[1] 20w Serotonergic modulation of the responses to excitatory amino acids of rat dorsal horn neurons in vitro: implications for somatosensory transmission

[1] 261w Serotonin (5-HT) is one of the major transmitters involved in supraspinal control of somatic sensation and nociception. The aim of the present study was to investigate if the 5-HT-induced modulation of sensory transmission in the dorsal horn could be due to regulation of neuronal responses to excitatory amino acids. Experiments were performed in an in vitro preparation of the young rat spinal cord. Responses to dorsal root stimulation (DR-EPSP) and to droplet application of N-methyl-D-aspartic acid (NMDA) and α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) were obtained by means of intracellular recordings of dorsal horn neurons. Bath applications of 5-HT (50 µM) generally caused reductions in amplitude and integrated area of DR-EPSPs and of responses to NMDA but the responses to AMPA were unaltered. A linear correlation was found between the effects of 5-HT on the DR-EPSP and on the NMDA response measured as percentage change in amplitude (r 2 ϭ 0.45; P ഛ 0.01) and integrated area (r 2 ϭ 0.77; P ഛ 0.001). The NMDA receptor antagonist d-AP5 (50 µM) completely abolished NMDA responses and caused a depression of the DR-EPSP similar to that of 5-HT. The 5-HT 1 receptor agonist 5-carboxamidotryptamine (5-CT; 1 µM) mimicked the depressant effects of 5-HT but had a stronger depressant action on the DR-EPSP than 5-HT. The depression of NMDA responses induced by 5-HT and 5-CT was tetrodotoxin (1 µM) resistant. It is concluded that 5-HT-induced depression of NMDA responses explains partially the depressant action of 5-HT on dorsal horn synaptic transmission activating a postsynaptic site sensitive to 5-CT. The possible activation of coadjuvant mechanisms is discussed.

[1] 108w The involvement of serotonin (5-HT) in the descending control of somatosensation is generally accepted on the basis of extensive experimental evidence including the presence in the spinal cord of the serotonergic terminals of descending fibres (Dahlstrom & Fuxe, 1965), the spinal release of 5-HT after peripheral injury (Sorkin et al., 1990) and the analgesic effect of 5-HT directly applied to the cord (Yaksh & Wilson, 1979). Consistent with this analgesic effect, 5-HT iontophoresed in the dorsal horn causes mostly depression of neuronal responses to noxious stimulation (Headley et al., 1978), although a small proportion of neurons, thought to be inhibitory interneurons, show potentiated responses (Todd & Millar, 1983).

[2] 230w The serotonergic mechanisms involved in modulating synaptic transmission through the dorsal horn are not fully understood due to the multiplicity of receptors present in the cord and the variety of effects that these may mediate. Different effects of 5-HT have been reported on presynaptic afferent terminals as well as on postsynaptic dorsal horn neurons. At a presynaptic level 5-HT has been shown to cause depolarization and hyperpolarization of primary afferents (Hentall & Fields, 1983;Carstens et al., 1987), inhibition of calcium currents in dorsal root ganglion neurons (Del Mar et al., Correspondence: Dr J. A. Lopez Garcia, as above. E-mail: FFJALG@ALCALA.ES Received 15 September 1997, revised 5 December 1997, accepted 8 December 1997 1994; Cardenas et al., 1995) and modulation of neurotransmitter release (Hori et al., 1996). At a postsynaptic level multiple effects of 5-HT have been reported including membrane depolarization and hyperpolarization, changes in membrane resistance and excitability (Grudt et al., 1995;Lopez-Garcia & King, 1996) and modulation of responses to excitatory amino acids (Murase et al., 1990). It has been shown previously (Lopez-Garcia & King, 1996) that 5-HT-induced depression of spinal synaptic transmission and 5-HT-induced primary afferent depolarization (PAD) have different concentration-effect relationships such that at high concentrations 5-HT produces maximal depression of dorsal root evoked EPSPs (DR-EPSPs) but only submaximal PAD. Furthermore, depression can occur with or without changes in membrane potential, resistance or excitability of the postsynaptic element.

[3] 185w An interesting and so far unexplored hypothesis proposes that 5-HT-induced depression of synaptic transmission, and the resulting behavioural analgesia, could be due to a modulatory action on neuronal responses to excitatory amino acids (Besson & Chaouch, 1987). In fact, serotonergic modulation of neuronal responses to excitatory amino acid mediated transmission has been demonstrated in cerebellum, ventrobasal thalamus, hippocampus, neocortex and entorhinal cortex (Nedergaard et al., 1987;Maura et al., 1988;Eaton & Salt, 1989;Sizer et al., 1992). In these areas both potentiatory and depressant actions have been described for one or more excitatory amino acids indicating the existence of different and complex regulatory mechanisms. In the dorsal horn in vivo 5-HT was shown to depress neuronal responses to glutamate (Willcockson et al., 1984). Murase et al. (1990) reported a selective depression of N-methyl-D-aspartic acid (NMDA)-induced currents by 5-HT acting at a site with the pharmacological profile of a 5-HT 1 receptor in isolated dorsal horn neurons. Furthermore, in the hemisected spinal cord 5-HT causes depression of the late components of the DR-EPSP as would be expected from the blockade of NMDA-mediated polysynaptic transmission (Lopez-Garcia & King, 1996).

[4] 144w The aim of the present investigation was to test the modulatory effects of 5-HT on amino acid-induced responses and to evaluate the contribution of these effects to the depression of sensory transmission in the dorsal horn in an attempt to gain more information about the cellular mechanisms mediating serotonergic analgesia. To this end an in vitro preparation of the rat hemisected cord was used. The effects of 5-HT on dorsal horn transmission and on the responses to exogenous excitatory amino acids were analysed by means of intracellular recordings and compared with the effects of d-AP5, an antagonist of the NMDA receptor, and 5-carboxamidotryptamine (5-CT), a 5-HT 1 receptor agonist known to mimic the depressant actions of 5-HT in the dorsal horn (Ali et al., 1994;Lopez-Garcia & King, 1996). Some of the results included in this report have been presented in abstract form (Lopez-Garcia, 1996).

[1] 62w A total of 119 dorsal horn neurons were intracellularly recorded but the results reported below are based on 65 long lasting recordings (over 1 h) during which a significant part of the experimental protocol was carried out. The mean resting potential of this latter neuronal population was -65.3 Ϯ 1.3 mV and their mean input resistance was 87.4 Ϯ 7.4 MΩ .

[2] 80w Application of 35 µL droplets of NMDA (n ϭ 26) and 7 µL droplets of AMPA (n ϭ 18) directly into the recording chamber produced long-lasting neuronal depolarizations (range 80-520 s) of variable amplitude (range 3-20 mV). The integrated area of depolarization was similar for NMDA (1397 Ϯ 392 mV•s) and AMPA (1678 Ϯ 340 mV•s). Sixty-six per cent of neurons excited with NMDA and 43% of the neurons excited with AMPA fired action potentials (respective ranges: 13-270 and 16-330).

[3] 119w The antagonism of d-AP5 (50 µM) on neuronal responses to NMDA was studied in three neurons. The amplitude of the response to droplet applications of NMDA was reduced from 8.9 Ϯ 1.9 mV to 1 Ϯ 0.7 mV in the presence of d-AP5 (P ഛ 0.05) and the integrated area was reduced by a 97% (P ഛ 0.05; control value 1010 Ϯ 304 mV•s) (see Fig. 1). Thirty minutes after wash-out of d-AP5 the amplitude of the response recovered to 7.7 Ϯ 1.3 mV. In two neurons the effects of d-AP5 on neuronal responses to AMPA were tested. The mean amplitude of the response changed from 9 mV to 7 mV and the integrated area was reduced by 14%.

[4] 46w CNQX 10 µM abolished the responses to AMPA in two neurons and had very little effect on responses to NMDA in two further neurons (not shown). The integrated area of responses to AMPA was reduced by 97% whereas the responses to NMDA were reduced by 20%.

[5] 240w Responses to application of exogenous NMDA in control conditions and in the presence of 5-HT (50 µM) were recorded from 15 neurons; five of these neurons were depolarized by 5-HT (amplitude range: 3-8 mV) but none was hyperpolarized. During 5-HT superfusion the responses to NMDA were depressed in 13 neurons and potentiated in the remaining two. In the group of 13 neurons showing depressed responses to NMDA in the presence of 5-HT, the mean amplitude of the response decreased from 10.4 Ϯ 1.5 mV in control ACSF to 6.4 Ϯ 1 mV during 5-HT superfusion (P ഛ 0.005), the mean integrated area from 1450 Ϯ 386 mV.s to 705 Ϯ 158 mV.s (P ഛ 0.01), the mean duration from 217 Ϯ 30 s to 176 Ϯ 23 s (P ഛ 0.01) and the spiking was abolished (five of eight) or reduced (three of eight) (see Fig. 1). Clear signs of recovery were obtained in eight of 10 neurons where long-term inpalements permitted a recovery test. For these 10 neurons the amplitude of response after 30 min wash-out recovered to 85 Ϯ 11% of control value. Two neurons showed an increased amplitude in the response to NMDA during 5-HT superfusion (from 6.6 mV to 13.5 mV in 5-HT) and recovery was observed after 5-HT © 1998 European Neuroscience Association, European Journal of Neuroscience, 10, 1341-1349 wash-out in the one case where long recordings permitted a recovery test (shown in Fig. 6).

[6] 102w Responses to AMPA in control and in 5-HT (50 µM) containing ACSF were recorded from 11 neurons. Control responses to AMPA had a mean duration of 293 Ϯ 26 s, a mean amplitude of 10.2 Ϯ 2 mV and an integrated area of 1605 Ϯ 318 mV•s. During 5-HT superfusion, responses to AMPA were similar to those obtained in control ACSF (Fig. 2). The mean amplitude of the response was 9.3 Ϯ 1.3 mV, the mean duration was 276 Ϯ 24 s and the mean integrated area was 1465 Ϯ 219 mV/s. None of these values differ significantly from their respective controls.

[7] 130w Effects of 5-carboxamidotryptamine on responses to NMDA and AMPA 5-CT superfusion (1 µM) caused a depressant action on the responses to NMDA droplets in seven neurons tested but did not change the resting membrane potential. The mean amplitude of the responses to NMDA was reduced from 12.8 Ϯ 2.8 mV to 6.7 Ϯ 1.3 mV (P ഛ 0.05) and the depolarization area by 43% (control value 1760 Ϯ 479 mV.s; P ഛ 0.05). An example of this depressant effect is shown in Figure 3(A). In contrast to 5-HT, signs of recovery after 5-CT superfusion in the responses to NMDA were obtained only rarely after long wash-out periods. Sixty to 90 min after wash-out the amplitude of the response was only 62 Ϯ 18% of the control value (n ϭ 4).

[8] 50w Superfusion of 5-CT had very little effect on responses to AMPA in three neurons tested. The mean amplitude of the responses to AMPA in control ACSF was 9.2 Ϯ 3 mV and after 5-CT superfusion it was 8.2 Ϯ 2 mV (see Fig. 3B). The difference was not statistically significant.

[9] 85w In order to determine whether the site of action of 5-HT was pre-or postsynaptic to the recorded neuron, nine neurons were tested during continuous superfusion of tetrodotoxin (TTX; 1 µM). After 8-10 min of TTX superfusion spontaneous activity disappeared, the responses to dorsal root stimulation were abolished and the neurons failed to produce action potentials to direct injection of current pulses. Under these conditions slightly greater amounts of NMDA were administered (40-50 µL droplets) to compensate for the loss of amplitude due to TTX superfusion.

[10] 85w The effects of 5-HT were studied in five neurons. The mean amplitude of depolarization in response to NMDA changed from 12.4 Ϯ 7.2 mV in TTX-containing medium to 8.8 Ϯ 6.2 mV after addition of 5-HT to the superfusate. Similarly, 5-CT produced a depression of the responses to NMDA in four neurons tested. The amplitude of the response was reduced from 9.3 Ϯ 5.9 mV to 4.3 Ϯ 2.1 mV. An example of the depressant actions of 5-HT and 5-CT is shown in Figure 4.

[11] 217w Responses to high intensity dorsal root stimulation (DR-EPSP) (n ϭ 39) typically consisted of a compound EPSP characterized by a fast rising phase with one or more action potentials and a slow decay. Numerical values of the rate of rise, amplitude and duration © 1998 European Neuroscience Association, European Journal of Neuroscience, 10, 1341-1349 can be found in Table 1. The mean integrated area was 38.6 Ϯ 14.9 mV.s. The great majority of neurons (33 of 39) fired one or more short latency (Ͻ 50 ms) action potentials (2.2 Ϯ 0.5) but only 16 of 39 displayed longer latency spikes (15.5 Ϯ 6.7). Three neurons displayed strong inhibitory responses. One or more drugs were tested in each of these neurons. Table 1 compares the inhibitory effects of these three drugs on the DR-EPSP. All of them shared a similar profile of inhibition which affected the longer latency components and this resulted in significant reductions for amplitude and duration but not for rise-rate. The greatest reductions in integrated area were seen with 5-CT (78%; control value 49.1 Ϯ 13.1 mV•s; P ഛ 0.01) but 5-HT and d-AP5 superfusion caused significant reductions in this parameter as well (respectively: 59%, control value 44.7 Ϯ 4.5 mV•s, P ഛ 0.001 and 60%, control value 33.5 Ϯ 7.9 mV•s, P ഛ 0.001).

[12] 137w In four neurons, d-AP5 was superfused until a stable reduction of the DR-EPSP was observed and then 5-HT was added to the superfusate. In all four neurons, the addition of 5-HT caused further depression of the DR-EPSP. This secondary depression manifested as significant (P ഛ 0.05) reductions in duration (61 Ϯ 22%), amplitude (59 Ϯ 5.7%) and integrated area (81 Ϯ 11%) with respect to the corresponding values obtained in d-AP5. An example of one of these experiments is shown in Figure 5. Neurons with a facilitated response in 5-HT showed increases in duration, number of spikes and integrated area, although no statistical significance was attained due to the low number of observations and the large variations in control values. For example, the mean integrated area increased from 4.8 Ϯ 11.5 mV•s to 12.3 Ϯ 10.5 mV•s.

[13] 346w All neurons showing a depressed response to NMDA during 5-HT superfusion (n ϭ 13) showed a depressed DR-EPSP as well. In addition, the two neurons with enhanced responses to NMDA during 5-HT superfusion also showed facilitated responses to dorsal root © 1998 European Neuroscience Association, European Journal of Neuroscience, 10, 1341-1349 stimulation. Two separate linear correlation analyses were run in order to establish whether the 5-HT-induced modulation of responses to NMDA was related to the 5-HT-induced modulation of responses to dorsal root stimulation (see Fig. 6). One neuron facilitated by 5-HT was excluded from the analysis due to the inhibitory nature of its response to dorsal root stimulation (see Fig. 6). The first analysis showed a statistically significant correlation between the change in amplitude of both responses (slope ϭ 0.75 Ϯ 0.23 and r 2 ϭ 0.45; P ഛ 0.01). The second one showed a statistically significant correlation between the change in integrated area of both responses (slope ϭ 1.02 Ϯ 0.16 and r 2 ϭ 0.77; P ഛ 0.001). Although weaker, the correlation was still significant for the group of 13 neurons with depressed responses to both NMDA and dorsal root TABLE 1. Quantitative effects of 5-hydroxytryptamine (serotonin or 5-HT), D(-)-2-amino-5-phosphonopentanoic acid (d-AP5) and 5-carboxamidotryptamine (5-CT) on rise rate, amplitude and duration of the excitatory postsynaptic potential recorded from dorsal horn neurons in response to high-intensity dorsal root stimulation Rise rate (mV/ms) Amplitude (mV) Duration (s) Control 8.0 Ϯ 1.2 (22) 6.6 Ϯ 0.6 (22) 12.6 Ϯ 1.3 (22) 5-HT (50 µM) 7.8 Ϯ 1.6 4.7 Ϯ 0.6*** 7.2 Ϯ 0.8*** Control 8.8 Ϯ 3 (8) 6.1 Ϯ 1.2 (9) 19.0 Ϯ 4.5 (9) d-AP5 (50 µM) 7.9 Ϯ 1.7 3.1 Ϯ 0.7** 11.2 Ϯ 4.1* Control 7.2 Ϯ 2.8 (6) 7.8 Ϯ 1.1 (7) 17.2 Ϯ 4.8 (7) 5-CT (1 µM) 6.8 Ϯ 2.4 4.8 Ϯ 1.2** 4.9 Ϯ 1.4** Values expressed as mean Ϯ SEM and statistical significance from respective controls is denoted by asterisks (*P ഛ 0.05, ** P ഛ 0.01, *** P ഛ 0.005). Number of observations is given in parentheses.

[14] 15w stimulation (r 2 values were 0.33 for amplitude and 0.37 for area; P ഛ 0.05).

[15] 61w No such relation could be found for AMPA in the 11 neurons tested. Although most of these neurons (nine of 11) showed a significant depression of the DR-EPSP during 5-HT superfusion (see Fig. 2), only small and insignificant variations of responses to AMPA were detected. In the one neuron with enhanced DR-EPSPs there was no corresponding enhancement of the AMPA response.

[1] 51w Intracellular recordings from dorsal horn neurons have been performed using the in vitro hemisected cord preparation of young rats in order to investigate the modulatory action of 5-HT on the excitations produced by NMDA and AMPA and to evaluate the weight of this factor on the 5-HT-induced modulation of somatosensory transmission.

[2] 100w Droplet applications of NMDA and AMPA caused excitations of dorsal horn neurons which were selectively antagonized by the classic antagonists d-AP5 and CNQX. 5-HT exerted a depressant action on the excitations induced by NMDA in a majority of cases, although a small proportion of neurons showed potentiated responses to NMDA in the presence of 5-HT. The depressant action of 5-HT on NMDA responses persisted almost intact in the presence of TTX indicating the involvement of a postsynaptic site of action for 5-HT and did not extend to neuronal responses to AMPA indicating the activation of a mechanism selective for NMDA.

[3] 143w These observations reinforce previously reported results on acutely isolated spinal dorsal horn neurons (Murase et al., 1990). It was reported that 5-HT had a depressant action on responses to NMDA from very small concentrations (i.e. 10 -15 M) but in the presence of glycine higher concentrations of 5-HT were required to cause depression of responses to NMDA. At higher concentrations (i.e. 10 -3 M) 5-HT lost its selectivity for NMDA receptors and depressed responses to kainate as well. The hemisected cord is known to contain active re-uptake sites for 5-HT (Wallis & Elliot, 1991) and levels of endogenous glycine enough to saturate the glycine site of the NMDA receptor (Thomson, 1989;King et al., 1992); therefore, the concentration of 5-HT used in this study is likely to have caused close to maximal effect on the responses to NMDA while still keeping a good selectivity.

[4] 178w 5-CT mimicked the depressant action of 5-HT on NMDA responses and this effect was TTX persistent. Similarly to 5-HT this agonist © 1998 European Neuroscience Association, European Journal of Neuroscience, 10, 1341-1349 had a small, non-significant effect on the responses to AMPA. 5-CT is highly selective for 5-HT 1 receptors and it is two to five times more potent than 5-HT itself (Hoyer et al., 1994). Such characteristics will ensure a clear-cut and potent activation of 5-HT 1 (-like) receptors. This is in keeping with previous observations on isolated dorsal horn neurons where depression of NMDA-mediated responses by 5-HT had a similar pharmacology to that of 5-HT 1A receptors (Murase et al., 1990). From a pharmacological point of view the serotonergic modulation of responses to NMDA in the CNS seems to be a complex phenomenon mediated by several receptor types and involving different molecular mechanisms (Murase et al., 1990;Zhang et al., 1994;Blank et al., 1995;Chesnoy-Marchais & Barthe, 1996). Further experimental work focused on pharmacological issues will be required to determine precisely the relevant pathways in the dorsal horn.

[5] 147w The main task undertaken in the present study was to investigate whether serotonergic modulation of responses to excitatory amino acids, and specifically to NMDA, could account for the modulation of synaptic responses to afferent input in the dorsal horn. The experiments reported here present new evidence in favour of this hypothesis. Neurons showing a depressed response to NMDA in the presence of 5-HT showed depressed DR-EPSPs and in those neurons where 5-HT potentiated the responses to NMDA it potentiated the DR-EPSP as well. The data supported a significant positive correlation between the modulation caused by 5-HT on the NMDA-induced excitations and on the DR-EPSPs. The two measurements used to analyse this correlation, i.e. changes in amplitude and integrated areaindicated r 2 values of 0.45 and 0.77, respectively, suggesting that serotonergic modulation of NMDA receptors may be a key mechanism to channel 5-HT actions in dorsal horn transmission.

[6] 426w The next question is to elucidate whether NMDA modulation is the only factor required to explain modulation of synaptic transmission or whether other factors are involved. The qualitative profile of depression caused by 5-CT and 5-HT on the DR-EPSPs was similar to that of d-AP5. Long-latency amplitude, duration and integrated area, all of them measurements of transmission across polysynaptic pathways, were reduced. In contrast the rising phase of the DR-EPSP, more likely to reflect monosynaptic transmission, was unaltered. This is consistent with the blockade of sensory transmission along polysynaptic pathways in which NMDA receptors play an important part according to work by Davis & Watkins (1983) and others (see King et al., 1992) and with the view that the serotonergic modulation of responses to NMDA may modulate somatosensory transmission. Yet, a quantitative approach reveals that the actions of 5-HT, 5-CT and d-AP5 on both DR-EPSPs and responses to NMDA are relatively different. The antagonist d-AP5, while virtually abolishing responses to NMDA, caused only partial depression of DR-EPSPs. In contrast, 5-HT and 5-CT achieved a similar or greater degree of depression of the DR-EPSPs causing a much smaller blockade of the responses to NMDA. Furthermore, 5-HT caused significant depression of the longlatency components of the DR-EPSP even after blockade of the NMDA receptors with d-AP5. All together these results clearly support the idea that 5-HT can activate other mechanisms not dependent upon NMDA receptor modulation. It seems very likely that both NMDA-dependent and NMDA-independent mechanisms were activated in parallel by 5-HT and 5-CT during the bulk of the experiments reported here. The nature and the 5-HT receptors involved in this hypothetical NMDA-independent mechanism are not known. In the present experiments, 5-CT showed a similar depressant effect on the responses to NMDA but a stronger depressant effect on the DR-EPSP than 5-HT. As 5-CT is a powerful agonist at 5-HT 1 receptors it is possible that it acted on slowly conducting primary afferents to decrease the release of sensory transmitters via the inhibition of calcium entry at the terminals or other mechanisms (Del Mar et al., 1994;Hori et al., 1996). 5-HT could employ this same mechanism although, under the present conditions, it could have activated a variety of receptors present in the dorsal horn some of which could work in opposite directions. For example 5-HT 2 receptors have been shown to mediate a long-lasting facilitation of synaptic transmission in the superficial dorsal horn by directly stimulating the exocitotic machinery of presynaptic terminals (Hori et al., 1996). Further experimental analysis will be required to test these and other possibilities.

[7] 112w The relevance of these mechanisms in intact animals, i.e. NMDAdependent vs. NMDA-independent, may be critically related to the way that 5-HT is physiologically released in the dorsal horn. According to available data, descending serotonergic fibres contact dorsal horn neurons but only rarely afferent terminals (Maxwell et al., 1983;Marlier et al., 1991), therefore it might be expected that the action of synaptically released 5-HT would be channelled mainly via modulation of responses to NMDA. However, there is experimental evidence supporting the idea that 5-HT can be extrasynaptically released and conduced via volume transmission in dorsal horn (Ridet et al., 1993). Consequently, a physiological action of 5-HT directly on primary afferents cannot be excluded.

[8] 66w In conclusion, the data obtained in this study supported the hypothesis under scrutiny. 5-HT-induced modulation of responses to NMDA, was clearly related to the modulation of transmission in the dorsal horn. Specific depression of responses to NMDA was mediated by a postsynaptic site sensitive to 5-CT and can explain partially the depression of synaptic transmission observed during 5-HT administration. However, the simultaneous involvement of other mechanisms,

[1] 93w Spinal cords were extracted from urethane anaesthetized (2 g/kg, i.p.) 10-14-day-old rats weighing less than 30 g. A dorsal laminectomy exposed the lumbar segments of the cord which, together with the attached segmental roots, was rapidly excised, placed in artificial cerebrospinal fluid (ACSF) and hemisected. The hemisected cord was pinned securely in the recording chamber and continuously superfused with warm (23-24 °C) and oxygenated ACSF containing (mM): NaCl, 128; KCl, 1.9; KH 2 PO 4 , 1.2; MgSO 4 , 1.3; CaCl 2 , 2.4; NaHCO 3 , 26; glucose, 10; pH 7.4.

[2] 50w A lumbar dorsal rootlet (L4-L5) was drawn into a tight suction electrode and electrically stimulated to provide afferent input to the dorsal horn. Intracellular recordings from dorsal horn neurons were performed with conventional glass micropipettes filled with 3 M potassium acetate (80-120 MΩ), placed in the preparation under visual guidance.

[3] 157w NMDA or α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) (both purchased from Sigma) were applied in droplets (35 µL of NMDA and 7 µL of AMPA, both at 10 -2 M dissolved in saline) directly into the ACSF inlet of the recording chamber. Pilot experiments (n ϭ 7) using this application procedure showed that repeated application of NMDA at 20-30 min intervals produced stable neuronal responses with a mean difference in amplitude between the first and second application of 1 Ϯ 0.3 mV and no clear signs of desensitization in up to five consecutive applications. Repeated applications of AMPA led to clear signs of desensitization in three of five neurons but their responses were stable during the first two to four applications with a difference in amplitude between applications of 1.7 Ϯ 0.6 mV. 5-HT, 5-CT, D(-)-2-amino-5-phosphonopentanoic acid (d-AP5) and 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX), all purchased from R.B.I. (Natick, MA, USA), were dissolved in ACSF and superfused during fixed time periods (see below).

[4] 140w For the intracellular studies, neurons with stable membrane potentials and overshooting spikes of amplitude greater than 60 mV were selected. After impalement, neurons were challenged with high intensity dorsal root stimulation (between 100 µs, 100 µA and 300 µs, 300 µA to activate both myelinated and unmyelinated afferents) followed by a droplet of either NMDA or AMPA. After a 25 min wash-out, 5-HT (50 µM) or 5-CT (1 µM) were superfused for 3-min periods and both the electrical and the chemical stimuli were repeated. The respective concentrations used were selected to produce maximal or near maximal effects according to previous studies using the same preparation (Lopez-Garcia & King, 1996). Recovery was tested at 30-min intervals. Studies with the antagonists d-AP5 (50 µM) and CNQX (10 µM) were made following the same protocol, although the superfusion time was longer (8-10 min).

[5] 121w The intracellular recordings were digitised and stored on magnetic tape for off-line computer-assisted analysis using 'Spike 2' (Cambridge Electronic Design Ltd). Excitatory amino acidinduced depolarizations were quantified in terms of: (i) maximal amplitude (mV); (ii) total number of spikes per response; and (iii) integrated area of depolarization (mV•s). The compound EPSPs elicited by dorsal root stimulation were characterized in terms of: (i) depolarization-rate measured in the first 2 ms of the response (mV/ms); (ii) maximal amplitude measured in a window between 200 ms and 800 ms from stimulus artefact; (iii) number of spikes per response; and (iv) integrated area of depolarization (mV/s). All data are expressed as mean Ϯ SEM and for statistical analysis, linear correlation and Student's t-tests were used.