Hidemasa Furue

Capsaicin facilitates excitatory but not inhibitory synaptic transmission in substantia gelatinosa of the rat spinal cord

Actions of capsaicin were examined on synaptic transmissions in the substantia gelatinosa (SG) of adult rat spinal cord slices using the whole-cell patch-recording technique. Bath-applied capsaicin at a concentration of 2 microM activated a slow inward current (having an amplitude of 33 pA at -70 mV), which was accompanied by an increase in the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs; by 234%); these actions were blocked by a capsaicin-receptor antagonist, capsazepine (10 microM). The capsaicin-induced increase in sEPSC frequency was resistant to tetrodotoxin (0.5-1 microM). On the other hand, capsaicin (2 microM) did not affect either glycine- or gamma-aminobutyric acid-mediated spontaneous synaptic transmission. The results indicate that capsaicin enhances excitatory but not inhibitory synaptic transmission, possibly through a direct action on primary afferent terminals in the SG. As the SG has been thought to participate in nociceptive pathway, it is suggested that such a presynaptic action of capsaicin contributes to nociceptive transmissions.

α2 adrenoceptor-mediated presynaptic inhibition of primary afferent glutamatergic transmission in rat substantia gelatinosa neurons

Background Although intrathecal administration of norepinephrine is known to produce analgesia, cellular mechanisms for this action have not yet been fully understood. Methods The actions of norepinephrine (50 &mgr;m) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat spinal cord slice with an attached dorsal root. Results Norepinephrine inhibited the amplitude of monosynaptically evoked A&dgr;-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A&dgr;-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 ± 4%, n = 20) was significantly larger than the latter one (28 ± 4%, n = 8). Both actions of norepinephrine were mimicked by an &agr;2 adrenoceptor agonist, clonidine (10 &mgr;m), and an &agr;2A agonist, oxymetazoline (10 &mgr;m), but not by an &agr;1 agonist, phenylephrine (10 &mgr;m), and a &bgr; agonist, isoproterenol (40 &mgr;m). The inhibitory actions were antagonized by an &agr;2 antagonist, yohimbine (1 &mgr;m), all of the results of which indicate an involvement of &agr;2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin. Conclusions Norepinephrine inhibits A&dgr;-fiber– and C-fiber–mediated sensory transmission to substantia gelatinosa neurons through the activation of the &agr;2 adrenoceptor (possibly &agr;2A type, based on the current, published behavioral and anatomical data) existing in primary afferent terminals; this action of norepinephrine is more effective in A&dgr;-fiber than C-fiber transmission. This could contribute to at least a part of inhibitory modulation of pain sensation in the substantia gelatinosa by intrathecally administered norepinephrine.

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording

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 (VH, −70 mV), the application of NA produced an outward current that was blocked by Cs+ and GDP‐β‐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 (EC50, 20 μm), and the reversal potential was −88 mV. The outward current was mimicked by an α2‐adrenoceptor agonist, clonidine, and suppressed by an α2‐adrenoceptor antagonist, yohimbine, but not by α1‐ and β‐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 α2‐adrenoceptors, thereby producing an antinociceptive effect.

Selective activation of primary afferent fibers evaluated by sine-wave electrical stimulation

Transcutaneous sine-wave stimuli at frequencies of 2000, 250 and 5 Hz (Neurometer) are thought to selectively activate Aβ, Aδ and C afferent fibers, respectively. However, there are few reports to test the selectivity of these stimuli at the cellular level. In the present study, we analyzed action potentials (APs) generated by sine-wave stimuli applied to the dorsal root in acutely isolated rat dorsal root ganglion (DRG) preparations using intracellular recordings. We also measured excitatory synaptic responses evoked by transcutaneous stimuli in substantia gelatinosa (SG) neurons of the spinal dorsal horn, which receive inputs predominantly from C and Aδ fibers, using in vivo patch-clamp recordings. In behavioral studies, escape or vocalization behavior of rats was observed with both 250 and 5 Hz stimuli at intensity of ~0.8 mA (T5/ T250), whereas with 2000 Hz stimulation, much higher intensity (2.14 mA, T2000) was required. In DRG neurons, APs were generated at T5/T250 by 2000 Hz stimulation in Aβ, by 250 Hz stimulation both in Aβ and Aδ, and by 5 Hz stimulation in all three classes of DRG neurons. However, the AP frequencies elicited in Aβ and Aδ by 5 Hz stimulation were much less than those reported previously in physiological condition. With in vivo experiments large amplitude of EPSCs in SG neurons were elicited by 250 and 5 Hz stimuli at T5/ T250. These results suggest that 2000 Hz stimulation excites selectively Aβ fibers and 5 Hz stimulation activates noxious transmission mediated mainly through C fibers. Although 250 Hz stimulation activates both Aδ and Aβ fibers, tactile sensation would not be perceived when painful sensation is produced at the same time. Therefore, 250 Hz was effective stimulus frequency for activation of Aδ fibers initiating noxious sensation. Thus, the transcutaneous sine-wave stimulation can be applied to evaluate functional changes of sensory transmission by comparing thresholds with the three stimulus frequencies.

Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording

1 Synaptic responses of 46 substantia gelatinosa (SG) neurones in the spinal dorsal horn to cutaneous mechanical and/or thermal stimuli were investigated in an in vivo rat preparation with whole‐cell patch‐clamp recordings. The clamped neurones were identified as being in the SG based on either their morphological features by intrasomatic injection of biocytin or the depth of the neurones from the surface of the spinal cord. 2 In all SG neurones examined where spontaneous EPSCs occurred, pinch (noxious) and air (innocuous) stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSCs (some of which initiated an action potential under current‐clamp conditions), which subsided just after cessation of the stimuli without any residual slow current (or after‐discharge). The spontaneous and evoked EPSCs were reversibly abolished by a non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist, CNQX (20 μm). 3 Noxious (≥ 45 °C) or innocuous (≤ 40 °C) thermal stimuli did not elicit any synaptic responses in all 18 SG neurones tested which were sensitive to mechanical stimuli. Noxious cold stimulation (≤ 10 °C) also failed to produce any responses (n= 6). 4 It is concluded that both noxious and innocuous mechanical information to SG neurones are transmitted primarily by activation of non‐NMDA receptors, probably without any involvement of slow synaptic transmission, and that thermal information is conveyed to areas of the dorsal horn other than SG.

Direct GABAergic and Glycinergic Inhibition of the Substantia Gelatinosa from the Rostral Ventromedial Medulla Revealed by In Vivo Patch-Clamp Analysis in Rats

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.

Cell‐type‐specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from Aδ‐ 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 Aδ‐fibres, while those of central cells were mediated by both Aδ‐ and C‐fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both Aδ‐ 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.

Actions of brain-derived neurotrophic factor on spinal nociceptive transmission during inflammation in the rat

The aim of the current study was to investigate whether, and if so how, brain‐derived neurotrophic factor (BDNF) acts to develop the spinal sensitization underlying inflammation‐induced hyperalgesia. In spinal cord slice preparations from rats with inflammation induced by complete Freunds adjuvant (CFA), BDNF, but not nerve growth factor (NGF) or neurotrophin‐3 (NT‐3), acted presynaptically to increase the frequency of excitatory miniature EPSCs in substantia gelatinosa (SG) neurones of the CFA‐treated, but not untreated rats, through activation of lidocaine (lignocaine)‐sensitive, TTX‐resistant Na+ channels. This effect was observed in the spinal cord slices of the CFA‐treated rat only 2–4 days after the CFA injection. On the other hand, the number of monosynaptic Aβ afferent inputs to the SG significantly increased 1 week after the onset of the inflammation, and this increase was significantly suppressed by treatment with anti‐BDNF antiserum administered 1 day before and just after the CFA injection. In addition, the treatment with anti‐BDNF antiserum significantly attenuated the CFA‐induced hyperalgesia and/or allodynia. These findings, taken together, suggest that BDNF, which is considered to be released from the sensitized primary afferents, increases the excitability of SG neurones through its action on the presynaptic terminals. BDNF may thereafter induce monosynaptic Aβ afferents to the SG, thereby developing hyperalgesia and/or allodynia during inflammation.

Action of capsaicin on dorsal root-evoked synaptic transmission to substantia gelatinosa neurons in adult rat spinal cord slices

An action of capsaicin was investigated on dorsal root-evoked synaptic transmission to substantia gelatinosa (SG) neurons in adult rat spinal cord slices by use of the whole-cell voltage-clamp technique. In 79% of neurons examined, superfusing capsaicin (1 microM) for 30 s depressed a C-fiber-evoked excitatory synaptic current in a manner sensitive to a capsaicin-receptor antagonist, capsazepine (10 microM). On the contrary, Adelta-fiber-evoked excitatory and inhibitory synaptic currents were unaffected by capsaicin in all of cells tested. It is concluded that capsaicin specifically acts on C-afferents, resulting in an inhibition of evoked excitatory transmission to the SG; this may contribute to, at least in part, an acute analgesic action of capsaicin.

Sensory processing and functional reorganization of sensory transmission under pathological conditions in the spinal dorsal horn

The superficial dorsal horn, particularly substantia gelatinosa (SG) in the spinal cord, receives inputs from small-diameter primary afferents that predominantly convey noxious sensation. This sensory information via the high-threshold Adelta and C afferents is modified and integrated in SG, and consequently regulates the outputs of projection neurons located in lamina I and laminae IV-V. Recent studies using slice and in vivo patch-clamp recordings indicate that the sensory inputs to SG are functionally reorganized during post-natal development. Even in the mature state, the synaptic connectivity and receptor expression in SG can be altered easily following peripheral tissue damage. In addition, the descending pain inhibitory system to SG is also modified under certain pathological conditions. Considering that the pain system is phylogenetically primitive, it is, therefore, not surprising that the system easily exhibits a plastic change in response to inflammation or nerve damage. Because such plastic changes in the neuronal circuit or receptor expression in SG are now generally accepted to be one of the explanations for the induction of pathological pain, SG is thought to be a primary therapeutic target for chronic pain. We review here recent results demonstrating plastic changes in SG under pathological conditions.