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1 Center for Brain Research, Department of Neurophysiology, Medical University of Vienna, Vienna, Austria
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Abstract |
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(Received 18 April 2005;
accepted after revision 29 April 2005;
first published online 5 May 2005)
Corresponding author J. Sandkühler: Center for Brain Research, Department of Neurophysiology, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria. Email: juergen.sandkuehler{at}meduniwien.ac.at
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Introduction |
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5%) of the total lamina I neuronal population sends projections to the brain (Spike et al. 2003), so that unidentified lamina I neurones can be assumed to mostly represent intrinsic spinal neurones. Unidentified neurones did not show a synaptic LTP after conditioning stimulation of C-fibres (Ikeda et al. 2003; Sandkühler & Ikeda, 2003). It is presently not clear why lamina I projection neurones are prone to become sensitized. Intrinsic cellular properties such as membrane excitability and/or network features such as type or strength of afferent input could have a decisive impact. Recently, we reported that lamina I projection neurones have intrinsic membrane properties that set them apart from unidentified lamina I neurones (Ruscheweyh et al. 2004). Here, we investigated both the primary afferent excitatory input and the action potential-independent inhibitory and excitatory input onto lamina I projection neurones and compared it with that of lamina I unidentified neurones. |
Methods |
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All procedures used in this study conformed to the European Communities Council Directive (86/609/EEC) and were approved by the guidelines of the Austrian Federal Ministry for Education, Science and Culture. Young (postnatal day 18–37) Sprague-Dawley rats were anaesthetized with a mixture of ketamine and xylazine (75 mg kg–1 and 7.5 mg kg–1 I.P., respectively) and placed in a stereotaxic apparatus. A small (< 1 cm) scalp cut was made and a hole was drilled into the skull bone to allow insertion of the needle of a 500 nl Hamilton syringe into the targeted area. The animals then received a single injection of 50–100 nl fluorescent tracer (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC12), 1.25–2.5%) into either the right lateral parabrachial area or the right ventrolateral and lateral PAG according to the atlas of Swanson (1992). The head wound was sewed with two stitches. Daily inspection revealed no signs of infection. After recovery from anaesthesia, the animals fed and drank normally. No pain-related behaviour was observed in any of the animals. After a 3- to 4-day survival period, preparation of spinal cord slices was carried out as described below and the rats were then killed by an overdose of ether. The brain was removed, cooled to –20°C in isopentan and cryostat sections (30 µm thick) of the brainstem were obtained in order to allow histological verification of the injection site (Fig. 1A and B). Only recordings from animals where the injection site was clearly confined to either the parabrachial area or the PAG were included in the present study.
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The lumbar spinal cord was removed under deep ether anaesthesia and the rat was killed by an overdose of ether. Transverse 500 µm thick lumbar spinal cord slices were cut on a microslicer (DTK-1000, Dosaka EM, Kyoto, Japan). In some slices a long (7–15 mm) dorsal root was preserved to allow stimulation of primary afferents. For part of the miniature inhibitory postsynaptic current (mIPSC) recordings, parasagittal slices of 400 µm thickness were made. Slices were incubated in a solution that consisted of (mM): NaCl 95, KCl 1.8, KH2PO4 1.2, CaCl2 0.5, MgSO4 7, NaHCO3 26, glucose 15 and sucrose 50, and was oxygenated with 95% O2–5% CO2; pH 7.4, osmolarity 310–320 mosmol l–1. A single slice was then transferred to the recording chamber and continuously superfused with oxygenated recording solution at 3 ml min–1. The recording solution was identical to the incubation solution except for (mM): NaCl 127, CaCl2 2.4, MgSO4 1.3 and sucrose 0. Experiments were conducted at room temperature (21–25°C).
Patch-clamp recording
Dorsal horn neurones were visualized with ‘Dodt’ infrared optics (Dodt et al. 1998). Retrogradely labelled lamina I projection neurones were detected by epifluorescence. They were found to lie within a distance of maximally 20 µm from the dorsal white/grey matter border (Fig. 1Ca and Da), corresponding to the zone where the tangential orientation of the neuropil typical of lamina I was observed. Randomly selected unlabelled neurons were labelled ‘unidentified’. (Fig. 1Ea). Spino-parabrachial, spino-PAG and unidentified lamina I neurones were recorded in the whole-cell patch-clamp configuration (Fig. 1Cb–Eb). Patch pipettes (2–6 M
) from borosilicate glass (Hilgenberg, Malsfeld, Germany) were made on a horizontal micropipette puller (P-87, Sutter Instruments, Novato, CA, USA) and filled with a solution that consisted of (mM): potassium gluconate 120, KCl 20, MgCl2 2, Na2ATP 2, NaGTP 0.5, Hepes 20 and EGTA 0.5; pH adjusted to 7.28 with KOH, measured osmolarity 300 mosmol l–1. Neurones were recorded in current- and voltage-clamp modes using an Axopatch 200B patch-clamp amplifier and the pCLAMP 8 and 9 acquisition software (both Axon Instruments, Union City, CA, USA). Signals were low-pass filtered at 2–10 kHz, sampled at 10–100 kHz and analysed off-line using pCLAMP 8 and 9 and Mini Analysis (v.5.6.3, Synaptosoft, Inc., Decatur, USA). Membrane potentials were held at –70 mV during voltage-clamp experiments. No correction for the liquid junction potential was made. At the end of the experiments the mediolateral locations of the recorded lamina I neurones were assessed by visually inspecting the position of the pipette tip under a 4 x objective.
Passive membrane properties
Passive membrane properties were investigated as previously described (Ruscheweyh et al. 2004). The membrane potential measured immediately after establishing the whole-cell configuration was called the ‘resting membrane potential’. Neurones with a resting membrane potential less negative than –50 mV were excluded from further investigation. The membrane capacitance was calculated from the area under the capacitive transient of the averaged reaction to 20 100 ms-long hyperpolarizing voltage steps from –70 to –80 mV.
Miniature excitatory and inhibitory postsynaptic currents
For investigation of AMPA/kainate receptor-mediated miniature excitatory postsynaptic currents (mEPSCs), voltage-clamp recordings were conducted in the presence of tetrodotoxin (TTX, 0.5 µM), D-2-amino-5-phosphonovaleric acid (D-AP5, 50 µM) (–)-bicuculline methiodide (bicuculline, 10 µM) and strychnine (4 µM). Samples of 2 to 5 min length were obtained to analyse the frequency, amplitude and the kinetics of mEPSCs in spinal lamina I neurones. Samples (4 min long) of mIPSCs were recorded in the presence of TTX (0.5 µM), D-AP5 (50 µM), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM) and either strychnine (300 nM or 4 µM; for recording of GABAergic mIPSCs) or bicuculline (10 µM; for recording of glycinergic mIPSCs). Due to the ratio of intracellular to extracellular Cl– concentrations set by our solutions, Cl– currents (e.g. currents through GABAA and glycine receptors) are inwardly directed at a holding potential of –70 mV. Antagonists used to investigate miniature synaptic currents were added to the bath 
10 min before starting the experiment. Only recordings with access resistances of less than 25 M (mean 17.4 ± 0.9 M, n
= 44) that were stable throughout the entire recording (< 20% change) were considered acceptable for analysis of mPSCs. In some neurones, high initial mPSC frequencies were observed that declined rapidly over the course of the first few minutes. These neurones were excluded from analysis. Detection threshold was set at 9 pA for mEPSCs and at 10 pA for mIPSCs. Each event was inspected visually and any noise that spuriously met the trigger specifications was rejected. For the analysis of mEPSC kinetics, the events were selected on the basis of the following criteria: superimposed events were discarded and only events that had stable baselines before the rise and after the end of decay were kept for analysis. The peak amplitude was calculated as the mean of the individual peak amplitudes of all events detected in an investigated neurone. For analysis of kinetics, the mEPSCs were averaged after aligning them at 50% rise time. The rise time of the resulting current was determined from 10% to 90%, the decay time from 90% to 37% of its amplitude.
Evoked excitatory postsynaptic currents
To assess both AMPA/kainate and NMDA receptor-mediated currents, evoked excitatory postsynaptic currents (eEPSCs) were investigated in a modified recording solution that was nominally free of magnesium and in most experiments was supplemented with glycine (10 µM). Data obtained in the presence and absence of glycine were pooled as no difference in the NMDA receptor-mediated currents was detected between the two groups. N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314, 5 mM) was added to the pipette solution to prevent generation of action potentials in the investigated neurone. EPSCs were evoked in labelled and non-labelled lamina I neurones by stimulating the dorsal root with supramaximal intensities (3–5 mA at 0.1 ms) in order to recruit C-fibres. Test pulses were given at 30-s intervals through a suction electrode with an isolated current stimulator (A360, World Precision Instruments, Sarasota, FL, USA). Afferent input was classified as C-fibre-evoked based on a combination of response threshold and conduction velocity (Chen & Sandkühler, 2000). C-fibre-evoked EPSCs were considered monosynaptic by the absence of failures during 10 consecutive pulses at 1 Hz and low jitter in response latencies (Fig. 2Aa and Ba; Nakatsuka et al. 2000). Only cells with access resistances of less than 40 M and < 50% change during the experimental recording were included into the analysis of eEPSCs. Neurones were discarded when leak currents at a holding potential of –70 mV exceeded ± 60 pA.
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Statistical analysis
All values are expressed as mean ± S.E.M. One-way ANOVA, the non-parametric Mann-Whitney U rank sum test, Wilcoxon signed rank test, Student's paired and unpaired t test, linear regression, analysis of covariance and the Kolmogorov-Smirnov test were used for statistical comparison where appropriate. The critical value for statistical significance was set at P < 0.05 (*) or P < 0.01 (**).
Application of drugs
Drugs were applied to the perfusion solution at known concentrations. The drugs used were bicuculline (10 µM), strychnine (300 nM, 4 and 8 µM), glycine (10 µM and 1 mM), GABA (1 mM), kainate (100 µM; all from Sigma, Deisenhofen, Germany), D-AP5 (50 µM), CNQX (10 µM), TTX (0.5 and 1 µM; all from Alexis, Grünstadt, Germany), picrotoxin (100–200 µM), GYKI 52466 (100 µM) and (2S,4R)-4-methylglutamate (SYM 2081, 1–10 µM; all from Tocris, Bristol, UK). DiIC12 (1.25–2.5%, Molecular Probes, Oregon, USA) was used as fluorescent tracer for neuronal labelling. QX-314 (5 mM, Sigma) was added to the pipette solution to investigate dorsal root-evoked EPSCs.
Stock solutions were prepared by dissolving bicuculline, glycine, GABA, kainate, D-AP5 and GYKI 52466 in distilled water, strychnine, CNQX and DiIC12 in dimethyl sulfoxide (DMSO, Sigma), SYM 2081 in NaOH, picrotoxin in ethanol and TTX in acidic buffer (pH 4.8). Stock solutions were stored in aliquots at –20°C except for DiIC12 which was stored at 4°C.
For exogenous application of kainate (100 µM), GABA (1 mM) and glycine (1 mM), oxygenated recording solution containing the respective drug was applied by means of a gravity-fed tube system operated by a manual switch that had its outlet opening directly onto the surface of the slice.
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Results |
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Characteristics of primary afferent, monosynaptic C-fibre input to lamina I projection and unidentified neurones
Activation of different ionotropic glutamate receptors may mediate monosynaptic C-fibre-evoked EEPSCs. Here, we quantitatively evaluated the contribution of NMDA, AMPA and kainate receptor-mediated currents to the eEPSC in projection and unidentified neurones in lamina I spinal dorsal horn. In slices from Sprague-Dawley rats between 18 and 37 days old, supramaximal stimulation of dorsal roots (3–5 mA, 0.1 ms) monosynaptically evoked C-fibre responses (threshold, 0.36 ± 0.03 mA; conduction velocity, 0.32 ± 0.03 m s–1; n = 23) in lamina I neurones which were recorded in nominally Mg2+-free solution (Fig. 2A and B).
Age dependence of the eEPSC area and its NMDA and AMPA/kainate receptor-mediated portions. NMDA receptors mainly contribute to the falling phase, not the peak of the eEPSC (Forsythe & Westbrook, 1988; Yoshimura & Jessell, 1990). Consequently, we determined the area under the current (see Methods) to compare NMDA and non-NMDA receptor-mediated portions of the eEPSC. Inspection of the total eEPSC areas suggests that they decrease with increasing age of the animal (Fig. 2C), following a non-linear dependence. To determine what kind of non-linear dependence is adequate to describe the data, a multiple of the existing data points would have been required, which was beyond the scope of the study. Therefore, it was not possible to determine whether the groups of projection neurones and unidentified neurones were homogeneous with respect to the total eEPSC area, but the distributions of eEPSC areas clearly overlapped among these groups.
The NMDA receptor antagonist D-AP5 (50 µM) was washed into the bath to investigate the NMDA receptor-mediated proportion of eEPSCs (Fig. 2A and B). Averaged over all age groups, D-AP5 (50 µM) reduced the eEPSC area of the investigated neurones to 51 ± 4% of control (postnatal day 26 ± 1, n = 23; P < 0.01). A display of the NMDA receptor-mediated current area (Fig. 2D) shows a developmental current decay that at the first sight looks similar to that of the total eEPSC area (Fig. 2C). However, if the NMDA receptor-mediated proportion of the eEPSC area (in percent) is illustrated, it becomes clear that the NMDA receptor-mediated current area decays more strongly with increasing age than the total eEPSC area (Fig. 2E). Between postnatal day 18 and 27, there was a significant decrease in the NMDA receptor-mediated proportion of the eEPSC area that could be fitted by a linear function (Fig. 2E, n = 18; P < 0.01 for the linear dependence). The linear dependence was tested together with the homogeneity of the groups in an analysis of covariance setting. The result was that projection and unidentified neurones are homogenous with respect to the developmental changes between postnatal day 18 and 27 (the homogeneity could not be rejected with P = 0.211). Obviously, the linear dependence cannot hold true for age values outside the studied age range, as the NMDA receptor-mediated current proportion would become negative around postnatal day 34. The additional data points at postnatal day 35–37 suggest that a steady state is reached around postnatal day 26 (Fig. 2E). In line with the results of other studies (Forsythe & Westbrook, 1988; Yoshimura & Jessell, 1990), the peak amplitude of the monosynaptic eEPSC was only slightly reduced by D-AP5 (50 µM; to 87 ± 2% of control, postnatal day 26 ± 1, n = 23; P < 0.01). The NMDA receptor-mediated current was determined by subtraction of the eEPSC after application of D-AP5 from the eEPSC before D-AP5 application as described in the Methods. Where possible, the decay of the NMDA receptor-mediated current was fitted with a single exponential. The resulting decay time constant (Fig. 2F) decreased with increasing age of the animal.
The eEPSC current remaining after application of D-AP5 was abolished by the AMPA/kainate receptor antagonist CNQX (10 µM, n = 7; data not shown). Therefore, the AMPA/kainate receptor-mediated proportion of the eEPSC area exhibited an age-dependent increase from postnatal day 18–27 in projection and unidentified neurones (data not shown) reciprocal to the decrease in the NMDA receptor-mediated proportion described above.
Kainate receptor-mediated portion of the eEPSC. Recent electrophysiological studies showed that besides NMDA and AMPA receptors, postsynaptic kainate receptors in superficial dorsal horn neurones are activated following stimulation of high-threshold primary afferents, presumably C-fibres (Li et al. 1999). In the present study, we tested the contribution of kainate receptor-mediated currents to C-fibre-evoked EPSCs in lamina I neurones. We applied the selective non-competitive AMPA receptor antagonist GYKI 52466 (100 µM) which nearly abolished the eEPSC amplitude remaining after application of D-AP5 (reduction from 90.6 ± 3.2% to 4.4 ± 1.1% of control value before application of D-AP5, postnatal day 22.8 ± 1, n = 11, projection and unidentified neurones data pooled; P < 0.01, Figs 2A and B, and 3Aa). All residual currents were eliminated by CNQX (10 µM; n = 10, Figs 2A and B, and 3A). To test whether the current remaining after application of D-AP5 (50 µM) and GYKI 52466 (100 µM) was truly kainate receptor-mediated or due to incomplete blocking of AMPA receptors by GYKI 52466, we used the kainate receptor desensitizing compound SYM 2081 (1–10 µM). It reduced the eEPSC amplitude only marginally (from 7.8 ± 4.1% to 5.5 ± 3.3% of control, n = 2; Fig. 3A, traces 3 and 4) whereas, in contrast, bath application of CNQX (10 µM) eliminated the residual currents (reduction to 0.7 ± 0.2% of control, n = 2; Fig. 3A, trace 5).
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In conclusion, the present experiments did not reveal a major contribution of kainate receptors to responses of lamina I neurones following stimulation of primary afferent C-fibres or bath application of kainate.
Higher mEPSC frequency in lamina I projection neurones than in unidentified neurones
The action potential-independent excitatory input to projection and unidentified neurones in lamina I was investigated by mEPSC recordings in animals between postnatal day 20 and 26. Individual traces and averaged miniature currents are shown in Fig. 4A–C. Results are summarized in Table 1.
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Recently, an age-dependent increase in mEPSC frequencies was described in superficial spinal dorsal horn from postnatal day 0–10 (Baccei et al. 2003). We did not observe any age dependence of mEPSC frequency between postnatal day 20 and 26.
Low frequency of mIPSCs in lamina I projection and unidentified neurones
To investigate the action potential-independent inhibitory input of lamina I neurones, we recorded glycinergic and GABAergic mIPSCs in rats between postnatal day 20 and 26. Both types of mIPSCs were very scarce. In traces of 4 min duration, only zero to four out of the nine to 12 neurones investigated per group (spino-parabrachial, spino-PAG and unidentified neurones; GABAergic and glycinergic mIPSCs) showed one to 37 mIPSCs. This corresponds to GABAergic and glycinergic mIPSC frequencies between 0 and 0.15 events s–1 with a median of 0 events s–1. Original traces are shown in Fig. 5C. Due to the low occurrence of GABAergic and glycinergic mIPSCs, neither an analysis of kinetics nor a comparison between groups was attempted. The amplitudes ranged between 10.1 and 53.3 pA. These recordings were made in transversal spinal cord slices. Many lamina I neurones extend their main dendrites in rostro-caudal direction, so that transsection of major parts of the dendritic tree in the transversal slice might be a cause of the low mIPSC frequencies. Another concern was that the relatively high strychnine concentration might have partially blocked GABAergic mIPSCs (Jonas et al. 1998). Therefore, we conducted a series of experiments, recording GABAergic mIPSCs in the presence of a lower strychnine concentration (300 nM) in unidentified lamina I neurones of parasagittal spinal slices. Under these conditions, the mIPSC frequency was still very low (one to nine GABAergic mIPSCs in four of the eight recorded neurones) and not significantly different from the results reported above.
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Discussion |
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Previous studies have revealed fundamental functional differences between lamina I projection neurones and interneurones. Projection neurones are necessary for the full expression of hyperalgesia in the rat and susceptible to LTP at their synapses with primary afferent C-fibres, while interneurones are not (Nichols et al. 1999; Ikeda et al. 2003). Retrograde labelling with DiI enabled us to record from lamina I neurones with an identified projection site in the brainstem and elucidate their differential intrinsic membrane (Ruscheweyh et al. 2004) and synaptic input (present study) properties. Concerning the specificity of projection areas, one has to keep in mind that many lamina I projection neurones send their axons to more than one area in the brainstem. For example, most spino-PAG neurones possess collaterals to the parabrachial area, making the spino-PAG neurones a subgroup of the spino-parabrachial neurones, accounting for about 30% of them (Spike et al. 2003). In addition, it has to be considered that the site of dye injection may not be identical to the final projection area of some of the studied neurones because fibres passing through the injected structures may take up the dye.
Low action potential-independent GABAergic and glycinergic inhibitory control of lamina I neurones
Exogenous application of GABA and glycine demonstrated that all investigated types of lamina I neurones expressed both functional GABAA and glycine receptors which is in line with a previous report (Chéry & De Koninck, 1999). Nonetheless, frequencies of GABA- and glycinergic mIPSCs were very low in lamina I unidentified and projection neurones. This may be due to a low spontaneous release probability of GABA and glycine and/or to a predominantly extrasynaptic location of GABAA and glycine receptors.
Evidence exists for perisynaptic and/or extrasynaptic GABAA receptors in adult rat dorsal horn (Chéry & De Koninck, 1999). Glycine receptors in cultured spinal neurones diffuse between synaptic, perisynaptic and extrasynaptic sites within seconds to minutes (Dahan et al. 2003). The synaptic recruitment of GABAA and glycine receptors, as well as the release probability of GABA and glycine, might be affected by different recording conditions or slice preparations (Lim et al. 2003) and this possibly contributes to the great variability of mIPSC frequencies in the superficial dorsal horn in the literature (0.1–5 events s–1, Chéry & De Koninck, 1999; Müller et al. 2003). It is well established that the mIPSC frequency is not a stable feature of a spinal neurone but rather susceptible to modulation. Glycinergic and GABAergic mIPSC frequencies in spinal dorsal horn are reduced after peripheral inflammation or nerve injury (Moore et al. 2002; Müller et al. 2003) and increased by application of transmitters that may be released by descending pathways in the intact animal (noradrenaline (norepinephrine), acetylcholine; Baba et al. 1998; 2000b,). It has also been reported that the frequency of GABAA receptor-mediated mIPSCs in lamina I neurones decreases with age (Keller et al. 2001). In the present study, mIPSC frequencies were too low to detect any age-related shift in the observation period (postnatal day 20–26).
Our finding that action potential-independent excitatory input to lamina I neurones was much more pronounced than inhibitory input could indicate that in superficial dorsal horn either excitatory synapses outnumber inhibitory synapses or the release probability is higher for excitatory than for inhibitory transmitters. Dominance of excitatory over inhibitory action potential-independent input has also been found in lamina II neurones (Iyadomi et al. 2000; Wu et al. 2003). GABA and glycine are not the only inhibitory neurotransmitters in superficial spinal dorsal horn. Lamina I neurones may be inhibited by activation of multiple types of postsynaptic receptors, among them µ-opioid receptors, 
-adrenergic receptors, cholinergic receptors and serotoninergic receptors (Aicher et al. 2000; Li & Zhuo, 2001).
Differences in evoked and action potential-independent excitatory synaptic input between lamina I projection and unidentified neurones
A major finding of the present study was that lamina I projection neurones exhibited significantly higher mEPSC frequencies, both absolute and in relation to their size, than unidentified neurones. Spino-PAG neurones exhibited an even higher mEPSC frequency than spino-parabrachial neurones, of which they constitute a subgroup (Spike et al. 2003). The most probable explanation would be that the density of excitatory synapses on the cell surface of these neurones is higher. However, the possibility cannot be excluded that projection neurones, especially spino-PAG neurones, receive synapses with a higher probability of spontaneous transmitter release. Previous recordings in the superficial dorsal horn revealed mean mEPSC frequencies between 0.14 and 12.6 events s–1 (Iyadomi et al. 2000; Baccei et al. 2003). We found mEPSC frequencies of 0.18 events s–1 in unidentified and 0.86 events s–1 in projection lamina I neurones. The variance between the different studies may be due to the dependence of mEPSC frequency on recording temperature (Simkus & Stricker, 2002), potassium content of the recording solution (Bao et al. 1998) and postnatal age of the investigated animal (Baccei et al. 2003). Wu et al. (2003), who recorded under conditions comparable to ours but from lamina II neurones, reported mean mEPSC frequencies of 1.08 events s–1.
The induction of C-fibre-mediated synaptic LTP in lamina I projection neurones requires an increase in intracellular Ca2+ levels. The Ca2+ rise during conditioning repetitive stimulation is significantly stronger in lamina I projection neurones than in unidentified neurones that do not undergo LTP (Ikeda et al. 2003). As NMDA receptors are Ca2+-permeable channels, and blockade of NMDA receptors prevents induction of LTP in projection neurones, one could suspect a difference of the NMDA receptor-mediated currents between projection and unidentified neurones. However, during the non-repetitive C-fibre stimulation which was applied here, projection and unidentified neurones had similar contributions of NMDA receptor-mediated currents to the eEPSC.
The contribution of ionotropic glutamate receptor subtypes to the C-fibre-evoked EPSC is developmentally regulated
Slice preparations from rat spinal cord are often obtained from animals which are at least 18 days old under the assumption that at this age, most of the spinal neuronal development is complete and the results obtained are therefore representative for the situation in the adult animal. However, in the present study, we describe a steep age-dependent decrease in the NMDA receptor contribution to C-fibre-evoked EPSCs in lamina I neurones between postnatal day 18 and 27. We performed our experiments in a nominally Mg2+-free recording solution, to release the voltage-dependent Mg2+ block of the NMDA receptor (Mayer et al. 1984), and in the presence of glycine. Therefore, the observed developmental changes could either reflect the conversion of purely NMDA receptor-containing synapses into mixed NMDA and AMPA receptor-containing synapses or a shift in the relative proportions of AMPA receptor-mediated and NMDA receptor-mediated currents at mixed synapses. Synapses containing only NMDA receptors but no functional AMPA receptors (‘silent synapses’), are present in the rat superficial dorsal horn during the first and second postnatal weeks (Li & Zhuo, 1998; Bardoni et al. 1998) and strongly decrease in number at later stages of development (Baba et al. 2000a). Interestingly, recent immunohistochemical results suggest that in adult rat lamina I, a relatively high number of synapses contain AMPA receptors but no NMDA receptors (Nagy et al. 2004).
Besides changes in the number of functional glutamate receptors, developmental modifications of the receptor properties could also account for the presently observed alterations. The developmental decrease in the decay time of NMDA receptor-mediated currents we observed points to a change in the receptor subunit composition. The results on NMDA receptor subunit distribution in spinal dorsal horn are highly controversial (see discussion in Nagy et al. 2004), but it has been suggested that in lamina I, the expression of the NMDA receptor subunits NR2B and NR2D decreases with postnatal age whereas subunit NR2A expression increases (Watanabe et al. 1994). NMDA receptors containing the NR2A subunit have significantly shorter decay times than those containing NR2B or NR2D (Monyer et al. 1994; Vicini et al. 1998), and a developmental shift towards increased NR2A expression leads to shorter decay times in rat neocortex (Flint et al. 1997). Similarly, the AMPA receptor subunit composition changes between postnatal day 7 and 28, accompanied by a decrease in overall AMPA receptor expression (Jakowec et al. 1995). Thus, the subunit composition of both NMDA and AMPA receptors undergoes developmental changes that might influence their ligand binding affinity and/or mean channel conductance and/or opening kinetics. This may lead to a shift in the relative contribution of NMDA and AMPA receptors to dorsal root-evoked EPSCs.
Kainate receptors make no major contribution to monosynaptic C-fibre-evoked responses in lamina I
It has been reported that kainate receptors are involved in primary afferent-evoked responses of superficial dorsal horn neurones at postnatal day 4–21, contributing about 30% to the combined AMPA/kainate receptor-mediated current evoked by single-shock high-intensity, presumably C-fibre, stimulation (Li et al. 1999). In addition, cultured dorsal horn neurones respond to exogenous application of kainate in the presence of selective AMPA receptor antagonists (Li et al. 1999; Kerchner et al. 2001). In the present study, the amplitude of the C-fibre-evoked response of lamina I projection and unidentified neurones was reduced to < 5% of control by the AMPA receptor antagonist GYKI 52466, demonstrating that if there is a kainate receptor-mediated component of the C-fibre signal, it is very small. In addition, this small residual current was resistant to the kainate receptor desensitizing compound SYM 2081 but sensitive to the mixed AMPA/kainate receptor antagonist CNQX. Equivalent results were obtained with the current evoked by exogenous kainate application. Thus, it seems most probable that the residual current was due to incomplete blocking of AMPA receptors by the AMPA receptor antagonist. At the concentration used in this study, GYKI 52466 has been reported to block about 80–90% of the AMPA receptor-mediated current. It also inhibits 20–30% of the kainate receptor-mediated current (Paternain et al. 1995). However, it is improbable that detection of kainate receptors was precluded by GYKI 52466, as SYM 2081 also had no effects on currents evoked by exogenous kainate application in the absence of GYKI 52466.
It can be concluded that kainate receptors make no major contribution to the C-fibre-evoked EPSC of lamina I neurones at postnatal day 21–26. Similarly, no significant contribution of kainate receptors to A
- or C-fibre-evoked EPSCs was found in lamina II of the adult mouse (Youn & Randi
, 2004). This is consistent with the result that expression of kainate receptor subunits in spinal dorsal horn strongly decreases during development. At postnatal day 22, only moderate expression of a single subunit (KA2) is found, and it is restricted to lamina II (Stegenga & Kalb, 2001). In adult dorsal horn, expression of kainate receptors is very low compared to the expression of AMPA receptors (Tölle et al. 1993).
Conclusions
The present results provide further evidence for the specific role of lamina I projection neurones in the processing of nociceptive stimuli. Their prominent action potential-independent excitatory input probably has an impact on membrane excitability and discharge probability. The balance between excitation and inhibition is critical for activity-dependent plasticity and the predominance of the former may facilitate induction of synaptic LTP in projection neurones.
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Footnotes |
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References |
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, spino-PAG neurones; 
, unidentified neurones.
