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. 1998 Feb 1;506(Pt 3):795–808. doi: 10.1111/j.1469-7793.1998.795bv.x

Inhibitory control of plateau properties in dorsal horn neurones in the turtle spinal cord in vitro

Raúl E Russo *, Frédéric Nagy †, Jørn Hounsgaard *
PMCID: PMC2230747  PMID: 9503338

Abstract

  1. The role of inhibition in control of plateau-generating neurones in the dorsal horn was studied in an in vitro preparation of the spinal cord of the turtle. Ionotropic and metabotropic inhibition was found to condition the expression of plateau potentials.

  2. Blockade of γ-aminobutyric acid (GABAA) and glycine receptors by their selective antagonists bicuculline (10-50 μM) and strychnine (5-20 μM) enhanced the excitatory response to stimulation of the dorsal root and facilitated the expression of plateau potentials.

  3. Bicuculline and strychnine also facilitated the generation of plateau potentials in response to depolarizing current pulses, suggesting the presence of tonic ionotropic inhibitory mechanisms in turtle spinal cord slices.

  4. Activation of GABAB receptors also inhibited plateau-generating neurones. The selective agonist baclofen (5-50 μM) inhibited wind-up of the response to repeated depolarizations induced synaptically or by intracellular current pulses.

  5. Baclofen reduced afferent synaptic input. This effect was not affected by bicuculline or strychnine and was blocked by the selective GABAB receptor antagonist 2-hydroxysaclofen (2-OH-saclofen, 100-400 μM).

  6. Postsynaptically, baclofen inhibited plateau properties. Activation of GABAB receptors produced a hyperpolarization (7.0 ± 0.5 mV, mean ± s.e.m., n= 29) with an associated decrease in input resistance (22.7 ± 3.1 %, n= 24). These effects were blocked by extracellular Ba2+ (1-2 mM).

  7. When the baclofen-induced hyperpolarization and shunt were compensated for by adjusting the bias current and the strength of the stimulus, baclofen still inhibited generation of plateau potentials. Wind-up and after-discharges were also inhibited by baclofen. These effects remained in the presence of tetrodotoxin (1 μM) and were antagonized by 2-OH-saclofen.

  8. The inhibition of plateau properties was observed even when the baclofen-induced hyperpolarization and shunt were blocked by Ba2+ and when potassium channels were blocked by Ba2+ (3 mM), tetraethylammonium (TEA, 15 mM) and apamin (0.25-0.5 μM). The baclofen-sensitive component of the plateau potential was reduced by nifedipine (10 μM), suggesting a modulation of postsynaptic L-type Ca2+ channels.

  9. We suggest that inhibitory regulation of plateau properties plays a role in somatosensory processing in the dorsal horn. The inhibitory control of wind-up and after-discharges may be particularly significant in physiological and therapeutic control of central sensitization to pain.

The prominence of plasticity in information processing in the somatosensory system is illustrated by nociception-related phenomena such as sensitization, hyperalgesia and allodynia (Coderre, Katz, Vaccarino & Melzack, 1993). The favoured spinal mechanisms contributing to these types of plasticity are modifications in synaptic efficacy produced by activity-dependent changes (Dubner & Ruda, 1992) and by modulatory effects of neurotransmitters (Zieglg änsberger & Tölle, 1993; Urban, Thompson & Dray, 1994). In addition to changes in synaptic strength, the intrinsic response properties of neurones provide a postsynaptic site of plasticity (Harris-Warrick, Nagy & Nusbaum, 1992). In the turtle, the intrinsic response properties of dorsal horn neurones play an important role in synaptic integration of primary afferent input (Russo & Hounsgaard, 1996a, b). In a particular subset of dorsal horn neurones, a plateau potential mediated by low threshold, dihydropyridine-sensitive L-type Ca2+ channels favours synaptic interactions on a slow time scale (Russo & Hounsgaard, 1996a). Indeed, the plateau potential mediates both the wind-up of the response to repetitive sensory stimulation and the associated after-discharges (Russo & Hounsgaard, 1994).

In the spinal cord, neuromodulation by substances released by primary afferents is thought to have a central role in the development of plastic changes related to pain mechanisms (Coderre et al. 1993; Zieglgänsberger & Tölle, 1993). We have recently shown that the plateau properties are upregulated by metabotropic pathways activated by primary afferent activity (Russo, Nagy & Hounsgaard, 1997). On the other hand, the spinal cord is under control of both phasic and tonic, supraspinal and segmental inhibition (Lundberg, 1982; Willis & Coggeshall, 1991). Failure of tonic or phasic inhibition may lead to states of hyperexcitability in some forms of pain (Hao, Xu, Yu, Seiger & Wiesenfeld-Hallin, 1992; Woolf & Doubell, 1994). It is possible that downregulation of plateau properties is important in shaping the activity of plateau-generating neurones. In the present study, we investigated the inhibitory control of plateau-generating neurones exerted by the two main inhibitory transmitters in the spinal cord: the amino acids γ-aminobutyric acid (GABA) and glycine. We found that both ionotropic and metabotropic inhibition regulate the information transfer from primary afferents to plateau-generating neurones. Based on the hypothesis that the wind-up and after-discharges, mediated by plateau potentials (Russo & Hounsgaard, 1994; Morisset & Nagy, 1996) are important elements in spinal sensitization to pain (Coderre et al. 1993; McMahon, Lewin & Wall, 1993) we suggest that inhibitory control of plateau-generating neurones may be particularly significant in physiological and therapeutic control of central sensitization to pain.

METHODS

Preparation

The preparation used in this study has been described elsewhere (Russo & Hounsgaard, 1996a). Briefly, adult turtles (Pseudemys scripta, 15-20 cm carapace length) were anaesthetized with pentobarbitone (100 mg kg−1i.p.). After complete unresponsiveness to painful stimuli was achieved, a window in the plastron was opened with an oscillating saw (Aesculap) and the blood removed by intraventricular perfusion with Ringer solution (6-10°C) of the following composition (mM): 120 NaCl, 5 KCl, 15 NaHCO3, 3 CaCl2, 2 MgCl2, 20 glucose. The lumbar enlargement was dissected and thick (1.5-2.0 mm) transverse slices with dorsal roots attached were cut with a home-made slicing machine. After dissection the animal was killed by decapitation. For recording, the slice was glued to a piece of filter paper with cyanoacrylate and fixed to the bottom of the recording chamber (1 ml volume) with pieces of silver wire. The preparation was kept at room temperature (20-22°C) and continuously superfused at a rate of 1 ml min−1 with Ringer solution which was saturated with a gas mixture containing 2 % CO2 and 98 % O2 to obtain a pH of 7.6.

Electrophysiological recordings and stimulation

Conventional intracellular recordings in current clamp mode were performed with an Axoclamp-2A. The electrodes for recording were pulled in a Flaming-Brown P-87 puller (Sutter Instruments) from borosilicate thin-walled glass capillaries (1.5 o.d, 1.17 i.d.; Clark Electromedical Instruments) and filled with 1 M potassium acetate and 1 % biocytin (50-80 M Ω). The electrodes were positioned under visual guidance using a dissecting microscope (Leica Wild M3Z). In order to characterize the passive and active properties of neurones, rectangular pulses of current were injected through the electrode, driving the amplifier with a programmable stimulator (Master-8; AMPI). The bridge was balanced by cancelling the fast voltage jump at the onset and offset of the current pulse, and bridge balance was continuously monitored throughout the experiment. Extracellular stimulation was performed by applying brief (0.5 ms) constant current pulses (Iso-Flex; AMPI) to the ipsilateral dorsal root by means of a suction electrode.

Database and processing

The criteria for selection of cells were an action potential of more than 60 mV (measured from peak to peak) and the ability to sustain increasing firing frequency of action potentials in response to long-lasting depolarizing current pulses, indicating the presence of a plateau potential (Russo & Hounsgaard, 1996a).

The recordings were stored on tape (Racal, 0-5 kHz bandwidth) for off-line analysis. The recorded data were digitized at 800 kHz using the envelope function of a digital oscilloscope (Hioki 8851) and subsequently transferred to a personal computer. Hard copies were obtained with a laser printer (Hewlett Packard Laserjet IIIp). Numerical values are expressed as means ±s.e.m., n is the number of observations in independent experiments.

Drugs

In some experiments, the following drugs were added to the normal medium in the indicated quantities: tetrodotoxin (TTX, 1 μM; Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 μM; RBI), strychnine (5-20 μM; Sigma), bicuculline (10-50 μM; Sigma), (±)-2-amino-5-phosphonopentanoic acid (AP-5, 100 μM; RBI), baclofen (5-50 μM, RBI), 2-hydroxysaclofen (2-OH-saclofen, 100-400 μM, RBI), tetraethylammonium (TEA, 10-15 mM; Sigma), apamin (0.25-0.5 μM, Sigma) and nifedipine (10 μM, Sigma). BaCl2 (1-2 mM) was added to the normal medium or replaced CaCl2.

RESULTS

Inhibitory control of plateau-generating neurones mediated by GABAA and glycine receptors

Plateau-generating neurones show incrementing frequency of action potentials (APs) at the onset of a sustained suprathreshold depolarization due to activation of a plateau potential (Russo & Hounsgaard, 1996a and Fig. 1Ab). In these cells, stimulation of the dorsal root (DR) produces synaptic responses that can include a long-lasting, nifedipine-sensitive component mediated by the plateau potential (Russo & Hounsgaard, 1994, 1996a). In some cases, however, a single shock to the DR was unable to activate the plateau potential. In the cell shown in Fig. 1A, a DR stimulus produced a short-delayed excitatory postsynaptic potential (EPSP; Fig. 1Aa, open arrow) followed by a polysynaptic component to which barrages of inhibitory postsynaptic potentials (IPSPs) contributed (Fig. 1Aa, filled arrows). Addition of bicuculline (50 μM) to the superfusate potentiated the excitatory response (n= 7). As shown in Fig. 1B, the previously subthreshold, short-delayed EPSP produced spike firing and was followed by a polysynaptic component that kept the cell depolarized (Fig. 1Ba). However, the most dramatic effect of bicuculline was observed about 400 ms after the DR stimulation, when a powerful increase in firing frequency appeared (Fig. 1Bb). This late increase in firing frequency was produced by activation of the plateau potential as suggested by the similarity with the profile of activity in response to a depolarizing current pulse (compare Ab and Bb). Addition of strychnine (10 μM) also increased the response to DR stimulation (n= 9; Fig. 1C). Spike generation was inactivated by the strong depolarizing response. The overall duration of the response, however, was not changed (Fig. 1B and C).

Figure 1. Effects of bicuculline and strychnine on responses provoked by DR stimulation.

Figure 1

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A, stimulation of the DR (20 μA, at ▴) in a plateau-generating neurone produced a short-delayed EPSP (open arrow) followed by a polysynaptic component (a). The filled arrows point to IPSPs. In the same cell a depolarizing current pulse produced an increasing frequency of APs (b). Here and in subsequent figures the instantaneous frequency plot and current pulse protocol are shown above and below the main trace, respectively. B, in the presence of bicuculline (50 μM), the same DR stimulus as in Aa produced a stronger response with firing of APs. Inspection of the response at a slower sweep speed (b) reveals a similar firing pattern to that observed in Ab, with incrementing frequency of APs. The synaptic response was followed by a slow after-hyperpolarization (sAHP). C, addition of strychnine (10 μM) further increased the synaptic response. The vertical voltage calibration in A also applies in B and C. All data are from the same cell.

The blockade of segmental inhibitory mechanisms also facilitated the induction of prolonged synaptic responses and of wind-up. As shown in Fig. 2A, strychnine (20 μM) strongly potentiated the response to a single DR stimulus (Fig. 2Aa). The duration of the response in the presence of strychnine decreased drastically on hyperpolarization of the cell (Fig. 2Ab), indicating a contribution of the plateau potential to the synaptic response at resting membrane potential. In the same cell, repetitive stimulation of the DR at 0.3 Hz in the control medium generated weak wind-up (Fig. 2Ba). After application of strychnine a strong wind-up developed upon repetition of the stimulus (Fig. 2Bb). Addition of AP-5 (100 μM) reduced the excitability but did not block the wind-up of the response (Fig. 2Bc). In the presence of nifedipine (10 μM), however, the response winds down (Fig. 2Bd).

Figure 2. Blockade of inhibition promoted prolonged after-discharges and wind-up.

Figure 2

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A, in normal medium, a single shock to the DR (40 μA) produced a brief burst of APs followed by polysynaptic IPSPs (a, upper trace). After addition of strychnine (20 μM), the initial burst increased and was followed by a sustained firing (a, lower trace). The time course of the overall response is shown in b. The duration of the response decreased when the cell was hyperpolarized from -55 mV (b, upper trace) to -105 mV (b, lower trace). B, repetitive stimulation of DR (30 μA at 0.3 Hz) in control medium elicited a weak wind-up of the synaptic response (a). A clear wind-up of the response to DR stimulation (12 μA) was seen after blockade of glycinergic receptors with strychnine (20 μM) (b). AP-5 (100 μM) decreased the response to DR stimulation but did not block wind-up (c). Addition of nifedipine (10 μM) completely blocked wind-up (d). All data are from the same cell.

Because plateau potentials underlie both the prolonged responses and wind-up to sensory stimulation, we also explored a possible tonic inhibitory influence on plateau properties (n= 7). The cell in Fig. 3A fired tonically during a moderate depolarization (Fig. 3Aa). Inspection of the instantaneous frequency plot (IFP) revealed a slightly incrementing firing frequency (Fig. 3Aa) that became more clear with stronger current pulses (not illustrated). In the presence of bicuculline (50 μM) the same stimulus as in Fig. 3A produced a powerful activation of the plateau potential generating a sharp increase in firing frequency (Fig. 3Ab). Notice, however, that the firing frequency at the onset of the stimulus was similar to that of the control. A tiny increase in input resistance was observed in the presence of bicuculline (Fig. 3Ab, inset). Similar results were obtained when glycinergic receptors were blocked (Fig. 3Bb). Strychnine (20 μM) promoted plateau properties as evidenced by a clearer increment in firing frequency during the pulse and by the appearance of a long-lasting after-discharge (Fig. 3Bb).

Figure 3. Bicuculline and strychnine facilitated the activation of plateau potentials by depolarizing current pulses.

Figure 3

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A, in normal medium a tonic firing pattern was produced by a weak depolarizing current pulse (a). When GABAA receptors were blocked by bicuculline (50 μM) the same pulse elicited a marked incrementing frequency of APs (b). A mild increase in input resistance was produced by bicuculline (inset). B, responses to the same depolarizing current pulse before (a) and after (b) addition of strychnine (20 μM). An increase in firing frequency during the pulse and a long-lasting after-discharge were observed when glycine receptors were blocked by strychnine. Vertical calibrations in B also apply in A. A and B are from different cells.

Inhibitory mechanisms mediated by GABAB receptor activation

In addition to ionotropic receptors, GABA also activates metabotropic GABAB receptors (Sivilotti & Nistri, 1991). Baclofen, a selective agonist for the GABAB receptor, blocked the wind-up induced both synaptically (n= 5) and intracellularly (n= 24). In Fig. 4A, repetitive stimulation of the DR produced action potential wind-up and a slow cumulative depolarization (Fig. 4Aa). The wind-up was blocked by baclofen which strongly inhibited the synaptic response (Fig. 4Ab). The response to DR stimulation partly recovered after washout of baclofen. A weak cumulative depolarization reappeared, but was not sufficient to generate AP wind-up (Fig. 4Ac). In the same cell, the wind-up induced by intracellularly injected current pulses (Fig. 4Ba) was also blocked by baclofen (Fig. 4Bb). Therefore, baclofen inhibited wind-up by reducing the synaptic input and the postsynaptic plateau properties. The effect of baclofen on synaptic input was even clearer when ionotropic inhibitory transmission was blocked (n= 7). In the cell shown in Fig. 5A the response to DR stimulation was boosted by blocking inhibitory ionotropic transmission with bicuculline (25 μM) and strychnine (10 μM) (Fig. 5Aa). Baclofen (5 μM) strongly inhibited the synaptic response. Only a small component of the response with a longer delay and a duration of 40 ms was resistant to this dose of baclofen (Fig. 5Ab). The effect of baclofen on synaptic input was partly antagonized by 2-OH-saclofen (100-400 μM, n= 7; Fig. 5Ac).

Figure 4. Baclofen inhibited synaptically and intracellularly induced wind-up.

Figure 4

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A, the wind-up induced by repetitive stimulation of the DR (a) was completely eliminated by 50 μM baclofen (b). The first response of the sequence is shown on the right. In the presence of baclofen the burst of APs was reduced to a tiny depolarization (b). After washout, a partial recovery of the synaptic response and a weak cumulative depolarization in response to repetitive stimulation were observed (c). B, the intracellularly induced wind-up (a) was reduced by baclofen (50 μM) in spite of the higher intensity of stimulation used (b). Vertical calibrations in B also apply in A. All data are from the same cell.

Figure 5. 2-OH-Saclofen blocked the baclofen-induced inhibition of the synaptic response.

Figure 5

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A, the response to a single shock (20 μA) applied to the DR in the presence of bicuculline (25 μM) and strychnine (10 μM) consisted of a powerful burst of APs (a). The same response is shown at two sweep speeds. Baclofen (5 μM) inhibited the synaptic response (b). A small polysynaptic EPSP remained. The effect of baclofen was partly antagonized by 2-OH-saclofen (200 μM) (c). B, the synaptic response produced by DR stimulation (50 μA) in the presence of TTX (1 μM) was inhibited by baclofen in a dose-dependent manner (a). Note that the earliest part of the response (arrow) was more sensitive to baclofen. The effect of baclofen (10 μM) on the synaptic input was effectively antagonized by 2-OH-saclofen (200 μM) (b). A and B are from different cells.

The inhibition of the earliest part of the synaptic response suggested an inhibitory action of baclofen on primary afferent terminals (monosynaptically connected to plateau-generating neurones). This possibility is supported by the experiment illustrated in Fig. 5B. In the presence of TTX (1 μM) it is possible pharmacologically to isolate a monosynaptic input probably mediated by Aδ and C fibres (Yoshida, Matsuda & Samejima, 1978). Baclofen inhibited the TTX-resistant EPSP in a dose-dependent manner (Fig. 5Ba) and was effectively antagonized by 2-OH-saclofen (Fig. 5Bb). Note that, as in control conditions, the earliest component of the TTX-resistant EPSP (Fig. 5Ba, arrow) was more sensitive to the action of baclofen.

Inhibitory modulation of plateau properties mediated by GABAB receptors

The suppression of wind-up by baclofen had an important postsynaptic component (see Fig. 4). In plateau-generating neurones, baclofen produced a slow hyperpolarization (7.0 ± 0.5 mV, mean ±s.e.m., n= 29) with an associated decrease in input resistance (Fig. 6A). The baclofen-induced shunt was observed in 77 % of the cells in which it was tested. The decrease in input resistance ranged from 8.4 to 55 % (22.7 ± 3.1 %, n= 24). Baclofen decreased the slope of the membrane potential-current relationship which intersected around -80 mV (Fig. 6B), suggesting a possible involvement of a K+ conductance in the baclofen-induced shunt.

Figure 6. Postsynaptic effects of baclofen.

Figure 6

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A, addition of baclofen (20 μM, arrow) to normal medium containing TTX (1 μM) produced a slow hyperpolarization. Hyperpolarizing current pulses (200 ms duration, 0.2 nA in amplitude) revealed a decrease in input resistance during the baclofen-induced hyperpolarization (inset, taken from corresponding points on the main trace). B, membrane potential-current relationships obtained under different conditions. Baclofen (20 μM) decreased the slope of the plot. Note that the third order regression lines intersect around -80 mV. The effect was reversible upon washout of the drug. The experiment was performed in the presence of TTX (1 μM). C, a powerful and sharp increase in firing frequency was observed in response to a depolarizing current pulse in medium containing bicuculline (25 μM) and strychnine (10 μM) (a). The firing stopped during the stimulus which was followed by a sAHP. Baclofen suppressed the increase in firing frequency due to activation of the plateau potential (b). The bias current and the strength of the pulse were adjusted to keep the same membrane potential and initial firing frequency as those of control. D, instantaneous frequency plots (IFPs) of a cell showing that the late increase in firing frequency due to activation of the plateau potential was eliminated by baclofen (10 μM). Note, however, that initially the two plots overlapped. E, a depolarizing pulse produced delayed firing and a prolonged after-discharge under normal conditions (a). After addition of baclofen (20 μM) a subthreshold response was obtained (b). Note that the same resting membrane potential and depolarization at the onset of the pulse (inset) were obtained in both conditions by adjusting the bias current and the strength of the pulse. A further increase of the stimulus could induce delayed firing and a mild acceleration but neither after-discharge nor after-potential was observed. A and B are from the same cell.

Plateau properties were strongly inhibited by baclofen (Fig. 6C-E). In Fig. 6C a plateau-generating cell displayed an incrementing frequency of APs in response to a depolarizing current pulse. The time course of the underlying plateau potential is suggested by the IFP (Fig. 6C). In the presence of baclofen the cell displayed an adapting firing pattern without evidence of plateau potential activation. Note that the baclofen-induced hyperpolarization was compensated by adjusting the bias current and stimulus strength was increased to attain the sme initial firing frequency as in control conditions. A similar result from another cell is illustrated graphically in Fig. 6D. The superimposed IFPs show that the firing pattern for the first 2 s was not changed by baclofen (Fig. 6D). However, in the presence of baclofen the cell failed to develop the late acceleration of firing observed under control conditions.

Another aspect of the inhibition of plateau properties by baclofen is illustrated in Fig. 6E. In this cell, the plateau potential activated just below the threshold for the Na+ spike producing a delayed firing followed by a progressive increase in firing frequency. A robust after-discharge was observed after termination of the current pulse (Fig. 6Ea). In the presence of baclofen (20 μM) a subthreshold response was obtained (Fig. 6Eb). Note that the baclofen-induced hyperpolarization and shunt were compensated for to keep the cell at the same membrane potential as that for control and to reach the same level of depolarization at the onset of the pulse (Fig. 6Eb, inset). A stronger depolarization produced delayed firing and a moderate increment in firing frequency but neither after-discharge nor after-potential could be elicited (Fig. 6Ec).

The inhibition of plateau properties by baclofen was also observed in the presence of TTX (n= 9), indicating that the baclofen-induced effect was not due to the removal of a tonic facilitatory influence. Figure 7A shows the characteristic firing pattern of a plateau-generating neurone, with delayed firing followed by a depolarizing after-potential. In the same cell, a robust plateau potential was still recorded after blockade of spikes with TTX (1 μM; Fig. 7B, control). The plateau potential was abolished by baclofen (20 μM) although the same initial depolarization was reached at the onset of the pulse (Fig. 7B, baclofen).

Figure 7. Baclofen-induced inhibition is resistant to TTX and blocked by 2-OH-saclofen.

Figure 7

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A, delayed firing followed by a depolarizing after-potential was recorded in normal medium. B, the robust plateau potential observed after blockade of fast spikes with TTX (1 μM) was abolished by baclofen (20 μM). Note the same trajectory of the membrane potential at the beginning of the depolarizing pulse. C, the wind-up-like activation of the plateau potential observed in the presence of TTX (1 μM) was blocked by baclofen (a). The effect disappeared after washout (b). D, IFPs of a plateau-generating neurone show that baclofen (5 μM) reduced the rate of acceleration in firing, an effect that was antagonized by 2-OH-saclofen (200 μM). E, 2-OH-saclofen (200 μM) partly antagonized the effect of 5 μM baclofen. The response was initially recorded in the presence of TTX (1 μM) and 2-OH-saclofen (a). Addition of baclofen shortened the plateau potential. Removal of 2-OH-saclofen led almost to the complete blockade of the plateau potential (a). The response partially recovered under the initial conditions (b). Vertical calibrations in E also apply in A-C. A-C are from the same cell.

The postsynaptic inhibitory effect of baclofen on wind-up was clearly observed in the presence of TTX. In control conditions an increasing, wind-up-like activation of the plateau potential was easily generated (Fig. 7Ca, control). Baclofen (20 μM) completely suppressed the wind-up of the plateau potential (Fig. 7Ca, baclofen). Notice the superimposition of the depolarization produced by the first pulse (0.15 nA, control and 0.25 nA, baclofen) under both conditions. The wind-up-like activation of the plateau potential gradually recovered during 1 h in normal medium (Fig. 7C).

The postsynaptic effects of baclofen were also antagonized by 2-OH-saclofen (Fig. 7D and E, n= 6). Figure 7D shows the IFPs of a plateau-generating neurone under different pharmacological conditions. In normal medium, a clear increment in firing frequency occurred during the first second and stabilized for the rest of the pulse (Fig. 7D, •). In the presence of baclofen (5 μM) the rate of increase in firing was slower and the discharge stabilized at a lower level, despite a similar initial firing frequency (Fig. 7D, ○). This effect was blocked by 200 μM 2-OH-saclofen (Fig. 7D, ▵). As shown in Fig. 7E, similar results were obtained when the postsynaptic cell was pharmacologically isolated by addition of TTX (1 μM). In this case, the control response was recorded in the presence of 200 μM 2-OH-saclofen (Fig. 7Ea). Addition of baclofen (5 μM) to the superfusate did not inhibit the plateau during the stimulus but decreased the duration of the after-potential. When 2-OH-saclofen was removed from the bath, however, baclofen almost eliminated the after-potential produced by the plateau (Fig. 7Ea). The response partly recovered on return to initial conditions (Fig. 7Eb).

Ba2+ blocks the shunt but not the inhibition of plateau properties produced by baclofen

In the hippocampus (Gähwiler & Brown, 1985) and spinal cord (Allerton, Boden & Hill, 1989) the hyperpolarization mediated by GABAB receptors is sensitive to low concentrations of Ba2+. In agreement, extracellular Ba2+ (1-2 mM, n= 7) decreased the baclofen-induced hyperpolarization (not illustrated) and shunt (Fig. 8A) in plateau-generating neurones. However, the inhibition of plateau properties by baclofen was not blocked by Ba2+. Figure 8B shows the response of a cell to a depolarizing current pulse in the presence of 1 mM Ba2+. A clear incrementing firing frequency followed by an after-discharge that subsided over several seconds was elicited by a depolarizing current pulse (Fig. 8Ba). Baclofen (50 μM) produced a moderate delay in firing and decreased the acceleration of firing frequency in response to the depolarizing pulse (Fig. 8Bb). The after-potential was strongly reduced and could not generate an after-discharge (Fig. 8Bb). In the same cell stronger stimuli produced the same rate of increase in initial firing frequency in both conditions, but in the presence of baclofen the plateau phase of the response was reduced (Fig. 8C).

Figure 8. Effects of Ba2+ on baclofen-induced modulation.

Figure 8

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A, I-V plot obtained in the presence of TTX (1 μM) showing that in the presence of 1 mM Ba2+ baclofen failed to produce a significant shunt. Same cell as that shown in Fig. 6B. B, incrementing firing frequency and an after-discharge were produced by a depolarizing pulse in the presence of 1 mM Ba2+. The normal medium also contained CNQX (10 μM), AP-5 (100 μM), strychnine (10 μM) and bicuculline (20 μM) (a). In the presence of baclofen the activation of the plateau was reduced and the after-discharge was blocked (b). C, the IFPs of the same cell as shown in B in response to stronger stimuli are compared. Despite the same initial rate of increase in firing frequency, in the presence of baclofen a clear adaptation appeared after reaching the peak frequency. D, the inhibition of the wind-up-like activation of the plateau potential by baclofen was not prevented by 1 mM Ba2+ (a), although this ion blocked the baclofen-induced decrease of input resistance (b). Note that Ba2+ by itself increased the input resistance of the cell (b). Vertical calibrations in B also apply in D.

The experiment in Fig. 8D shows that powerful inhibition of plateau properties can be produced by baclofen without a concomitant shunt. The strong wind-up-like activation of the plateau potential in medium containing 1 mM Ba2+ was completely suppressed by baclofen (Fig. 8Da). Note that in both conditions the responses to the first current pulse were identical. The input resistance increased by 50 % after addition of Ba2+ and was not further modified by addition of baclofen (Fig. 8Db). These data suggest that baclofen modifies conductance(s) directly involved in plateau potential generation. In Fig. 9, Ca2+ was replaced by Ba2+ and K+ conductances were blocked (n= 9) with apamin (0.25-0.5 μM) and TEA (15 mM) added to TTX containing medium. Under these conditions a high-amplitude, long-lasting plateau potential was observed (Fig. 9Aa, control). The amplitude and particularly the duration of the plateau potential were progressively reduced after addition of baclofen (Fig. 9Aa). The response recovered after washout of baclofen (Fig. 9Bb). As previously reported (Russo & Hounsgaard, 1996a), nifedipine (10 μM, n= 3) inhibited the plateau potential recorded under these conditions (Fig. 9Bb, nifedipine). Addition of baclofen induced almost no further reduction of the plateau potential (Fig. 9Bb, nifedipine + baclofen). Therefore, nifedipine substantially reduced the effect of baclofen on the plateau potential (Fig. 9Ba and Bb). These data suggest a modulation of L-type Ca2+ channels by GABAB receptors.

Figure 9. Baclofen inhibited the plateau potential.

Figure 9

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A, in the presence of TTX (1 μM) replacement of Ca2+ with Ba2+ and the blockade of K+ conductances with TEA (15 mM) and apamin (0.5 μM) resulted in a high-amplitude, long-lasting plateau (a, control). Under these conditions baclofen (50 μM) reduced significantly the duration of the plateau potential (a, baclofen). The response to the 3rd, 5th, 7th and 8th responses after baclofen illustrate the progressive decrease in the duration of the plateau. The baclofen-induced inhibition of the plateau potential reversed upon washout of the drug (b, wash). B, in another cell, the plateau potential recorded under the same conditions as in A was reduced by baclofen (25 μM). The response recovered after washout of baclofen (b) and was subsequently inhibited by nifedipine (10 μM). Further addition of baclofen (25 μM) did not significantly affect the plateau potential (b).

DISCUSSION

Our study shows that the two main inhibitory transmitters in the spinal cord, GABA and glycine (Willis & Coggeshall, 1991), regulate information processing in plateau-generating neurones. Inhibitory modulation of the plateau potential resulted in a decreased ability to generate wind-up and to sustain after-discharges. Given the hypothesized relation of these phenomena to nociception (Coderre et al. 1993; McMahon, Lewin & Wall, 1993) the inhibitory modulation of plateau properties may be particularly relevant for spinal processing of nociceptive information.

Ionotropic inhibition

In plateau-generating neurones the blockade of GABAA and glycine receptors by their selective antagonists potentiated the excitatory responses mediated by primary afferents, facilitating the activation of the plateau potential. GABA is present throughout the central nervous system (Sivilotti & Nistri, 1991) and has been identified by immunohistochemical techniques in the mammalian dorsal horn (Sivilotti & Nistri, 1991; Mitchell, Spike & Todd, 1993). In the turtle, immunostaining against GABA reveals its presence in small to medium-sized dorsal horn neurones and abundant GABA stained punctae, particularly concentrated in a region of neuropil with abundant contacts between primary afferent terminals and postsynaptic elements (A. Fernández, M. Radmilovich & O. Trujillo-Cenóz, personal communication).

Anatomical (Pershon, Malherbe & Richards, 1991) and electrophysiological (Désarmenien, Feltz, Occhipinti, Santangelo & Schlichter, 1984) evidence indicates that GABA acts at both pre- and postsynaptic sites in the spinal cord. In the dorsal horn, a presynaptic action is believed to play a major role (Sivilotti & Nistri, 1991). In turtle dorsal horn cells with plateau properties spontaneously occurring IPSPs were effectively antagonized by strychnine while bicuculline had little effect (data not shown). Therefore, although we cannot rule out an effect on postsynaptic inhibition, it seems more likely that the potentiation of the synaptic response mediated by bicuculline was primarily due to blockade of presynaptic inhibition.

Glycinergic neurones, terminals and receptors are present throughout the dorsal horn (van den Pol & Gorcs, 1988). Although glycine is co-localized with GABA in axo-axonic synapses in the dorsal horn (Mitchell et al. 1993), there is no evidence favouring a presynaptic action of glycine. By contrast, postsynaptic glycinergic inhibition in the spinal cord is well documented (Curtis, Hosli & Johnston, 1968). Our results indicate that glycinergic inhibition exerts a powerful control on postsynaptic responses mediated by primary afferents and inhibits the activation of plateau potentials, thereby reducing wind-up and after-discharges.

Phasic inhibition can contribute to termination of plateau potentials. We have previously shown that hyperpolarizing pulses or a barrage of IPSPs can terminate an ongoing plateau potential (Russo & Hounsgaard, 1996a). In rats, in vivo after-discharges in convergent dorsal horn neurones can be terminated by a transient inhibitory input (Cadden, 1993). In these cells, activity was readily restored by applying a stimulus in the excitatory field. The most parsimonious explanation for these observations is that convergent dorsal horn neurones in the rat also display plateau potentials, which can be terminated by phasic inhibitory mechanisms. In agreement with this possibility, plateau potentials have recently been found in deep dorsal horn neurones in the rat (Morisset & Nagy, 1996).

Blockade of GABAA and glycine receptors also promoted the plateau potential elicited by depolarizing current pulses. This fact indicates that in addition to a phasic inhibitory control, tonic ionotropic inhibition was present in thick transverse slices of the turtle spinal cord. The promotion of plateau properties by bicuculline and strychnine may be explained by removal of a shunting effect produced by a background activation of GABAA and glycinergic receptors. However, in all cases the shunt was small or absent. Another possibility is that the tonic inhibition controls neurones which are presynaptic for plateau-generating neurones and whose activity facilitates the plateau potential. This possibility remains to be tested experimentally.

Metabotropic inhibition

In addition to the ionotropic actions mediated by the GABAA receptor, GABA exerts modulatory effects via metabotropic GABAB receptors (Dolphin & Scott, 1987; Sivilotti & Nistri, 1991). A role for GABAB receptor-mediated inhibition in the spinal cord is suggested by the depressant effect of the selective GABAB agonist baclofen (Curtis, Lodge, Bornstein & Peet, 1981). GABAB receptors are found in the dorsal horn both at postsynaptic sites (Price, Kelly & Bowery, 1987) and in primary afferents (Désarmenien et al. 1984).

In the turtle, activation of GABAB receptors inhibited the activity of plateau-generating neurones in two ways. First, baclofen strongly reduced the postsynaptic response to dorsal root stimulation. A presynaptic mechanism for this effect is likely since a presynaptic action of baclofen on rat dorsal root ganglion cells has been observed (Désarmenien et al. 1984), and baclofen is believed to reduce the release of excitatory amino acids in the dorsal horn by a presynaptic mechanism (Kangrga, Jiang & Randic, 1991).

Second, baclofen also had a prominent postsynaptic effect. As described previously in the hippocampus (Newberry & Nicoll, 1984) and rat dorsal horn (Allerton et al. 1989), the hyperpolarization induced by baclofen in plateau neurones in the turtle was associated with decreased input resistance. In hippocampal pyramidal neurones, the baclofen-induced hyperpolarization is produced by a K+ conductance (Newberry & Nicoll, 1984) which is blocked by low concentrations of Ba2+ (Gähwiler & Brown, 1985). A similar Ba2+ sensitivity of the baclofen-induced hyperpolarization was also observed in unidentified dorsal horn neurones of the rat (Allerton et al. 1989). By analogy, it seems likely that a similar K+ conductance is responsible for the hyperpolarization observed in this study.

The activation and time course of the plateau potential is very sensitive to small changes in resting membrane potential (Russo & Hounsgaard, 1996a). Taking this into account, the baclofen-induced hyperpolarization alone is bound to have an important inhibitory influence on the activation and time course of the plateau potential. However, the change in membrane potential and the decrease in input resistance were not the only mechanisms mediating the baclofen-induced inhibition of plateau properties. Even when these factors were compensated for, in the presence of baclofen the plateau potential had a substantially higher threshold and shorter duration. Furthermore, under conditions where the baclofen-induced hyperpolarization and shunt were blocked (i.e. in the presence of Ba2+), baclofen still inhibited the plateau properties, wind-up and after-discharges. This suggests that baclofen either activated another outward current or directly inhibited the plateau potential. The fact that baclofen still reduced the plateau potential when K+ conductances were blocked and that the effects of baclofen and nifedipine, which also reduced the plateau potential, were not additive indicates a modulation of L-type Ca2+ channels by activation of GABAB receptors. The direct inhibitory effect of GABAB receptors on Ca2+ currents has been well characterized in dorsal root ganglion cells (Dolphin & Scott, 1987) where it is thought to be the basis of the presynaptic effect of baclofen on primary afferents. A direct effect of baclofen on currents mediated by L-type Ca2+ channels has been observed by Scholz & Miller (1991) in hippocampal neurones (but see Gähwiler & Brown, 1985). Inhibition of Ca2+ currents by baclofen in spinal neurones has been also observed (Sah, 1990; Matsushima, Tegnér, Hill & Grillner, 1993).

Segmental inhibition and nociceptive mechanisms

An important role for fast synaptic inhibition in nociceptive information processing is suggested by the finding that blockade of either GABAA or glycine receptors increase the nociceptive flexion withdrawal reflex (Sivilotti & Woolf, 1994). Furthermore, activation of GABAA receptors by the selective agonist muscimol has antinociceptive effects both in acute pain models (Hammond & Drower, 1984) and in the formalin pain model (Dirig & Yaksh, 1995). It has been suggested that a failure in segmental inhibitory control contributes to central sensitization to pain (Sivilotti & Woolf, 1994). Our results show that in the case of failure of fast synaptic inhibition, plateau properties would be facilitated leading to increased firing frequency and after-discharges which are well-known elements in nociceptive coding (Coghill, Mayer & Price, 1993; Price, 1984).

The inhibitory control via GABAB receptors may also play an important part in processing nociceptive information in the dorsal horn. Antinociceptive effects of baclofen have been observed after intrathecal injection at the level of the lumbar spinal cord (Wilson & Yaksh, 1978) and in the formalin pain model (Dirig & Yaksh, 1995). In addition, baclofen reverses the hypersensitivity of wide dynamic range neurones in the rat produced by a transient ischaemia of the cord (Hao et al. 1992).

It is believed that the main action of GABAB receptors in the spinal cord is presynaptic. The present study shows that a subpopulation of dorsal horn neurones is strongly affected by GABAB-mediated inhibition through a postsynaptic mechanism. Indeed, the inhibitory control of plateau-generating cells by baclofen is achieved by a dual mechanism: the activation of a hyperpolarizing, Ba2+-sensitive conductance and probably by modulation of Ca2+ channels. The main function of GABAergic inhibition of plateau potential generation may be the fine-tuning of the integrative properties of dorsal horn cells. Inhibition of the Ca2+-mediated plateau potential and wind-up could have other important implications. Intracellular Ca2+ is a major second messenger that can interact with a variety of metabolic pathways. Indeed, Ca2+ is believed to trigger cellular events (i.e. stimulation of protein kinase C, activation of proto-oncogenes) that produce the long-term modifications observed in chronic pain states (Coderre et al. 1993). The higher threshold and shorter duration of the plateau potential produced by activation of GABAB receptors will reduce Ca2+ entry and inhibit a possible pathway of sensitization.

The GABAergic neurones that mediate the neuromodulation described here are intrinsic elements of the dorsal horn network. Intrinsic neuromodulation is well known in some neural networks devoted to the processing of sensory information and provides a mechanism for local activity to regulate the sensitivity and dynamic range of the system (Katz & Frost, 1996). In the turtle dorsal horn, plateau properties are facilitated by glutamate and substance P released from primary afferents (Russo, Nagy & Hounsgaard, 1997). The upregulating effects produced by activation of metabotropic glutamate receptors and NK-1 tackykinin receptors are counteracted by the downregulation of the plateau potential produced by activation of GABAB receptors. These modulatory actions are likely to be part of a more complex intrinsic neuromodulatory system (Willis & Coggeshall, 1991) that dynamically sets the electrophysiological phenotype of dorsal horn neurones to match the requirements of changing internal and external environments.

Acknowledgments

This work was kindly funded by the European Economic Community, The Danish MRC, The Lundbeck Foundation, The NOVO-Nordisk Foundation, and the MDRI Département des Sciences de la Vie du CNRS.

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