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Adenosine Inhibits GABAergic and Glycinergic Transmission in Adult Rat Substantia Gelatinosa Neurons

Department of Physiology, Saga Medical School, Saga 849-8501, Japan

Submitted 26 April 2004; accepted in final form 14 June 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The effect of adenosine on inhibitory postsynaptic currents (IPSCs) was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Adenosine reversibly reduced the amplitude of GABAergic and glycinergic electrically evoked IPSCs (eIPSCs) in a dose-dependent manner (EC₅₀ = 14.5 and 19.1 µM, respectively). The A₁ adenosine-receptor agonist N⁶-cyclopentyladenosine also reduced the eIPSCs, whereas the A₁ antagonist 8-cyclopentyl-1,3-dimethylxanthine reversed the inhibition produced by adenosine. In paired-pulse experiments, the ratio of the second to first GABAergic or glycinergic eIPSC amplitude was increased by adenosine, whereas the response of SG neurons to exogenous GABA or glycine was unaffected. Adenosine reduced the frequency of GABAergic and glycinergic spontaneous IPSCs without changing their amplitude. This reduction in frequency disappeared in the presence of a K⁺-channel blocker (4-aminopyridine) but not in the absence of Ca²⁺. The inhibition by adenosine disappeared in the presence of cyclic-AMP analog (8-Br-cyclic AMP) and adenylate-cyclase activator (forskolin) but not protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate). We conclude that adenosine suppresses inhibitory transmission in SG neurons by activating presynaptic A₁ receptors and that this action is mediated by K⁺ channels and cyclic AMP but not by Ca²⁺ channels and PKC. This inhibitory action of adenosine probably contributes to the modulation of pain transmission in the SG.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
In the spinal dorsal horn, adenosine is released from neurons and glial cells or produced from ATP and modulates synaptic transmission. Adenosine is also involved in various physiological functions including protecting neurons from the excitotoxic effects of L-glutamate during ischemia and regulating nociceptive transmission (for review, see Brundege and Dunwiddie 1997; Dunwiddie and Masino 2001; Salter et al. 1993; Sawynok 1998). Adenosine usually inhibits excitatory transmission (e.g., see Bagley et al. 1999; Deuchars et al. 2001; Lambert and Teyler 1991; Oliet and Poulain 1999; Scanziani et al. 1992; Shen and Johnson 2003; Thompson et al. 1992; Wu and Saggau 1994), whereas its actions on inhibitory transmission vary among different types of neurons. Adenosine receptors have been classified into A₁, A_2a, A_2b, and A₃ types (Brundege and Dunwiddie 1997). A₁ agonists inhibit GABAergic transmission in the periaqueductal gray (PAG) (Bagley et al. 1999), the hypothalamic supraoptic nucleus (Oliet and Poulain 1999), the subthalamic nucleus (Shen and Johnson 2003), and the immature hippocampal CA1 area (Jeong et al. 2003). This inhibition, however, is not in the adult CA1 area (Lambert and Teyler 1991), the CA3 area (Thompson et al. 1992), and the lateral horn (Deuchars et al. 2001). In contrast, A_2a agonists increase GABAergic transmission in the globus pallidus (Shindou et al. 2001, 2002). With respect to glycinergic transmission, Umemiya and Berger (1994) found an A₁ receptor-mediated inhibition and A₂ receptor-mediated potentiation in the brain stem. However, there are few studies comparing the actions of adenosine on glycinergic and GABAergic transmission.

The superficial dorsal horn, particularly the substantia gelatinosa (SG; lamina II of Rexed), plays an important role in modulating nociceptive transmission (for review, see Mason 1999; Melzack and Wall 1965; Willis and Coggeshall 1991). Glutamatergic transmission to SG neurons is inhibited by analgesics such as opioids (Kohno et al. 1999), baclofen (Ataka et al. 2000; Iyadomi et al. 2000), serotonin (Ito et al. 2000), nociceptin (Luo et al. 2002), and noradrenaline (Kawasaki et al. 2003). Adenosine A₁ receptors are present at a high density in the SG (Ackley et al. 2003; Choca et al. 1988; Geiger et al. 1984; Goodman and Snyder 1982; Schulte et al. 2003). We have previously reported that adenosine inhibits glutamatergic transmission by activating presynaptic A₁ receptors (Lao et al. 2001, 2004; also see Ackley et al. 2003; Li and Perl 1994; Park et al. 2002; Patel et al. 2001), an action also found in other areas of the CNS. This may mediate the antinociception produced by adenosine analogs administered intrathecally (Sawynok 1998). GABAergic and glycinergic inhibitory interneurons are involved in polysynaptic pathways originating in primary-afferent terminals (Coggeshall and Carlton 1997; Iyadomi et al. 2000; Kohno et al. 1999, 2000; Luo et al. 2002; Todd et al. 1996; Willis and Coggeshall 1991), and probably serve to modulate nociceptive transmission (e.g., see Coull et al. 2003; Moore et al. 2002). A facilitatory action of adenosine on inhibitory transmission may contribute to its antinociceptive effect; for example, midazolam facilitates GABAergic transmission (Kohno et al. 2000). Adenosine receptors appear to be expressed predominantly on intrinsic dorsal horn neurons (perhaps GABAergic or glycinergic; see Schulte et al. 2003), since adenosine-binding sites were minimally reduced after dorsal root rhizotomy or neonatal capsaicin (Choca et al. 1988; Geiger et al. 1984). However, the effect of adenosine on GABAergic and glycinergic transmission in the spinal dorsal horn remains unclear. In this study, we use the whole cellpatch-clamp technique in spinal cord slices to investigate the effect of adenosine on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) in SG neurons.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
All animal experiments were approved by the Animal Care and Use Committee of Saga Medical School.

Slice preparation

Spinal cord slices from adult rats were prepared as described (Ito et al. 2000; Iyadomi et al. 2000; Yang et al. 2001). In brief, adult Sprague-Dawley rats (7–8 wk old; 250–300 g) were anesthetized with urethane (1.5 g/kg body weight, ip), and a laminectomy was performed to extract a lumbosacral spinal cord segment. The spinal cord was quickly immersed in ice-cold (1–3°C) Krebs solution containing (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl₂, 1.2 NaH₂PO₄, 1.2 MgCl₂, 25 NaHCO₃, and 11 glucose, bubbled with 95% O₂-5% CO₂. Rats were killed by exsanguination. A transverse slice (500 µm thick) was cut using a microslicer (DTK-1000, Dousaka, Kyoto, Japan) in oxygenated ice-cold Krebs solution. The slice was transferred to the recording chamber (volume: 1.5 ml) and continuously perfused with preheated (35 ± 1°C) and oxygenated Krebs solution for 1 h before recordings.

Whole cell voltage-clamp recordings and focal stimulation

The SG can be identified under a steromicroscope as a translucent band across the dorsal horn (Nakatsuka et al. 1999; Yang et al. 2001). Spinal cord slices could be maintained for 12 h when they were superfused at 10–15 ml/min with preoxygenated Krebs solution at 35 ± 1°C. Whole cell voltage-clamp recordings from single SG neurons were stable for 4 h. The recorded neurons were located at the center of SG to avoid recordings from laminae I and III neurons. Patch pipettes were filled with solution containing (in mM) 110 Cs₂SO₄, 0.5 CaCl₂, 2 MgCl₂, 5 EGTA, 5 HEPES, 5 Mg-ATP, and 5 tetraethylammonium (TEA)-Cl, and had a resistance of 5–8 M. The holding potential (V_H) used to record IPSCs was 0 mV, as reported previously (Iyadomi et al. 2000; Kohno et al. 1999, 2000; Luo et al. 2002), unless otherwise mentioned. In some experiments, another patch-pipette solution, where 110 mM Cs₂SO₄ in the preceding-mentioned solution was replaced by 80 mM Cs₂SO₄ and 30 mM CsCl, and tonicity was adjusted to an original one by adding sucrose, was also used to record IPSCs having a considerable amplitude at –70 mV. Cs⁺ and TEA were added to inhibit K⁺ channels located postsynaptically in the recorded SG neurons, which may be opened by adenosine receptors (Li and Perl 1994; Patel et al. 2001), and to easily shift V_H to 0 mV from resting membrane potentials (Yang et al. 2001).

Electrically evoked IPSCs (eIPSCs) were triggered at 0.1 Hz by stimulating SG neurons with rectangular pulses (duration, 0.1 ms) using an extracellular monopolar silver-wire electrode (50 µm diam; isolated except for the tip) located within 150 µm of the recorded neurons; the stimulus intensity was monitored with a digitized output isolator. Paired-pulse stimulation with a short time interval (30–80 ms) was also used to obtain eIPSCs. All signals were amplified by an AxoPatch 200B amplifier (Axon Instruments, Foster City, CA), digitized at 333 or 500 kHz with an A/D converter (Digidata 1200A or 1322, Axon Instruments) and stored on a personal computer using the pCLAMP 6 or 8 data acquisition program (Axon Instruments).

Data analysis

The signals were analyzed off-line using an AxoGraph 4.0 (Axon Instruments). Spontaneous IPSCs (sIPSCs) were automatically analyzed with a variable amplitude template and visually examined to find out whether erroneous sIPSC events were recorded; if so, the template was changed and the analysis was repeated. This process was repeated until erroneous sIPSC events were not noted. The frequency and amplitude of sIPSC was calculated from sIPSC events measured for 1 min. Data are means ± SE, and statistical significance was set at P < 0.05 using a paired Student's t-test (unless otherwise mentioned) or a Kolmogorov-Smirnov test. In all cases, n refers to the number of neurons studied.

Application of drugs

All drugs were applied by switching the perfusion solution to one containing the drug at a known concentration using a three-way tap. The perfusion rate or temperature was not altered. Drug-containing solutions reached to the recording chamber within 10 s. Drugs used were N⁶-cyclopentyladenosine (CPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Tocris Neuramin (Bristol, UK); GABA, glycine, (–)-bicuculline methiodide, strychnine, 4-aminopyridine (4-AP), 7-acetoxy-1,6,9-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), 8-Br-adenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP), phorbol 12,13-dibutyrate (PDBu), and N-ethylmaleimide (NEM) from Sigma (St. Louis, MO); and adenosine and TTX from Wako (Osaka, Japan). All drugs except TTX, GABA, glycine, bicuculline, strychnine, and 4-AP (where distilled water was used as a solvent) were first dissolved in dimethyl sulfoxide at 1,000 times the concentration to be used and diluted to the desired concentration in Krebs solution immediately before use. Adenosine and 8-Br-cyclic AMP were directly dissolved in Krebs solution. The osmotic pressure of nominally Ca²⁺-free, high-Mg²⁺ (5 mM) Krebs solution was adjusted by lowering the Na⁺ concentration.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
SG neurons had resting membrane potentials more negative than –55 mV (when measured in a current-clamp mode), and exhibited glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) at a V_H of –70 mV. No sIPSCs were observed at this V_H, because their reversal potential was near –70 mV. When V_H was shifted to 0 mV, sIPSCs were recorded, whereas sEPSCs disappeared because their reversal potential was close to 0 mV (Iyadomi et al. 2000; Kohno et al. 1999, 2000; Luo et al. 2002). The frequency and amplitude of sIPSCs was unaffected by TTX (0.5 µM; data not shown), as reported previously (see Iyadomi et al. 2000), indicating that the production of the sIPSCs was independent of the spontaneous activity of neurons presynaptic to SG neurons.

Adenosine reduces both GABAergic and glycinergic eIPSC amplitudes

GABAergic and glycinergic eIPSCs were evoked, respectively, in the presence of the glycine-receptor antagonist strychnine (1 µM) and the GABA_A-receptor antagonist bicuculline (10 µM). The non–N-methyl-D-aspartate (non-NMDA) receptor antagonist CNQX (10 µM) was added to block glutamatergic neurotransmission. GABAergic eIPSCs were longer in duration by about threefold than glycinergic ones, as reported previously (Kohno et al. 1999, 2000; Luo et al. 2002).

Adenosine (100 µM) superfused for 2 min reversibly reduced the peak amplitude of the GABAergic eIPSCs (Fig. 1A). This action was maximal 2 min after the application of adenosine and disappeared 8 min after washout. A reduction of >5% was observed in 11 of 12 neurons tested; the peak amplitude was 70 ± 4% (P < 0.01) of control (229 ± 33 pA, n = 11). The remaining neuron did not respond to adenosine. The peak amplitude of glycinergic eIPSCs was also reduced by adenosine (100 µM) with a similar time course to that of GABAergic ones (Fig. 1B). A reduction of >5% was observed in seven of eight neurons examined; the peak amplitude was 52 ± 5% (P < 0.01) of control (281 ± 70 pA, n = 7). The remaining neuron did not respond to adenosine. The effect of adenosine on GABAergic and glycinergic eIPSCs was examined in a concentration range of 0.1–1,000 µM. The reductions of GABAergic and glycinergic eIPSC amplitudes increased with adenosine concentration (Fig. 2, A and B). The effective concentrations of adenosine for half-reduction (EC₅₀s) of GABAergic and glycinergic eIPSC amplitudes were 14.5 and 19.1 µM, respectively.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Adenosine reversibly reduces the amplitude of electrically evoked inhibitory postsynaptic currents (eIPSCs) in substantia gelatinosa (SG) neurons. A and B: time courses of the peak amplitudes of GABAergic and glycinergic eIPSCs, respectively, under the action of adenosine (100 µM), relative to control. Top: averaged traces of 3 consecutive GABAergic and glycinergic eIPSCs, respectively, before (a), during the action of adenosine (b), and 8 min after washout (c); results of a–c correspond to those in the graphs given below traces. Krebs solution contained CNQX (10 µM) together with strychnine (1 µM; A) or bicuculline (10 µM; B). Holding potential (VH) = 0 mV.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Adenosine dose-dependently reduces eIPSC amplitude in SG neurons. A and B: peak amplitudes of GABAergic and glycinergic eIPSCs, respectively, in the presence of adenosine at various concentrations, relative to control, which were plotted against the logarithm of adenosine concentration. Results in A and B were obtained from 4–8 and 3–6 neurons, respectively; the continuous curve was drawn according to the Hill equation with an EC50 value of 14.5 (A) or 19.1 µM (B). VH = 0 mV.

A₁ receptors mediated the decreases of eIPSC produced by adenosine

First we examined whether the A₁ agonist CPA mimics the reductions of eIPSC amplitudes by adenosine. CPA (1 µM) superfused for 2 min reduced GABAergic eIPSC amplitudes by >5% in six of seven cells examined [to 53 ± 7% (P < 0.05) of control (172 ± 32 pA, n = 6); Fig. 3A]. The remaining cell was insensitive to CPA. Glycinergic eIPSCs were also reduced in amplitude to 57 ± 14% (P < 0.05) of control (164 ± 71 pA, n = 4; Fig. 3B). We then examined whether the A₁ antagonist DPCPX (1 µM) affects the reductions of eIPSC produced by adenosine (100 µM). DPCPX by itself did not affect GABAergic and glycinergic eIPSC amplitudes [100 ± 2% of control (233 ± 74 pA, n = 4) and 96 ± 2% of control (287 ± 147 pA, n = 3), respectively; Fig. 3, C and D, top left]. In neurons that responded to adenosine, pretreatment with DPCPX for 4 min reversed the inhibition of GABAergic and glycinergic eIPSC by adenosine [amplitude: 96 ± 3% of control (232 ± 71 pA, n = 4) and 94 ± 5% of control (270 ± 140 pA, n = 3), respectively; Fig. 3, C and D].

View larger version (18K): [in this window] [in a new window]   FIG. 3. Reductions of eIPSC amplitude by adenosine are mediated by A1 receptors in the SG. A and B: averaged traces of 3 consecutive GABAergic and glycinergic eIPSCs, respectively, before (Control), during the action of the A1 agonist CPA (1 µM; left), and 7 min after washout (right). C and D: averaged traces of 3 consecutive GABAergic and glycinergic eIPSCs, respectively, in the absence and presence of adenosine (100 µM) with (top) and without (bottom) pretreatment with the A1 antagonist DPCPX (1 µM). Note that DPCPX alone did not affect GABAergic and glycinergic eIPSCs (top left). Krebs solution contained CNQX (10 µM) together with strychnine (1 µM; A and C) or bicuculline (10 µM; B and D); VH = 0 mV.

The reduction of eIPSC by adenosine was studied in paired-pulse experiments. GABAergic eIPSCs exhibited paired-pulse depression with a ratio of the second to first eIPSC amplitudes of 0.882 ± 0.025 (n = 6; range, 0.833–0.969). Adenosine (100 µM) increased this ratio to 1.41 ± 0.17 (Fig. 4A), a value significantly larger than control (P < 0.02). This change in paired-pulse depression was observed for glycinergic eIPSCs as well (Fig. 4B); the ratio (1.21 ± 0.07) in the presence of adenosine was larger than control (0.819 ± 0.087, n = 4; P < 0.02).

View larger version (26K): [in this window] [in a new window]   FIG. 4. Reductions of eIPSC amplitude by adenosine in SG neurons are presynaptic. A and B: average traces of 3 consecutive GABAergic and glycinergic eIPSCs, respectively, by a paired stimulus with a time interval of 50 (A) or 30 ms (B) before (left), under the action of adenosine (100 µM; middle), and their superimposition (right; where the 1st eIPSC in the paired eIPSC in the presence of adenosine was scaled to control). C and D: current responses evoked by exogenous GABA (1 mM; C) or glycine (1 mM; D), respectively, in the absence (left) and presence (right) of adenosine (100 µM). In this and following figures, horizontal bars above records indicate the period of time during which the drugs were applied. VH = 0 mV.

Adenosine reduces GABAergic and glycinergic sIPSC frequency but not amplitude by activating A₁ receptors

Two kinds of GABAergic and glycinergic sIPSCs were recorded from SG neurons, as reported previously (Iyadomi et al. 2000; Kohno et al. 1999, 2000; Luo et al. 2002). Adenosine (100 µM) superfused for 2 min reversibly reduced the frequency of GABAergic sIPSCs, which are observed in the presence of strychnine (1 µM; Fig. 5A). When cumulative distributions of the inter-event interval and amplitude of GABAergic sIPSC were examined in the absence and presence of adenosine, a proportion of sIPSCs having a longer inter-event interval was increased by adenosine, while there was not a significant change in the cumulative distribution of sIPSC amplitude. GABAergic sIPSCs were reduced in frequency to 42 ± 7% (P < 0.01) of control (1.61 ± 0.32 Hz, n = 7), whereas the amplitude was unchanged [97 ± 3% (P > 0.05) of control (34.4 ± 5.4 pA, n = 7)].

View larger version (35K): [in this window] [in a new window]   FIG. 5. Adenosine reduces the frequency but not amplitude of spontaneous IPSCs (sIPSCs) in SG neurons. A and B: continuous chart recordings of GABAergic and glycinergic sIPSCs, respectively, in the absence and presence of adenosine (100 µM). Traces given below the recordings show sIPSCs, which are shown in an expanded scale in time, recorded consecutively for a period indicated by a bar shown just below the recordings. Bottom: cumulative distributions of the inter-event interval (left) and amplitude (right) of sIPSCs, before (straight line) and during (dotted line) the action of adenosine, which were obtained by analyzing events measured for 1 min (1,235 and 494 events in A; 2,672 and 595 events in B), respectively. Adenosine had no effect on the amplitude distribution (P > 0.3) while shifting the interval distribution to a longer one (P < 0.01; Kolmogorov-Smirnov test). Krebs solution contained strychnine (1 µM; A) or bicuculline (10 µM; B); VH = 0 mV.

With respect to pharmacological properties of the inhibition of sIPSCs by adenosine, pretreatment with DPCPX (1 µM) for 4 min reversed the reduction of GABAergic sIPSC frequency by adenosine (100 µM) [93 ± 3% (P > 0.05) of control (1.64 ± 0.33 Hz, n = 5) 2 min after its application; data not shown]. About 15 min after washout of DPCPX, when examined in the same cells, adenosine (100 µM) decreased the frequency to 60 ± 9% (P < 0.05) of control (1.59 ± 0.38 Hz, n = 5; data not shown). Glycinergic sIPSCs were also not reduced in frequency by adenosine in the presence of DPCPX [97 ± 3% (P > 0.05) of control (1.05 ± 0.23 Hz, n = 5); data not shown]. After washout of DPCPX, adenosine reduced the frequency to 73 ± 9% (P < 0.05) of control (1.03 ± 0.25 Hz: data not shown). Like adenosine, CPA (1 µM) superfused for 2 min reduced GABAergic and glycinergic sIPSC frequency [68 ± 9% (P < 0.05) of control (1.48 ± 0.37 Hz, n = 3) and 57 ± 11% (P < 0.05) of control (1.38 ± 0.44 Hz, n = 3), respectively; data not shown].

Role of presynaptic Ca²⁺ or K⁺ channels in the inhibition of sIPSC by adenosine

Since the activation of A₁ receptors is well known to suppress voltage-gated Ca²⁺ channels or open K⁺ channels (for review, see Brundege and Dunwiddie 1997), it is conceivable that these channels that may exist in presynaptic terminals are involved in the reductions of eIPSC and sIPSC produced by adenosine. In support of an involvement of action potential-dependent Na⁺/K⁺/Ca²⁺ channels in releasing GABA or glycine from nerve terminals, GABAergic eIPSCs were reversibly abolished by superfusing nominally Ca²⁺-free, high-Mg²⁺ (5 mM) Krebs solution with a fast onset and offset time (about 2 min each; n = 4; data not shown). TTX (0.5 µM) also blocked GABAergic eIPSCs in a quick and reversible manner (n = 4; data not shown). Similar results were obtained for glycinergic eIPSCs (n = 3; data not shown). Owing to such a blockade of eIPSCs in the absence of Ca²⁺, it was difficult to examine whether the eIPSC amplitude reductions by adenosine depend on extracellular Ca²⁺. We therefore examined a cellular mechanism for the sIPSC frequency reductions by adenosine. Although TTX does not affect sIPSC frequency and amplitude, the following experiments were done in the presence of TTX (0.5 µM) fearing that drugs applied may induce action potentials, the production of which affects presynaptic actions of adenosine.

The lack of extracellular Ca²⁺ reduced GABAergic sIPSC frequency by more than 5% in five of six neurons examined [to 72 ± 8% (P < 0.05) of control (1.42 ± 0.40 Hz, n = 5)], as observed for spontaneous glutamatergic transmission in the superficial dorsal horn (Hori et al. 1992). This result suggests that presynaptic Ca²⁺ channels are partially open at the resting state, resulting in a tonic Ca²⁺ entry in nerve terminals. Under this condition, adenosine (100 µM) still decreased the frequency to 53 ± 5% (P < 0.01) of control (0.99 ± 0.22 Hz, n = 5; Fig. 6A). A similar action in the absence of Ca²⁺ was observed for glycinergic sIPSCs. Removal of extracellular Ca²⁺ resulted in decreasing glycinergic sIPSC frequency [to 64 ± 12% (P < 0.05) of control (2.07 ± 0.44 Hz, n = 4)], under the condition of which adenosine (100 µM) still reduced the frequency to 51 ± 6% (P < 0.01) of control (1.98 ± 0.46 Hz, n = 4; Fig. 6A). The GABAergic and glycinergic sIPSC frequency reductions produced by adenosine in the absence of Ca²⁺ were not significantly different in extent from those in normal Krebs solution (see Fig. 7). A K⁺-channel blocker 4-AP (100 µM) increased GABAergic and glycinergic sIPSC frequency [151 ± 13% (P < 0.05) of control (1.30 ± 0.19 Hz, n = 4) and 130 ± 8% (P < 0.05) of control (0.80 ± 0.15 Hz, n = 6), respectively; Fig. 6B] as reported in other types of neurons (e.g., see Kumamoto and Kuba 1985). In the presence of 4-AP, adenosine (100 µM) did not affect GABAergic and glycinergic sIPSC frequency [102 ± 6% (P > 0.05) of control (2.05 ± 0.51 Hz, n = 4) and 102 ± 5% (P > 0.05) of control (1.04 ± 0.19 Hz, n = 6), respectively; Fig. 6B].

View larger version (27K): [in this window] [in a new window]   FIG. 6. Reductions of sIPSC frequency by adenosine are mediated by K+ channels and cyclic AMP but not by Ca2+ channels and protein-kinase C (PKC). A–D: effects of adenosine (100 µM) on GABAergic (left) and glycinergic sIPSC frequency (right) in Ca2+-free, 4-AP (100 µM)-, forskolin (100 µM)-, and PDBu (0.5 µM)-containing Krebs solution, respectively. In each bar graph, the 2nd bar indicates sIPSC frequency under each condition, relative to control, while the 4th bar shows sIPSC frequency in the presence of adenosine under each condition, relative to that before adenosine application. In this and subsequent figures, vertical (or horizontal) lines accompanied by bars show SE; statistical significance between data shown by bars is indicated by asterisks; *P < 0.05; **P < 0.01; n.s., not significant.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Summary of the effect of adenosine on sIPSC frequency under a variety of conditions. A and B: GABAergic and glycinergic sIPSC frequency reductions, respectively, by adenosine (100 µM) in normal Krebs (Adenosine), Ca2+-free Krebs (+Ca2+-free), Krebs solution containing 4-AP (100 µM; +4-AP), NEM (1 mM; +NEM), forskolin (100 µM; +Forskolin), 8-Br-cyclic AMP (100 µM; +8-Br-cAMP), and PDBu (0.5 µM; +PDBu). Number of neurons studied under each condition is indicated in parentheses. sIPSC frequency reductions produced by adenosine under the conditions except for those of +Ca2+-free and +PDBu were significantly smaller in extent than those in normal Krebs solution. **P < 0.01; n.s., not significant.

Since A₁ receptors are coupled to G-proteins (Brundege and Dunwiddie 1997), we examined the effect of adenosine (100 µM) on sIPSCs in the presence of the sulfhydryl-alkylating agent NEM, which is known to inhibit actions of G-protein–coupled receptors (Asano and Ogasawara 1986). In slice preparations pretreated for 5 min with NEM (1 mM), which itself unaltered GABAergic and glycinergic sIPSC frequency [92 ± 6% (P > 0.05) of control (3.01 ± 0.35 Hz, n = 3) and 104 ± 3% (P > 0.05) of control (2.04 ± 0.44 Hz, n = 6), respectively], adenosine did not affect their frequency [102 ± 5% (P > 0.05) of control (2.72 ± 0.17 Hz) and 97 ± 2% (P > 0.05) of control (2.12 ± 0.44 Hz), respectively; see Fig. 7].

Since A₁ receptors regulate adenylate-cyclase and protein-kinase C (PKC) pathway (Brundege and Dunwiddie 1997), the activation of which modulates synaptic transmission, we next examined whether their second messenger systems are involved in the sIPSC frequency reduction produced by adenosine. Adenylate-cyclase activator forskolin (100 µM) increased GABAergic and glycinergic sIPSC frequency [166 ± 21% (P < 0.05) of control (1.87 ± 0.44 Hz, n = 6) and 139 ± 15% (P < 0.05) of control (0.85 ± 0.19 Hz, n = 7), respectively; Fig. 6C]. In the presence of forskolin, adenosine (100 µM) superfused for 2 min failed to reduce GABAergic and glycinergic sIPSC frequency [81 ± 9% (P > 0.05) of control (2.96 ± 0.57 Hz, n = 6) and 100 ± 4% (P > 0.05) of control (1.13 ± 0.23 Hz, n = 7), respectively; Fig. 6C]. A membrane-permeable analog of cyclic AMP (8-Br-cyclic AMP, 100 µM) also exhibited a similar effect. GABAergic sIPSC frequency was not reduced by adenosine [100 µM; 109 ± 9% (P > 0.05) of control (1.65 ± 0.63 Hz, n = 3)] in the presence of this drug, which itself increased the frequency to 159 ± 13% of control (1.12 ± 0.54 Hz; data not shown). Glycinergic sIPSC frequency was also unchanged by adenosine [100 µM; 96% of control (4.02 Hz, n = 1)] in the presence of 8-Br-cyclic AMP, which itself increased the frequency to 127% of control (3.17 Hz; data not shown).

Like the cyclic AMP analog, PKC activator PDBu (0.5 µM) increased GABAergic and glycinergic sIPSC frequency [120 ± 5% (P < 0.05) of control (2.57 ± 0.22 Hz, n = 5) and 117 ± 5% (P < 0.05) of control (3.21 ± 0.44 Hz, n = 8), respectively; Fig. 6D]. Under these conditions, adenosine (100 µM) reduced their frequency [55 ± 10% (P < 0.05) of control (3.11 ± 0.36 Hz, n = 5) and 65 ± 6% (P < 0.05) of control (3.82 ± 0.57 Hz, n = 8), respectively; Fig. 6D], values not different from those in the absence of PDBu (see Fig. 7). Figure 7 summarizes the results of the effect of adenosine on sIPSC frequency under a variety of conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
This study showed that adenosine reduces GABAergic and glycinergic eIPSC amplitude and their sIPSC frequency in almost all of adult rat SG neurons examined. This was so, although SG neurons exhibit a diversity in morphology, action potential properties, and dorsal root-evoked synaptic responses (Grudt and Perl 2002). The EC₅₀ value (14.5 µM) for adenosine in reducing GABAergic eIPSC amplitude was comparable with that (12.7 µM) in the hypothalamic supraoptic nucleus (Oliet and Poulain 1999). This value was much smaller than EC₅₀ values (277 and 217 µM, respectively) for adenosine in reducing sEPSC frequency and dorsal-root evoked monosynaptic EPSC amplitude in the SG (Lao et al. 2001, 2004). Although a facilitation by adenosine of glycinergic transmission in the brain stem (Umemiya and Berger 1994) and of GABAergic transmission in the globus pallidus (Shindou et al. 2001, 2002) has been reported, such facilitatory actions were not observed in SG neurons.

Adenosine suppression of inhibitory transmission is mediated by presynaptic A₁ receptors

The reduction in sIPSC frequency by adenosine indicates a decrease in a probability of the release of neurotransmitters from nerve terminals. This was true for evoked transmission, because adenosine affected neither GABAergic and glycinergic sIPSC amplitude nor a response of SG neurons to exogenous GABA or glycine. The presynaptic action of adenosine is supported by the observation that a ratio of the second to first GABAergic or glycinergic eIPSC amplitude was increased by adenosine, because the ratio would remain to be constant if adenosine inhibits a sensitivity of postsynaptic neurons to GABA or glycine and as a result the first and the second eIPSC amplitudes are reduced by the same extent.

CPA as well as adenosine reduced GABAergic and glycinergic eIPSC amplitude and their sIPSC frequency, whereas DPCPX reversed the reductions produced by adenosine. These results, together with the presynaptic action of adenosine as mentioned above, indicate an involvement of A₁ receptors expressed in GABAergic and glycinergic neuron terminals. This idea is consistent with morphological observations that A₁ receptors are present at a high level in intrinsic, especially inhibitory (Schulte et al. 2003) SG neurons (Ackley et al. 2003; Choca et al. 1988; Geiger et al. 1984; Goodman and Snyder 1982). Similar presynaptic inhibition of GABAergic transmission by A₁ receptor activation has been reported in the PAG (Bagley et al. 1999), the hypothalamic supraoptic nucleus (Oliet and Poulain 1999), the subthalamic nucleus (Shen and Johnson 2003), and the hippocampal CA1 area (Jeong et al. 2003). Umemiya and Berger (1994) have shown A₁ receptor-mediated presynaptic inhibition of glycinergic transmission in the brain stem.

Cellular mechanisms for the depression of inhibitory transmission by adenosine

Activation of G-protein–coupled A₁ receptors modulates K⁺ or Ca²⁺ channels and second messenger systems such as the cyclic AMP pathway (Brundege and Dunwiddie 1997). Consistent with the involvement of G-proteins, the sIPSC frequency reduction produced by adenosine in SG neurons was not observed in the presence of NEM.

The release of neurotransmitters from presynaptic terminals is modulated by a variety of mechanisms (for review, see Miller 1998). The level of second messengers and also a modulation of K⁺ or Ca²⁺ channels in nerve terminals have been suggested to account for presynaptic actions of adenosine (Brundege and Dunwiddie 1997). It is well known that a modulation of presynaptic K⁺ channels affects the release of neurotransmitters (for review, see Meir et al. 1999). Under the condition of blockade of K⁺ channels by 4-AP, the sIPSC frequency reduction by adenosine was diminished in extent, indicating an involvement of K⁺ channels in the inhibition. Similar inhibitory mechanisms for the release of neurotransmitters have been shown in the actions of -opioid agonists on GABAergic transmission in the PAG (Vaughan et al. 1997) and on glutamatergic transmission in the hippocampal CA3 area (Simmons and Chavkin 1996). In addition to K⁺ channels, voltage-gated Ca²⁺ channels present in nerve terminals may be modulated by A₁ receptor activation, as shown in the brain stem (Umemiya and Berger 1994) and the CA1 area (Wu and Saggau 1994; for review, see Wu and Saggau 1997). Presynaptic inhibition of glutamatergic transmission by µ-opioid agonists in the superficial dorsal horn is due to a decrease in Ca²⁺ entry in nerve terminals (Hori et al. 1992). This was, however, not the case in SG neurons, because the sIPSC frequency reduction by adenosine was unaffected by the absence of Ca²⁺, which reduced the release of GABA and glycine from nerve terminals (see Fig. 6A).

With respect to second messenger systems, adenosine reduces cyclic AMP level by activating A₁ receptors (Brundege and Dunwiddie 1997). It is well known that this level in nerve terminals affects the release of various kinds of neurotransmitters, including GABA and glycine (Katsurabayashi et al. 2001; Shindou et al. 2002; for review, see Kuba and Kumamoto 1990). GABAergic and glycinergic neuron terminals in the SG are endowed with a cyclic AMP system, the activation of which results in increasing the release of GABA and glycine, as seen from the effects of forskolin and 8-Br-cyclic AMP on sIPSC frequency (see Fig. 6C; see Capogna et al. 1995 for similar actions in the hippocampus). The lack of the inhibitory action of adenosine under the activation of this cyclic AMP system indicates that adenylate-cyclase inhibition is involved in the adenosine actions. Similar results have been reported for A₁-receptor mediated inhibition of GABAergic transmission in the PAG (Bagley et al. 1999). Contrary to this cyclic AMP system, PKC pathway was not involved in the presynaptic depression by adenosine, because this action persisted under PKC activation by PDBu, which enhanced the release of GABA and glycine (see Fig. 6D; see Capogna et al. 1995 for similar actions in the hippocampus).

It is of interest to note that there is a similarity in cellular mechanisms between the inhibitory actions of adenosine on GABAergic and glycinergic transmission, as mentioned above. Their eIPSC amplitudes were reduced by adenosine with quite comparable EC₅₀ values (14.5 vs. 19.1 µM). These results might be due to the fact that these two kinds of synapses are controlled under a similar release machinery. Spinal dorsal horn, especially SG neurons, contain both GABA and glycine, which might be co-released into the dorsal horn (Coggeshall and Carlton 1997; Todd et al. 1996).

Physiological significance for the depression of inhibitory transmission by adenosine

The SG has been generally accepted as a pivotal site for the modulation of nociceptive transmission to the CNS since Melzack and Wall (1965) proposed the gate-control theory of pain. Behavioral studies have shown that intrathecally administered adenosine analogs elicit antinociception, presumably by activating A₁ receptors (for review, see Sawynok 1998; also see Bastia et al. 2002; Borghi et al. 2002 for similar results by intraperitoneal administration). A₁ agonists administered intrathecally inhibited an acute nociceptive response recorded from in vivo dorsal horn neurons (Reeve and Dickenson 1995). These results indicate that adenosine is a potential antinociceptive neuromodulator, which acts on A₁ receptors in the spinal dorsal horn. Previous studies have shown that A₁ receptor activation inhibits glutamatergic transmission in the spinal dorsal horn; this depression is thought to be a cellular mechanism for the antinociception produced by adenosine (Lao et al. 2001, 2004; Li and Perl 1994; Patel et al. 2001). However, our present results indicate that adenosine suppresses inhibitory transmission by activating A₁ receptors. Adenosine is known to postsynaptically activate A₁ receptors to open K⁺ channels, resulting in hyperpolarization in SG neurons (Li and Perl 1994; Patel et al. 2001). Thus when an adenosine analog is considered as an antinociceptive drug that acts on A₁ receptors in the spinal dorsal horn, its suppressive action on inhibitory transmission must be also taken into consideration together with its inhibitory action on excitatory transmission and its hyperpolarizing action. The inhibitory actions of adenosine on excitatory and inhibitory transmission were due to a decrease in the release of neurotransmitters from nerve terminals. This may lead to the reduction of background synaptic noise as a result of a decrease in opening of non-NMDA, GABA_A, and glycine receptor channels, which in turn could increase input resistance and thus make SG neurons electrically compact, contributing to the modulation of nociceptive transmission.

Under the condition of hypoxia, adenosine is released in the spinal dorsal horn; adenosine so released depresses an exaggeration of glutamatergic transmission (Park et al. 2002) and thus protects neurons from the excitotoxic effects of L-glutamate. At the same time, it is conceivable that adenosine also suppresses inhibitory transmission. Thus when a role of adenosine in neuronal protection under the condition of hypoxia is considered, its action on not only excitatory but also inhibitory transmission will have to be taken into consideration. As a purinergic P2X-receptor agonist, ATP facilitates the release of glycine in the spinal dorsal horn (Rhee et al. 2000) and the trigeminal nucleus pars caudalis (Wang et al. 2001), while as a purinergic A₁-receptor agonist, adenosine suppresses inhibitory transmission, as revealed in this study. Since adenosine is produced from ATP (see Salter et al. 1993), it is suggested that inhibitory transmission in the spinal dorsal horn may be modulated by ATP in a biphasic manner such as facilitation followed by inhibition.

In conclusion, when the actions of adenosine on synaptic transmission are considered in the SG, its presynaptic depressive action on inhibitory GABAergic and glycinergic transmission must be taken into consideration together with its presynaptic inhibitory action on excitatory transmission and its hyperpolarizing action, as revealed in opioid actions in the PAG (Vaughan and Christie 1997).

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The authors thank Dr. J.C.G. Marvizón for reading the manuscript.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: E. Kumamoto, Dept. of Physiology, Saga Medical School, Saga 849-8501, Japan (E-mail: kumamoto{at}post.saga-med.ac.jp).

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