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The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from A delta- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from A delta-fibres, while those of central cells were mediated by both A delta- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both A delta- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1-20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

Effects of neuropeptide Y (NPY) on substantia gelatinosa neurons were investigated in adult rat spinal cord slices using blind whole-cell patch-clamp technique. Bath application of NPY (1 microM) induced a membrane hyperpolarization, resulting in a suppression of the dorsal root stimulation-induced action potentials in 24% of the substantia gelatinosa neurons tested. In voltage clamp mode, NPY produced an outward current dose-dependently in about one third of substantia gelatinosa neurons at the holding potential of -60 mV, which was not affected by tetrodotoxin (1 microM). The NPY-induced current was suppressed by perfusion with a Ba2+-containing external solution and a Cs2SO4 or tetraethylammonium-containing pipette solution. In addition, The NPY-induced outward currents reversed its polarity near the equilibrium potential of K+ ions (-93 mV). The response to NPY recorded with guanosine-5'-O-(2-thiodiphosphate)-beta-S (GDP-beta-S) containing pipette solution was abolished 30 min after patch formation, suggesting that the response was mediated by the G-protein-coupled receptors. Application of an NPY-Y1 selective agonist, [Leu(31), Pro(-34)]-NPY (1 microM), for 30 s also induced an outward current with a similar time course and amplitude to that induced by NPY. On the other hand, the NPY response was blocked by a simultaneous application of NPY-Y1 selective antagonist, BIBP 3226 (1 microM). No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents and dorsal root evoked excitatory postsynaptic currents by NPY. In addition, NPY did not affect both of the miniature inhibitory postsynaptic currents and evoked inhibitory postsynaptic currents, mediated by either the GABA or glycine receptor. These findings, taken together, suggest that NPY produces an outward current in substantia gelatinosa neurons through G-protein coupled, and NPY-Y1 receptor-mediated activation of K+ channels without affecting presynaptic components. The inhibition of the synaptic transmission from the primary fibers to the substantia gelatinosa neurons is considered to contribute to the antinociceptive effects of NPY.

To study the functional projection patterns of the primary afferents in the spinal cord, the postsynaptic responses of substantia gelatinosa (SG) neurones evoked by L5 dorsal root stimulation (DRS) were examined from the neurones located at L2 to S1 in horizontal slices of the adult rat spinal cord using a blind whole-cell patch-clamp technique. In the voltage-clamp mode, the L5 DRS evoked the Adelta- and C-afferent-mediated excitatory postsynaptic currents (EPSCs) in more than 70% of the neurones tested at the L5 level. Both Adelta- and C-afferent EPSCs were also recorded in more than 50% of the neurones at L4. At L3 and L6, the number of neurones receiving the C-afferent EPSCs (> 40%) was significantly greater than that of Adelta-afferent EPSCs (< 20%). On the other hand, the Adelta- and C-afferent-mediated inhibitory postsynaptic currents (IPSCs) elicited by L5 DRS were almost equally observed from L2 to S1. In the current-clamp mode, L5 DRS evoked Adelta- and C-afferent-mediated EPSPs, some of which initiated action potentials (APs). Most of the Adelta-afferent-mediated APs were limited at the L5 level, while C-afferent-mediated APs were observed at L5 and L4. As the L2 DRS-evoked APs in the L2 SG neurones were suppressed by L5 DRS, the widespread distribution of the inhibitory inputs was considered to be functional. These findings suggest that the excitatory projection of the C afferents to the SG neurones was thus spread more rostrocaudally than that of the Adelta afferents, thereby contributing to more diffuse pain transmission. In addition, the widespread distribution of the inhibitory inputs may thus play a role as a lateral inhibitory network and thereby prevent the expansion of the excitatory inputs of noxious stimuli.

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current-clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0-9.5 mV) and suppressed the action potentials. In the voltage-clamp mode (V(H), -70 mV), the application of NA produced an outward current that was blocked by Cs(+) and GDP-beta-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre- and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC(50), 20 microM), and the reversal potential was -88 mV. The outward current was mimicked by an alpha(2)-adrenoceptor agonist, clonidine, and suppressed by an alpha(2)-adrenoceptor antagonist, yohimbine, but not by alpha(1)- and beta-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K(+) channels through alpha(2)-adrenoceptors, thereby producing an antinociceptive effect.