INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mechanical dynamic allodynia is a distressing symptom characterized by the inappropriate perception of tactile stimuli as being painful (Fromm 1993). This clinical condition is often encountered in trigeminal neuropathic syndromes. For instance, it has been found that weak and innocuous stimuli, such as light touch, hair movement, or chewing are the most effective triggers for eliciting attacks of trigeminal neuralgia. The trigger point is frequently located in the oral and perioral region (Fromm 1993). Several mechanisms have been advanced (see Rappaport and Devor 1994 for review). Fromm (1993) suggested that an alteration of segmental inhibitory mechanisms in the spinal trigeminal nucleus oralis (Sp5O) is implicated in the pathogenesis of trigeminal neuralgia. This hypothesis is based on the fact that the mechanoreceptive fields of the Sp5O nociceptive and non-nociceptive neurons frequently are localized to intraoral and perioral regions (see, e.g., Dallel et al. 1999) that encompass the usual sites of tactile trigger points of trigeminal neuralgia. Moreover, most of the drugs effective against trigeminal neuralgia (e.g., carbamazepine) have been shown to enhance the segmental inhibitory mechanisms that modulate the properties of Sp5O neurons (Fromm 1993). In agreement with this finding, altering glycinergic inhibition results in the miscoding of non-noxious inputs as being noxious. For instance, strychnine, a glycine receptor antagonist, causes innocuous tactile stimulation to evoke behavioral, cardiovascular, and neuronal responses comparable to those elicited by noxious stimuli (Sorkin and Puig 1996; Yaksh 1989).

The role of glycine in the Sp5O is unknown. This subnucleus has recently been implicated in the processing of nociceptive information coming mainly from the oral region (Dallel et al. 1999). It contains numbers of convergent (wide dynamic range) neurons that share most of the features of convergent neurons in the lamina V of the spinal dorsal horn and the spinal trigeminal nucleus caudalis (Sp5C). In the present study, we evaluated the effect of systemic administration of strychnine on the responses of Sp5O convergent neurons evoked by innocuous peripheral electrical and mechanical stimulation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The methods used for animal preparation and anesthesia, stimulation, neuronal recording, and classification were similar to those detailed previously (Dallel et al. 1999). Briefly, male Sprague-Dawley rats weighing 250-300 g were anesthetized with halothane (0.5%) in 67% N₂O and 33% O₂, immobilized with pancuronium bromide (0.5 mg/h), and artificially ventilated. The percentage of expired CO₂ and rectal temperature were maintained at 3.5-4.5% and 37°C, respectively. Single neuronal activity was recorded extracellularly from histologically confirmed sites in the Sp5O. Neurons were classified as convergent based on their responses both to mechanical and percutaneous electrical stimulation applied to their receptive fields (RFs). Innocuous mechanical stimuli included air puffs, brushing with a soft brush, gentle stroking, and light pressure with a blunt probe. Noxious mechanical stimuli consisted of heavy pressure, pinprick, and pinching with fine forceps. Electrical square-wave stimuli (0.1-2 ms, 0.1-0.5 Hz) were applied through a pair of stainless steel stimulating electrodes inserted subcutaneously into the center of the previously delineated RF.

The experimental procedure consisted of a sequence of 15 electrical shocks of 0.1 Hz frequency and 0.1 ms duration applied to the excitatory RF at the threshold for A-fiber activation. Each sequence was separated by a 5-min interval. The threshold was defined as the lowest value that elicited 1-2 spikes/trial in at least four to six trials. When two successive control sequences with a variation of <10% in the magnitude of A-fiber-evoked responses was recorded, a single dose of strychnine (0.2, 0.4, or 0.8 mg/kg) was slowly injected intravenously. Only one neuron per rat was studied.

The effects of strychnine on innocuous mechanical stimulation were also assessed. Stimulation consisted of 20 air puffs (50 ms duration, 2 Hz) delivered through a 19-gauge needle at 2 Hz. The frequency and duration of the air puffs were controlled with a stimulator connected to a CED 1401plus interface (Cambridge Electronic Design), a personal computer (Spike 2.09 software), and an electronically controlled valve (Sherman et al. 1997). The stimulus was applied tangentially to the surface of the hairy skin at sufficient force to deflect the hairs 0.5-1 cm from the tip of the needle.

The mean of the two controls was taken as the reference value for subsequent calculation of strychnine effect. The effect was then expressed as percentage increase or decrease in the number of spikes with reference to the control. The data were analyzed with analysis of variance (ANOVA) followed by the Neuman-Keuls test. The level of significance was set at P < 0.05. Results are expressed as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The effects of strychnine were assessed on 36 Sp5O convergent neurons. Most of the neurons had an ipsilateral RF that included the intraoral or perioral region. They were sensitive both to innocuous and noxious mechanical stimuli. When percutaneous electrical stimulation was applied, responses attributable to peripheral activation of A and C fibers could be observed. The A-fiber-evoked responses were obtained at a mean threshold of 0.98 ± 0.13 mA and a mean latency of 1.24 ± 0.05 ms. Based on an approximate distance of 50 mm between the stimulating electrode and the Sp5O, the conduction velocity was ~40 m/s, which corresponds to the A fibers.

The majority of neurons had no spontaneous activity before drug administration and strychnine did not produce any firing of the neurons in the absence of stimulation but produced a rapid, dose-dependent and reversible increase of the A-fiber-evoked responses for 83% (n = 30) of the Sp5O convergent neurons (Fig. 1). The cumulative results obtained from 25 neurons are presented in Fig. 2. The facilitatory effects of strychnine on A-fiber-evoked responses were apparent as soon as 5 min after the injection. At this time point, the responses were increased to 155 ± 22% (P < 0.03, n = 6), 218 ± 31% (P < 0.001; n = 11), and 243 ± 23% (P < 0.001; n = 8) of the initial value following 0.2, 0.4, and 0.8 mg/kg of intravenous strychnine, respectively. The effects of strychnine lasted for ~25 min with 0.2 and 0.4 mg/kg strychnine and ~35 min with 0.8 mg/kg strychnine.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Individual examples of the effects of three doses of strychnine (0.2, 0.4, and 0.8 mg/kg i.v. from top to bottom) on A-fiber-evoked responses of three spinal trigeminal nucleus oralis (Sp5O) convergent neurons induced by electrical stimulation. Poststimulus histograms (15 trials) were built before (control) and after strychnine administration. Note the dose-dependent effect on A-fiber-evoked responses and its reversal.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Cumulative results showing the time course of the effects of three doses of strychnine (0.2, 0.4, and 0.8 mg/kg i.v.) on A-fiber-evoked responses of Sp5O convergent neurons.

Strychnine (0.8 mg/kg i.v.) also produced profound changes in the responses of the Sp5O convergent neurons (n = 11) to air-jet stimulation (Fig. 3). At 5 min after the injection of strychnine, responses to air-jet stimulation were increased to 326 ± 88% of the control value (P < 0.007).

View larger version (12K): [in this window] [in a new window]   Fig. 3. A: individual example showing the effect of intravenous strychnine (0.8 mg/kg) on air-jet-evoked responses of one Sp5O convergent neuron. Poststimulus histograms (20 trials) were built before (control) and after strychnine administration. Note that after strychnine, air jet stimulation evoked a prolonged unit response with a prolonged afterdischarge. B: cumulative results showing the time course of the effects of intravenous strychnine (0.8 mg/kg) on air-jet-evoked responses of Sp5O convergent neurons.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present results are the first report that systemic strychnine increases the electrically evoked A fiber responses of Sp5O convergent neurons in a dose-dependent fashion and potentiates their responses to innocuous mechanical stimulations such as air-jet stimulation. A similar increase of the A-fiber-evoked responses of Sp5C (Yokota et al. 1979) and lumbar dorsal horn convergent neurons (Duggan et al. 1981) has also been reported after intravenous strychnine. In the spinal cord, local application of strychnine also enhanced the responses of motoneurons (Sivilotti and Woolf 1994) and convergent neurons to low-threshold cutaneous stimuli (Peng et al. 1996; Sorkin and Puig 1996). Interestingly, the blockade of glycinergic transmission in the spinal cord not only affects segmental responses but is also reflected supraspinally in the thalamus (Sherman et al. 1997). The time course of the changes in neuronal activity observed in the present experiment was consistent with previous behavioral (Sakai et al. 1979; Yaksh 1989) and physiological studies (Peng et al. 1996; Sorkin and Puig 1996).

Because strychnine was administered systemically, its exact site and mechanism of action cannot be determined but some hypotheses can be advanced. First, strychnine may act directly at the level of the Sp5O. The presence in the Sp5O of a high density of glycinergic receptors (Zarbin et al. 1981), glycinergic neurons, and glycinergic nerve endings (Rampon et al. 1996) is compatible with this idea. Thus strychnine could increase the excitability of Sp5O neurons or remove the inhibition exerted on Sp5O convergent neurons. It has been suggested that one control of the central neurons could originate from the tonic inhibition exerted by nociceptive primary afferents (A and C fibers) (Calford and Tweedale 1991). However, this hypothesis is unlikely because intrathecal opioids or neonatal treatment with capsaicin, which specifically block or destroy nociceptive primary afferents, did not affect the cardiovascular, motor (Sherman and Loomis 1996; Yaksh 1989), and spinal dorsal horn responses evoked by innocuous stimulation (Sorkin and Puig 1996) after strychnine treatment in rats. On the other hand, several arguments suggest that strychnine alters the inhibition mediated by A fibers. Indeed, anatomic findings have shown that glycine-containing cells receive significant synaptic inputs from low-threshold myelinated primary afferents (Todd and Spike 1993). Furthermore, Game and Lodge (1975) showed that strychnine blocked the early inhibition of deep dorsal horn neurons evoked by volleys in large myelinated cutaneous afferent fibers.

Consistent with this idea, physiological studies have shown that the RF of many cells, particularly the convergent neurons, is often characterized by a central excitatory RF activated both by noxious and non-noxious stimuli surrounded by an inhibitory area where most innocuous mechanical stimuli are able to inhibit the neuron's activity (Wall 1988). Strychnine may block this mechanism, presumably mediated by glycinergic neurons driven by low-threshold cutaneous afferents. In turn, these changes may influence convergent neurons to produce an expansion of the excitatory portion of their receptive fields (Sherman et al. 1997; Sorkin and Puig 1996). Thus a low-threshold stimulus will now be able to activate convergent neurons in the peripheral zone of their RF and then activate pathways normally activated by noxious stimuli only (Dubner et al. 1987). This mechanism may apply to Sp5O convergent neurons. In other respects, spinal and trigeminal convergent neurons have been shown to receive weak excitatory input from areas of skin immediately adjacent to or surrounding their RFs (Dostrovsky 1999). This area of skin constitutes the cell's subliminal fringe and natural stimulation within this area does not normally excite the cell to fire impulses. The strengthening of such input resulting from the alteration of the excitability of the Sp5O convergent neurons is likely to occur following strychnine administration. Such a possibility is indeed supported by earlier findings reporting extensive afferent convergence in Sp5O neurons (Hu et al. 1992).

Previous studies established that electrical stimulation of the the periaqueductal gray matter (PAG) and the nucleus raphe magnus (NRM) inhibits the Sp5O neurons (Chiang et al. 1989; Lovick and Wolstencroft 1979; Sessle and Hu 1981). It appears that the modulatory influence exerted by the PAG and the NRM on the Sp5O neurons is direct because anatomical (Basbaum et al. 1978; Li et al. 1993; Lovick and Wolstencroft 1983) and electrophysiological (Lovick and Wolstencroft 1982; Sessle and Hu 1981; Watabe et al. 1985) studies have shown that the PAG and the NRM project to the Sp5O in the rat and the cat. Thus strychnine may remove the supraspinal descending inhibitory action exerted on Sp5O glycinergic neurons. Consistent with this idea, microdialyzed strychnine attenuated the inhibition induced by NRM or PAG stimulation of the responses of spinal convergent neurons to mechanical stimulation (Peng et al. 1996; Sorkin et al. 1993). However, opposite results have been reported (Belcher et al. 1978; Johnston and Davies 1981) and intravenous or iontophoretic strychnine in amounts sufficient to reduce segmental inhibition failed to reduce tonic supraspinal inhibition (Duggan et al. 1981).

Alternatively, strychnine acting directly or indirectly on the Sp5C could decrease ascending inhibitory controls that exert on the Sp5O through intranuclear trigeminal pathways (Jacquin et al. 1990). In support of this hypothesis, immunohistochemical studies have demonstrated the presence of a high density of glycine receptors and glycinergic neurons in the Sp5C (Rampon et al. 1996; Zarbin et al. 1981) and strychnine application in the Sp5C has been shown to increase the responses of Sp5O convergent neurons both to innocuous tap and dental pulp stimulation (Khayyat et al. 1975).

In conclusion, our findings indicate that inputs conveyed to Sp5O nociceptive neurons by myelinated low-threshold mechanoreceptive afferents are under a glycinergic control, thus providing further support for the idea that alteration of the trigeminal glycinergic modulation may contribute to the dynamic mechanical allodynia that occurs in trigeminal neuropathies.