal SIN-1 administration, and sensitization after intradermal injection of CAP is NOS dependent. We believe that one mechanism underlying the reduced inhibitory effect of PAG stimulation when the NO level is increased is a functional change in both glycine and GABA receptors, which contribute to the inhibition of dorsal horn neurons following PAG stimulation (Lin et al. 1994; McGowan and Hammond 1993; Sorkin et al. 1993). This could be one of the mechanisms that helps develop and maintain central sensitization.

The reasons why NO did not change significantly the inhibition of most HT and superficial WDR STT cells mediated by PAG stimulation and IAA release remains unclear. We have reported a similar lack of effect of intraspinal administration of 8-bromo-cGMP on PAG-induced inhibition of superficial WDR and HT STT cells (Lin et al. 1997a). As discussed in the preceding paper (Lin et al. 1999), HT and superficial WDR STT neurons may normally have a different function from that of the deep WDR STT cells (Chung et al. 1986; Dougherty and Willis 1992; Ferrington et al. 1987). On the other hand, we have noticed that the changes in PAG- and IAA-induced inhibition of responses to peripheral stimulation due to SIN-1 administration seem to depend on the cell's responsiveness to SIN-1. This implies that NO probably influences the excitability of particular kinds of STT cells by modulating receptor functions and/or synaptic transmission to the cells. Others have found that action of NO depends on the cell type on which NO acts (Pehl and Schmid 1997; Schmid and Pehl 1996).

In summary, we find that the inhibition of STT cells mediated by activating the descending pathway from the PAG or by iontophoresis of glycine and GABA is reduced when STT cells are sensitized by perfusion of the spinal dorsal horn with a source of exogenous NO. This effect is mainly seen in WDR STT neurons located in the deep layers of the dorsal horn. Additionally, attenuation of the PAG-induced inhibition when STT cells are sensitized after intradermal injection of CAP is dependent on NO. It is suggested that the NO/cGMP pathway, when activated, may attenuate inhibitory synaptic transmission in the spinal cord and that this action contributes to the development and maintenance of central sensitization.


    ACKNOWLEDGMENTS

The authors thank K. Gondesen, G. Robak, and Drs. E. Al-Chaer and Yi Feng for technical and collegial assistance in preparation of the experimental animals, and G. Gonzales for expert assistance with the illustrations.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-09743 and NS-11255.

Present address of Y. B. Peng: PNMB/NIDR/NIH, Bldg. 49, Rm. 1WW14, 49 Convent Dr., Bethesda, MD 20892-4410.


    FOOTNOTES

Address for reprint requests: W. D. Willis, Dept. of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 April 1998; accepted in final form 4 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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