GABAA and 5-HT3 Receptors Are Involved in Dorsal Root Reflexes: Possible Role in Periaqueductal Gray Descending Inhibition

Yuan Bo Peng,1 Jing Wu,3 William D. Willis,3 and Daniel R. Kenshalo2

 1Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research and  2Center for Scientific Review, National Institutes of Health, Bethesda, Maryland 20892; and  3Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1069


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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES

Peng, Yuan Bo, Jing Wu, William D. Willis, and Daniel R. Kenshalo. GABAA and 5-HT3 Receptors Are Involved in Dorsal Root Reflexes: Possible Role in Periaqueductal Gray Descending Inhibition. J. Neurophysiol. 86: 49-58, 2001. The dorsal root reflex (DRR) is a measure of the central excitability of presynaptic inhibitory circuits in the spinal cord. Activation of the periaqueductal gray (PAG), a center for descending inhibition of spinal cord nociceptive transmission, induces release of variety of neurotransmitters in the spinal cord, including GABA and serotonin (5-HT). GABA has been shown to be involved in generation of DRRs. In this study, pharmacological agents that influence DRRs and their possible mechanisms were investigated. DRRs were recorded in anesthetized rats from filaments teased from the cut central stump of the left L4 or L5 dorsal root, using a monopolar recording electrode. Stimulating electrodes were placed either on the left sciatic nerve or transcutaneously in the left foot. Animals were paralyzed and maintained by artificial ventilation. Drugs were applied topically to the spinal cord. A total of 64 units were recorded in 34 Sprague-Dawley rats. Peripheral receptive fields were found for nine of these units. In these units, DRRs were evoked by brush, pressure, and pinch stimuli. Nine units were tested for an effect of electrical stimulation in the periaqueductal gray on the DRRs. In eight cases, DRR responses were enhanced following PAG stimulation. The background activity was 4.2 ± 1.9 spikes/s (mean ± SE; range: 0-97.7; n = 57). The responses to agents applied to the spinal cord were (in spikes/s): artificial cerebrospinal fluid, 7.1 ± 3.6 (range: 0-86.9; n = 25); 0.1 mM GABA, 16.8 ± 8.7 (range: 0-191.0; n = 22); 1.0 mM GABA, 116.0 ± 26.5 (range: 0.05-1001.2; n = 50); and 1.0 mM phenylbiguanide (PBG), 68.1 ± 25.3 (range: 0-1,073.0; n = 49). Bicuculline (0.5 mM, n = 27) and ondansetron (1.0 mM, n = 10) blocked the GABA and PBG effects, respectively (P < 0.05). Significant cross blockade was also observed. It is concluded that GABAA receptors are likely to play a key role in the generation of DRRs, but that 5-HT3 receptors may also contribute. DRRs can be modulated by supraspinal mechanisms through descending systems.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Somatosensory information is generally considered to originate in the peripheral terminals of primary afferent neurons and is then transmitted to the spinal cord or brain. The primary afferents make synapses with second-order neurons that relay the information to higher centers. However, under certain circumstances, activity in primary afferent neurons can be generated within the spinal cord and can travel antidromically toward the periphery along the sensory fibers, a phenomenon called the dorsal root reflex (DRR). DRRs were first described by Gotch and Horsley (1891), following which there was a detailed study by Barron and Matthews (1935a,b, 1938a-c). The DRR is triggered by suprathreshold depolarization of the central terminals of primary afferent fibers, or primary afferent depolarization (PAD) (Barron and Matthews 1935; Eccles et al. 1961; Schmidt 1971; Toennies 1938). The negative dorsal root potential (DRP) reflects the primary afferent depolarization and is considered to underlie one form of presynaptic inhibition (Eccles et al. 1962, 1963a; Lloyd 1952; Lloyd and McIntyre 1949; reviewed by Rudomin and Schmidt 1999). PAD can be detected by intracellular recordings from primary afferent fibers (Eccles and Krnjevic 1959; Eccles et al. 1963a; Koketsu 1956), by extracellular field potentials (Eccles et al. 1963a), and by measurement of enhanced excitability of the primary afferent fibers (Eccles et al. 1963a; Madrid et al. 1979; Rudomin et al. 1980; Wall 1958; Willis et al. 1976). PAD has been detected not only in large myelinated afferents but also in fine afferent fibers, including both Adelta and C fibers (Calvillo et al. 1982; Carstens et al. 1979; Fitzgerald and Woolf 1981; Hentall and Fields 1979; Whitehorn and Burgess 1973). While DRRs have usually been recorded from large myelinated fibers (Brooks and Koizumi 1956; Eccles et al. 1961; Toennies 1938), there is evidence that DRRs can also be conducted in Adelta fibers and in C fibers (Jänig and Zimmermann 1971; Lin et al. 2000; Toennies 1938). DRRs in fine afferents may contribute to neurogenic inflammation by releasing substance P and calcitonin gene-related peptide from sensory nerve endings and causing plasma extravasation and vasodilation (Lin et al. 1999; Rees et al. 1995; Sluka et al. 1993; reviewed by Willis 1999).

It has been suggested that PAD results from the action of GABA released from inhibitory interneurons and acting on GABAA receptors located on the synaptic terminals of primary afferent fibers because PAD can be blocked by administration of GABAA-receptor antagonists, such as bicuculline or picrotoxin (Curtis and Lodge 1982; Curtis et al. 1971, 1982; Duchen 1986; Eccles et al. 1963b; Levy 1974, 1977; Levy and Anderson 1972; Levy et al. 1971; Sivilotti and Nistri 1991). Behavioral and DRR studies using a rat inflammation model also demonstrated the involvement of GABAA receptors (Rees et al. 1995; Sluka et al. 1993, 1994). GABAA antagonists only reduce but do not abolish the DRP (Wall 1994). Following intra-arterial administration of either GABAA or 5-HT2 receptor antagonists, the DRP was reduced to 20% of the control level (Thompson and Wall 1996), whereas 5-HT1A and 5-HT3 receptor antagonists had no significant effect. Radioligand binding studies have demonstrated the presence of binding sites for 5-HT1, 5-HT2 and 5-HT3 receptor ligands in the dorsal half of the spinal cord (Glaum and Anderson 1988; Leysen et al. 1982; Monroe and Smith 1983; Pazos et al. 1985). A high density of 5-HT3 receptors is found in the superficial dorsal horn at all levels of the spinal cord (Hamon et al. 1989). They are mainly restricted to lamina I (Hamon et al. 1989; Kidd et al. 1993). These may reflect the presence of 5-HT3 receptors on primary afferent fibers because it is known that 5-HT3 receptors are present on dorsal root ganglion cells, including those belonging to C fibers (Todorovic and Anderson 1990a,b). 5-HT3 receptors are directly linked to the opening of nonselective monovalent cation channels (Derkach et al. 1989; Yaksh 1985). Opening of the channels by 5-HT3 permits the passage of Na+ and K+ and thus depolarizes the cell (Wallis and Elliott 1991).

GABAA and 5-HT3 receptors have been shown to be involved in descending inhibition of spinal cord nociceptive neurons following stimulation in the periaqueductal gray (PAG) (Peng et al. 1996c; cf. Proudfit et al. 1980). In a preliminary study, GABAA and 5-HT3 receptors were found to be involved in the initiation of the DRRs when agonists of those receptors are applied to the dorsal surface of the spinal cord (Peng et al. 1996d). The hypothesis of this study is that PAG activation leads to PAD and the generation of DRRs through release of GABA and serotonin, which activate GABAA and 5-HT3 receptors on the central terminals of primary afferent fibers. The aim of this study is to understand the mechanisms underlying the generation of DRRs by PAG stimulation.


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INTRODUCTION
METHODS
RESULTS
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Animal preparation

Adult Sprague-Dawley rats were initially anesthetized with pentobarbital sodium (50 mg/kg ip). A catheter was placed in the jugular vein for continuous administration of anesthetic and a tracheotomy allowed insertion of a tracheal cannula for artificial ventilation. In some animals, craniotomy was performed to allow placement of a PAG-stimulating electrode. The left sciatic nerve was also exposed for stimulation in some experiments. A 4-cm-long laminectomy was performed over the lumbosacral enlargement to expose the spinal cord. The rat was held in a stereotaxic frame to prevent any movement during recording. The skin over the laminectomy was elevated to form a pool that was filled with light mineral oil. Continuous anesthesia was accomplished by giving a mixture of 50 mg sodium pentobarbital in 9 ml 0.9% NaCl at a rate of 1.0 ml/h. Paralysis of the musculature was achieved by intravenous injection of 0.5 ml (1 mg/ml) pancuronium bromide every 2 h. The end tidal CO2 was maintained at around 30 mmHg. The animal's body temperature was maintained at 37°C by a feedback-controlled electric heating blanket.

All procedures used in this study were approved by the Animal Care and Use Committees of National Institute of Dental and Craniofacial Research and the University of Texas Medical Branch and followed the guidelines for the treatment of animals of the International Association for the Study of Pain (Zimmermann 1983).

Data acquisition

EXTRACELLULAR SINGLE-FIBER RECORDINGS FROM THE CENTRAL STUMP OF THE DORSAL ROOT. For electrophysiological recordings, a silver wire hook electrode was used to record extracellular single-unit discharges in filaments of the L4 or L5 dorsal roots. A small strand of the dorsal root was teased centrally from the main trunk and was further separated into a filament containing a single active fiber (Fig. 1). The dorsal root filament was wrapped around the recording electrode. In some cases, DRRs could be evoked by mechanical stimulation of a receptive field located in the skin. In those units, DRRs were recorded in response to graded intensities of mechanical stimuli delivered to the receptive field. Background activity and responses to brush, pressure, and pinch stimuli were recorded for 10 s each with a 20-s inter-stimulus interval. The rationale for examining receptive fields was to show that activation of the primary afferent fibers in the receptive field could evoke antidromic action potentials in different primary fibers, which implies an interaction between primary afferents.



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Fig. 1. Illustration of method for dorsal root reflex (DRR) recordings. A single strand of teased central stump of the dorsal root was placed over a recording electrode from which single fiber activity was recorded, as indicated by A, inset, where the top panel was the rate histogram of the spike wavemark seen in the bottom panel. Pharmacological agents were applied to the dorsal surface of the spinal cord. In a separate protocol, a stimulating electrode was placed around the sciatic nerve and evoked DRR activity was recorded from the central stump of the dorsal root filament, as indicated by B, inset, in which 2 different units were recorded at different latencies. black-triangle, the stimulus artifact.

The SPIKE2 computer software program and CED 1401Plus, a multi-channel data-acquisition system by Cambridge Electronic Design, were used to record the timing and amplitude of action potentials and to analyze data on- or off-line. The advantage of this program is that it can differentiate between action potentials of different dorsal root units by amplitude and assign a colored and numbered template to each spike. Each channel can be duplicated to demonstrate various plotting modes simultaneously. The most commonly used features of the SPIKE2 software in this experiment were wavemarks (the raw spike data) and frequency histograms.

ELECTRICAL STIMULATION. To evoke DRRs from the periphery, a tripolar electrode was used to stimulate the sciatic nerve. We used a tripolar electrode to minimize the stimulus artifact and to avoid current spread. The cathode was in the middle of the array, and two anodes, one on each side of the cathode, were separated from the cathode by 1 mm. The stimulus intensity was set at 1-2.5 times the threshold at various frequencies (0.1-50 Hz) and durations (0.1-1.0 ms).

PAG is well known to be a source of descending inhibition of spinal cord nociceptive neurons (Basbaum and Fields 1979; Fardin et al. 1984; Liebeskind et al. 1973; Liebman et al. 1970; Reynolds 1969; Richardson and Akil 1977). Stimulating in the PAG can cause a reduction of dorsal horn neuronal activity through the release of serotonin (5-HT), norepinephrine, GABA, glycine, and opioids (Peng et al. 1996a-c; Yaksh 1979), and both GABA and 5-HT produce PAD (Eccles et al. 1963b; Proudfit et al. 1980). To determine whether or not direct electrical stimulation of the PAG will elicit DRRs, a monopolar or a bipolar electrode was used to stimulate the PAG (7 mm posterior to bregma, 0.2 mm lateral, and 4.5-5 mm deep from the surface of the cerebral cortex; stimulus parameters: train of stimulus from 1 to 333 Hz at duration of 0.1-1 ms). A test of correct position of the PAG stimulating electrode was carried out by extracellular recording of single dorsal horn neurons; these were inhibited by PAG stimulation (Peng et al. 1996a-c).

PHARMACOLOGICAL MANIPULATIONS. Pharmacological agents were dissolved in artificial cerebrospinal fluid (ACSF, pH = 7.2). The ACSF contained (in mM) 151.1 Na+, 2.6 K+, 0.9 Mg2+, 1.3 Ca2+, 122.7 Cl-, 21.0 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 2.5 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>.

Since it is known that GABA and 5-HT are released by PAG stimulation (Cui et al. 1999), it is important to determine whether GABA and 5-HT3 agonists will generate DRRs following direct application to the dorsal surface of the spinal cord and whether receptor-selective antagonists of GABAA and 5-HT3 receptors will block the generation of DRRs elicited by the agonists. Pharmacological agents used in this study were: GABA, 1.0 mM (Sigma); 1-phenylbiguanide (PBG, 5-HT3 agonist), 1.0 mM (RBI); bicuculline, 0.5 mM (Sigma, GABAA antagonist); and ondansetron, 1.0 mM (5-HT3 antagonist), which was kindly provided by Glaxo Group Research. Each drug was released through a needle by dropping the drug-containing solution over the spinal cord around the dorsal root entry zone. Controls were done by releasing ACSF. The duration of drug action varies from experiment to experiment. Residual drugs were removed, along with cerebrospinal fluid, by gentle suction. The time between successive applications of drugs was based on the observation of the recovery of spontaneous activity to the control level.

Data analysis

The stored digital record of unit activity was retrieved and analyzed off-line. For single-fiber DRR recordings, responses to mechanical stimuli applied to the receptive field for 10 s were calculated by subtracting the preceding 10 s of background activity to yield a net increase in discharge rate. For responses to pharmacological applications, agonist or antagonist-induced activities were calculated in a fixed time interval of 10 s including the peak if the effect lasted longer than 10 s. If the effect was less than 10 s, it was averaged from the start to the end of the drug effect. The effect of PAG stimulation on DRRs was evaluated by calculating the ratio of the spike rate during PAG stimulation to the spike rate when PAG stimulation was turned off and reporting this as a percentage. Statistical significance was tested using one-way ANOVA followed by Dunn's test. A change was judged significant if P < 0.05. All values are presented as means ± SE.


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INTRODUCTION
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A total of 64 units were recorded in 34 Sprague-Dawley rats. Receptive fields were identified for nine units from nine animals (Fig. 2). Three of these units responded only to brush, one unit only to pressure and pinch, and five units to brush, pressure, and pinch. For these nine units with receptive fields, the average background activity and responses to brush, pressure and pinch were 0.19 ± 0.1 (range: 0-0.8 spikes/s), 12.0 ± 3.6 (range: 0-32.7 spikes/s), 17.6 ± 6.2 (range: 0-50.8 spikes/s), and 20.5 ± 7.2 (range: 0-57.5 spikes/s), respectively. In one unit, high-intensity mechanical stimulation by squeezing the nose, ears, front and hind paws elicited DRRs (Fig. 3).



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Fig. 2. Peripheral receptive fields were found for 9 units. DRRs were evoked by brush, pressure, and pinch stimuli. Top: the rate histogram (bars), spike wavemarks (the raw action potentials shown as vertical lines), and the receptive field of the DRR are shown. The wavemark is seen on a fast time base in D. B: the measurements of DRRs recorded from 9 individual units for baseline activity and responses to brush, pressure, and pinch of the receptive field. C: the responses of these 9 units. Activity is indicated as mean spike/s (±SE).



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Fig. 3. In 1 unit, high-intensity mechanical stimulation of the ears and front and hindpaws all elicited DRR responses, suggesting an involvement of descending systems. The 7 panels show the responses evoked by pinching at different places as indicated. Top: a rate histogram (spikes/s). Bottom: the wavemarks (vertical lines in mV).

Nine units from nine animals were tested for the effects of PAG stimulation on DRRs. The average antidromic spontaneous firing rate in dorsal root axons was 3.4 ± 2.1 (range: 0.25-19.5 spikes/s) when PAG stimulation was off, and the average antidromic discharge frequency during PAG stimulation was 20.4 ± 7.7 (range: 1.3-64.7 spikes/s), a statistically significant increase during PAG stimulation (paired t-test, P < 0.05; Fig. 4).



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Fig. 4. Nine units were tested for an effect of electrical stimulation of the periaqueductal gray (PAG) on the DRRs. In 8 cases, there were enhanced responses following PAG stimulation. One example is shown in A, in which the short bars above the figure indicate the period of PAG stimulation (1-s trains at 333 Hz, 800-µA intensity, 0.2 ms duration separated by 2 s). B: the sizes of the DRR discharges of 9 units are shown with (PAG-on) and without (PAG-off) PAG stimulation. The activity was generally lower when PAG stimulation was off than when it was on. C: a summary of the results and shows the mean firing rates in the absence and presence of PAG stimulation.

In 20 units, the minimum latency of DRRs evoked in the dorsal root filament was measured following stimulation of the ipsilateral sciatic nerve. There was typically a random shift of the latency of the responses to a series of sciatic nerve stimulations, as indicated by the two vertical lines in Fig. 5. The response latency in this circuit depends on three components: the latency of the primary afferent discharges from the stimulating electrode to the spinal cord, the central synaptic delay, and latency from the central terminals to the recording electrode.



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Fig. 5. DRRs evoked by stimulating the ipsilateral sciatic nerve. The minimum afferent conduction velocity was 30.1 ± 6.3 m/s (range: 3.7-118.3 m/s; n = 20). The DRRs showed a marked tendency toward latency shifting. Top: the spike evoked by single stimulation at the sciatic nerve. Bottom: the spikes evoked by repeated stimulation at 1.0 Hz in the same unit. Note the variation of the latencies over about 2 ms.

DRRs evoked by electrical stimulation in the periphery were frequency dependent (Fig. 6). Sometimes there was a wide range of stimulus intensities that could evoke DRRs. In this representative unit, ipsilateral sciatic nerve stimulation at 1.0-Hz, 0.1-ms duration, and 2.0-V intensity elicited a doublet DRR from a single unit. When the stimulation frequency was increased to 10 Hz, there was only one spike with a slightly delayed latency. However, when the stimulation frequency was increased to 20 Hz, the evoked spike was abolished. It reappeared immediately after the frequency was reset to 1.0 Hz.



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Fig. 6. The electrically evoked DRRs were frequency dependent. In this representative unit, ipsilateral sciatic nerve stimulation at 1.0-Hz, 0.1-ms duration, and 2.0-V intensity elicited a doublet discharge. When the stimulation frequency was increased to 10 Hz, there was only 1 spike with a delayed latency (the smaller spikes that appeared occasionally should be ignored). However, when the stimulation frequency was increased to 20 Hz, the evoked spike was abolished, and it reappeared immediately after the frequency was reset to 1.0 Hz.

When droplets containing GABA or 5-HT3 agonists and antagonists were applied to the dorsal surface of the spinal cord, antidromic discharges were recorded from primary afferent fibers. Typical antidromic dorsal root discharges evoked by release of GABA or PBG are shown in Fig. 7, A and B. The average spontaneous background activity was 4.2 ± 1.9 (range: 0-97.7; n = 57), and the response to ACSF was 7.1 ± 3.6 (range: 0-86.9; n = 25; Fig. 7C). There was a very large response to GABA application (Fig. 7C), which was dose dependent. The average responses to 0.1 and 1.0 mM GABA were 16.8 ± 8.7 (range: 0-191.0; n = 22) and 116.0 ± 26.5 (range: 0.1-1001.2; n = 49), respectively. The average response to 1.0 mM PBG, which is a 5-HT3-receptor specific agonist, was 68.1 ± 25.3 (range: 0-1073.0; n = 49; Fig. 7C). The increases in the antidromic dorsal root activity evoked by 1.0 mM GABA or 1.0 mM PBG were significant as compared with the background activity (1-way ANOVA, followed by Dunn's test, ***P < 0.001; *P < 0.05). No significant changes were detected in the ACSF and 0.1 mM GABA groups.



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Fig. 7. Representative antidromic dorsal root activity evoked by topical application of GABA (A) or PBG (B) over the spinal cord. A summary is shown in C. A statistically significant increase of spontaneous antidromic dorsal root activity occurred when 1.0 mM GABA or 1.0 mM PBG was applied.

When the GABAA receptor antagonist, bicuculline, was used at a concentration of 1.0 mM, in 27 units, the GABA-induced antidromic dorsal root activity was significantly attenuated from the control level (agonist only) of 166.3 ± 43.5 (range: 0.4-1,001.2; n = 27) to 16.9 ± 4.8 (agonist and antagonist; range: 0-72.3; n = 27, P < 0.05), and there was recovery (agonist only) to 81.3 ± 29.5 (range: 0.3-312.4; n = 15; Figs. 8A and 9). Similarly in 10 units, when the 5-HT3 receptor antagonist, ondansetron, was used at a concentration of 1.0 mM, the PBG-induced antidromic dorsal root activity was attenuated significantly from a control level (agonist only) of 210.4 ± 103.5 (range: 9.8-1,073.0; n = 10) to 21.4 ± 11.0 (agonist and antagonist; range: 0.1-96.3; n = 10, P < 0.05), and there was only a slight recovery (agonist only) to 34.2 ± 13.8 (range: 3.2-119.6; n = 8; Figs. 8C and 9). The effect of bicuculline on PBG-induced antidromic dorsal root activity for control (agonist only), drug (agonist and antagonist), and after washout (agonist only) was 146.6 ± 51.1 (range: 0.2-1,073.0; n = 24), 31.4 ± 12.3 (range: 0-269.5; n = 24, P < 0.05), and 21.9 ± 5.3 (range: 0.1-74.5; n = 19), respectively. The effect of ondansetron on GABA-induced antidromic dorsal root activity for control, drug, and after washout was 82.9 ± 31.5 (range: 5.4-318.7; n = 9), 16.6 ± 7.2 (range: 0-58.2; n = 9, P < 0.05), and 13.3 ± 6.4 (range: 0.2-47.9; n = 7), respectively. There were significant cross actions between the agonists and antagonists of these two receptors, i.e., bicuculline reduced PBG-induced antidromic dorsal root activity, and ondansetron markedly reduced GABA-induced antidromic dorsal root activity (Fig. 9). There was no obvious recovery.



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Fig. 8. Representative experiment showing the effects of bicuculline or ondansetron on GABA- or PBG-induced antidromic dorsal root activity. A: effect of bicuculline on GABA-induced antidromic dorsal root activity. B: effect of bicuculline on PBG-induced antidromic dorsal root activity. C: effect of ondansetron on PBG-induced antidromic dorsal root activity. D: effect of ondansetron on GABA-induced antidromic dorsal root activity. Control, agonist only; drug, agonist plus antagonist; recovery (the term indicates the process of recovery, whether or not recovery occurs), agonist only.



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Fig. 9. Summary of the effects of bicuculline or ondansetron on GABA- and PBG-induced antidromic dorsal root activity. Control, agonist only; drug, agonist plus antagonist; recovery, agonist only.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DRRs evoked by peripheral stimulation

In previous work, DRRs were evoked in the medial articular nerve in arthritic rats (Rees et al. 1994) as well as in cats and monkeys (Sluka et al. 1995) when the tissue around the joint or even on the lower limb was stimulated mechanically by firm pressure or repeated brisk taps. The same stimuli failed to evoke any activity in anesthetized animals that were not arthritic or on the contralateral side in acutely arthritic animals (Rees et al. 1994). DRRs have also been recorded from dorsal root filaments in response to cutaneous stimulation in rats following intradermal injection of capsaicin (Lin et al. 1999, 2000). Some DRR activity was seen prior to capsaicin injection, but the DRR responses were increased during the acute inflammation. In this study, we were also able to record DRRs from dorsal root filaments in noninflamed rats following stimulation of the skin or of the sciatic nerve. We could record responses following mechanical stimuli that were applied ipsilaterally (Fig. 2) or contralaterally (Fig. 3), although not from every single dorsal root fiber examined. Thus our results are consistent with cross talk between two sides of the body. This could occur at the spinal level or at a supraspinal level. There are other reports that DRRs can be induced by natural stimulation, including light touch, pressure, vibration, and muscle stretch (Millar 1979). Toennies (1938) described DRRs evoked in the saphenous nerve following stimulation of the saphenous nerve itself, adjacent dorsal roots, or other sensory nerves in the ipsilateral or contralateral limb. There was a 4-ms delay of the reflex, suggesting a polysynaptic pathway. Spread of the DRR both along and across the cord has been noted in several studies (Bagust et al. 1989, 1993; McCouch and Austin 1958; Toennies 1938). In the isolated hamster spinal cord, this spread extends over at least 16 segments from lower lumbar to upper thoracic roots, in both rostrocaudal and caudorostral directions from the stimulated dorsal root, and to the contralateral side of the cord (Bagust et al. 1989).

DRRs evoked by PAG stimulation

It has been reported that spinalization increases the antidromic discharge rate in sensory axons to a level about four times higher than the control rate; this change could last for 2 h, suggesting a tonic inhibition by supraspinal structures (Lin and Fu 1998). We did not observe a similar phenomenon in this study because we did not transect the spinal cord. On the contrary, we found that stimulation of the PAG, the origin of one of the major descending inhibitory systems, could evoke or enhance DRRs.

Latency shift and frequency dependency of peripherally evoked DRRs

The ipsilateral sciatic nerve was stimulated to determine the minimum conduction velocity of the afferents that evoked the DRRs, although the way we measured this might not be accurate because we were measuring the total latency of the reflex, and we assumed that the incoming and outgoing volleys propagated at the same conduction velocity. We found that the threshold was difficult to determine. Sometimes there was a wide range of stimulus intensities that could evoke DRRs. Usually the very first stimulus required a lower intensity than the subsequent ones. We also found that the latency of the DRR in a specific dorsal root fiber shifted from stimulus to stimulus (Fig. 5). Lin and Fu (1998) observed a similar phenomenon. They recorded DRRs at L5, and the threshold was determined by recording the afferent volley in the L6 dorsal root when the sciatic nerve was stimulated. When the stimulus intensity was changed between 2 and 40 times threshold, the latency of the afferent volley at L6 did not change, but the latency between the incoming volley and DRR at L5 changed from 7.5 ms at 5 times threshold to 3 ms at 20 times threshold. However, they did not indicate whether there was a smear shift or a jump of the latency.

In addition, we found that the DRRs evoked by stimulation of the sciatic nerve were frequency dependent. In the fiber illustrated in Fig. 6, stimulation at 1.0 Hz evoked a doublet DRR; at 10 Hz, only one spike was evoked with a slightly longer latency; at 20 Hz, DRRs were totally abolished. Previous studies reported similar findings. For example, Eccles and Willis (1962) found that DRRs in group Ia afferent fibers can release enough neurotransmitter to excite motoneurons in kittens. When a number of different peripheral nerves were stimulated at low (0.3 Hz) and high (10 Hz) frequencies, DRRs elicited at low frequencies evoked large excitatory postsynaptic potentials (EPSPs) that sometimes caused the motoneuron to discharge, but the EPSPs were subthreshold when elicited at a high frequency (Eccles and Willis 1962). This phenomenon suggests a dynamic change of the excitability of the spike generation sites near the central terminals of primary afferent fibers and is consistent with the involvement of polysynaptic circuitry in DRR generation. In a hemisected spinal cord in vitro preparation, it was found that stimulation of a lumbar dorsal root evokes a reflex in up to four adjacent spinal segments in both rostral and caudal directions, and a period of depressed activity was demonstrated following both evoked and spontaneous discharges, which could be as long as 600 ms (Bagust et al. 1985). Since the fascicles from which recordings were made were disconnected from the periphery, DRRs evoked by stimulating the sciatic nerve were speculated to be generated either by collaterals of afferent fibers interacting with the fiber from which recordings are made through an interneuronal pathway in the spinal cord or by activating a descending system from brain stem structures, such as the PAG. Both shifting of latency and frequency dependence of DRRs would suggest synaptic delays in the interneuronal circuitry within the dorsal horn or even the involvement of ascending and descending systems.

GABAA and 5-HT3 receptors are involved in DRRs

In this study, we have focused on two major neurotransmitter receptors, GABAA and 5-HT3. Pharmacological agents were applied by directly dropping them on the dorsal surface of the spinal cord near the dorsal root entry zone. We usually applied ACSF as a control first before other pharmacological agents were applied.

In one experiment, three separate units were recorded simultaneously when the sciatic nerve was stimulated at 1 Hz and 80 V. When 0.3 ml of 0.5 mM bicuculline was administered intravenously, the DRRs evoked in these three units were almost eliminated (a total of 12 evoked spikes after a series of 60 stimuli), as compared with control (58/63) and recovery (60/60). Although the effect of bicuculline was dramatic, we were not confident about the site of drug action. Instead of systemic administration of agents, later in our experiments, we chose to use local applications of the drugs. In agreement with other studies (Curtis and Lodge 1982; Curtis et al. 1971, 1982; Duchen 1986; Eccles et al. 1963b; Levy 1974, 1977; Levy and Anderson 1972; Levy et al. 1971; Sivilotti and Nistri 1991; Sluka et al. 1993, 1994), we found that GABA induced a large DRR response (Fig. 8A). This response was significantly, but not completely, blocked by the GABAA antagonist, bicuculline, applied locally to the surface of the spinal cord prior to GABA administration (Fig. 9). An explanation of the failure of bicuculline to eliminate the GABA responses completely would be that too low a dose reached the GABAA receptors.

Various 5-HT receptors have been demonstrated on dorsal root ganglion cells (Todorovic and Anderson 1990a,b) and in the dorsal half of the spinal cord (Glaum and Anderson 1988; Leysen et al. 1982; Monroe and Smith 1983; Pazos et al. 1985). Thompson and Wall (1996) found that the DRP was reduced to 20% of control level by a 5-HT2 receptor antagonist, whereas 5-HT1A and 5-HT3 receptor antagonists had no significant effect. The dose of granisetron (a 5-HT3 antagonist) used was 0.1 mg/kg with arterial injection of 0.1 ml, which seems to be a low dose as compared with the dose of ondansetron that we used. Furthermore, it is unclear where the site of action was since the drug was delivered systemically. The actual concentration at the spinal cord level would be much lower than the injected concentration. However, a high density of 5-HT3 receptors is found in the superficial dorsal horn at all levels of the spinal cord (Hamon et al. 1989; Kidd et al. 1993). It has been demonstrated that 5-HT3 receptors produce depolarization by opening nonselective monovalent cation channels (Derkach et al. 1989; Wallis and Elliott 1991; Yaksh 1985). At least some of the 5-HT3 receptors in the dorsal horn are likely to be on primary afferent terminals (Todorovic and Anderson 1990a,b).

Midbrain PAG-induced descending antinociception (Hayes et al. 1979; Oliveras et al. 1974; Reynolds 1969) has been shown to be mediated in part by descending serotonergic pathways (Carstens et al. 1981; Yezierski et al. 1982). We have implicated 5-HT3 and 5-HT1A receptors (Lin et al. 1996; Peng et al. 1996c), as well as alpha 2-adrenoreceptors (Peng et al. 1996b), and GABAA and glycine receptors in PAG inhibition of dorsal horn neurons (Lin et al. 1994; Peng et al. 1996a). The inhibition of dorsal horn neurons could be the result of both pre- and postsynaptic inhibition. In addition to GABAA receptors, 5-HT3 receptor activation is a possible contributor to this mechanism. A schematic drawing of the possible dorsal horn circuitry is shown in Fig. 10. The results of our study showed that, similar to the application of GABA, application of the 5-HT3 receptor agonist, PBG, induced large DRR responses. These were partially but significantly blocked by ondansetron, a 5-HT3 antagonist, applied to the surface of the spinal cord. Since there are both GABAA and 5-HT3 receptors on the central terminals of the primary afferent fibers, application of the GABAA receptor antagonist, bicuculline, significantly reduced GABA-induced antidromic dorsal root activity but not the tonic activity from direct activation of 5-HT3 receptors on the central terminals of the primary afferents. The reason why bicuculline significantly reduced PBG-induced antidromic dorsal root activity (Fig. 9) was because even though GABA may have been released by activation of 5-HT3 receptors on the GABAergic interneurons (Fig. 10), its effect on the central terminals of the afferents would have been reduced due to the presence of bicuculline. On the other hand, when 5-HT3 antagonist, ondansetron was applied, it would block 5-HT3 receptors on the central terminals to prevent the generation of DRRs. Furthermore, we speculate that ondansetron also had a secondary effect by blocking 5-HT3 receptors on GABAergic interneurons to reduce the release of GABA, which had acted on GABAA receptors on the primary terminals to generate DRRs (Fig. 10). This would explain how ondansetron could reduce the antidromic dorsal root activity, but externally applied GABA could still have some activity on the GABAA receptors on the primary afferent terminals. The fact that there was significant cross blockade of GABAA activity by a 5-HT3 receptor antagonist and vice versa suggests these two systems play synergistic roles in the generation of DRRs. While there was partial recovery of GABA-induced antidromic dorsal root activities following bicuculline, the absence of recovery following antagonist application in the other groups of animals might be attributed to tachyphylaxis from repeated agonist applications.



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Fig. 10. Illustration of the dorsal root circuitry that may underlie the generation of DRRs by GABA and serotonin.

Conclusion

GABAA and 5-HT3 receptors are present on the central terminals of primary afferent fibers. Activation of these receptors can induce antidromic action potentials that conduct toward the peripheral terminals of the sensory axons. Stimulation of the PAG will induce release of 5-HT in the spinal cord dorsal horn, which may directly activate 5-HT3 receptors on the central terminals of the primary afferents. Alternatively, PAG stimulation may indirectly activate GABAergic interneurons by excitation of postsynaptic 5-HT3 receptors, causing release of GABA, which in turn activates GABAA receptors on the central terminals of the primary afferents. Either mechanism could account for the generation of DRRs following PAG stimulation.


    ACKNOWLEDGMENTS

The authors thank G. Gonzales for assistance with the illustrations.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-09743 and by the National Institute of Dental and Craniofacial Research Division of Intramural Research.


    FOOTNOTES

Address for reprint requests: W. D. Willis (E-mail: wdwillis{at}utmb.edu).

Received 24 October 2000; accepted in final form 20 March 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society