Hiroaki Shiokawa

NR2 subunits and NMDA receptors on lamina II inhibitory and excitatory interneurons of the mouse dorsal horn

BackgroundNMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg2+ sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter.ResultsAnalysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg2+ sensitive receptors and the NR2C/D containing NMDA receptors with less Mg2+ sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations.ConclusionsStronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.

Direct excitation of deep dorsal horn neurones in the rat spinal cord by the activation of postsynaptic P2X receptors

ATP mediates somatosensory transmission in the spinal cord through the activation of P2X receptors. Nonetheless, the functional significance of postsynaptic P2X receptors in spinal deep dorsal horn neurones is still not yet well understood. Using the whole‐cell patch‐clamp technique, we investigated whether the activation of postsynaptic P2X receptors can modulate the synaptic transmission in lamina V neurones of postnatal day (P) 9–12 spinal cord slices. At a holding potential of −70 mV, ATPγS (100 μm), a nonhydrolysable ATP analogue, generated an inward current, which was resistant to tetrodotoxin (1 μm) in 61% of the lamina V neurones. The ATPγS‐induced inward current was accompanied by a significant increase in the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the majority of lamina V neurones. The ATPγS‐induced inward current was not reproduced by P2Y receptor agonists, UTP (100 μm), UDP (100 μm), and 2‐methylthio ADP (100 μm), and it was also not affected by the addition of guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) into the pipette solution, thus suggesting that ionotropic P2X receptors were activated by ATPγS instead of metabotropic P2Y receptors. On the other hand, α,β‐methylene ATP (100 μm) did not change any membrane current, but instead increased the mEPSC frequency in the majority of lamina V neurones. The ATPγS‐induced inward current was suppressed by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (10 μm), but not by trinitrophenyl‐ATP (TNP‐ATP) (1 μm). Furthermore, we found that ATPγS (100 μm) produced a clear inward current which was observed in all lamina V neurones over P16 spinal cord slices, in contrast to P9–12. These results indicate that distinct subtypes of P2X receptors were functionally expressed at the post‐ and presynaptic sites in lamina V neurones, both of which may contribute to the hyperexcitability of lamina V in a different manner. In addition, the data relating to the developmental increase in the functional P2X receptors suggest that purinergic signalling may thus be more common in somatosensory transmission with maturation.

Enhancement of GABAergic tonic currents by midazolam and noradrenaline in rat substantia gelatinosa neurons in vitro

Background:Substantia gelatinosa of the spinal dorsal horn is crucial for transmission and modification of noxious stimuli. Previous studies have demonstrated that intrathecal midazolam, a benzodiazepine agonist, enhanced perioperative analgesia. Not only synaptic but also extrasynaptic inhibitory currents contribute to modification of noxious stimuli. Thus, the effects of midazolam on extrasynaptic &ggr;-aminobutyric acid (GABA) type A receptors in substantia gelatinosa neurons and interaction with noradrenaline, a transmitter of the descending inhibitory systems, were investigated. Methods:Using whole cell patch-clamp technique in the adult rat spinal cord slices, extrasynaptic GABAergic currents were recorded in substantia gelatinosa neurons in the presence of gabazine (1 &mgr;m), which blocked synaptic GABAergic currents, and then midazolam (5 &mgr;m) and noradrenaline (20 &mgr;m) were applied. Results:Bath application of midazolam induced tonic outward currents in the presence of gabazine. Although the decay time of synaptic current was prolonged, neither frequency nor amplitude was affected by midazolam. In contrast, the application of noradrenaline markedly increased both frequency and amplitude of synaptic currents with a slight enhancement of tonic currents. Coapplication of noradrenaline and midazolam markedly increased tonic currents, and the increase was much greater than the sum of currents induced by noradrenaline and midazolam. Conclusions:Midazolam had much larger effects on extrasynaptic GABA type A receptors than the synaptic receptors, suggesting a role of the enhancement of GABAergic extrasynaptic currents in the midazolam-induced analgesia. Because noradrenaline is shown to increase extrasynaptic GABA concentration, simultaneous administration of noradrenaline and midazolam may enhance the increased GABA action by midazolam, thereby resulting in an increase in tonic extrasynaptic currents.

Direct Effect of Remifentanil and Glycine Contained in Ultiva® on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses.

Background Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord. Methods We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli. Results Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine. Conclusions We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations.

The Effect of Guidance regarding Home Exercise and ADL on Adolescent Females Suffering from Adverse Effects after HPV Vaccination in Japanese Multidisciplinary Pain Centers

Background. Two prophylactic papillomavirus (HPV) vaccines have been available for primary prevention of cervical cancer. Although serious adverse effects (AE) were rare, more than 230 women have been suffering from severe AEs such as persistent pain and headache in Japan. Our research group started to treat adolescent females suffering from the AEs. Objective. To survey the characteristics of and the effects of cognitive behavioral therapy on adolescent female suffering from the AEs in Japanese multidisciplinary pain centers. Methods. One hundred and forty-five patients suffering from the AEs were reviewed retrospectively and 105 patients of them were provided guidance on home exercise and activities of daily living based partially on a cognitive-behavioral approach. The intensity of pain was rated by the patients using a numerical rating scale (NRS). Furthermore, the Hospital Anxiety and Depression Scale (HADS) and the Pain Catastrophizing Scale (PCS) were used. Results. Eighty out of the 105 patients who received the guidance were followed up, 10 displayed a marked improvement, and 43 showed some improvement. Conclusions. Guidance on home exercise and activities of daily living based on a cognitive-behavioral approach alleviated the AEs that women suffered from after HPV vaccination in Japan.

In vivo patch clamp analysis of synaptic responses evoked in primary somatosensory cortex in inflamed rats

Previous studies on frequency modulation (FM) reported that the primary auditory cortex (A1) neurons in anesthetized cats transiently responded to instantaneous FM-frequency (Fi) only at the boundary of cell’s tonal frequency-receptive-field (FRF), and preferred fast FM-speeds. In the present study on awake cats, we found a group of A1 neurons that show sustained FM-responses and prefer slow FM-speeds. These cells showed sustained responses to pure tone stimuli. When the sweep-speed of FM stimuli slowly traversed a cell’s FRF, the cell continuously responded to the FM tone until it passed FRF. The peak response was triggered by Fi corresponding to the frequency, which evoked the best response in pure tone stimuli. Cells preferring upward FM direction tended to have dominant high frequency inhibitory-FRF (defined by two tone stimuli), suggesting contribution of inhibitory-FRF to the direction selectivity. Thus, the sustained response cells may play a role in continuously encoding FM-sweep trajectories of cat’s vocalizations.

156 Modification of bursting activities in somatosensory cortex in inflamed-rats in vivo

Aim. The aim was to differentiate the effects of sucrose loading on thermal pain sensitivity in diabetic and non-diabetic rats. Materials and methods. Five months of age male diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, and their age-matched non-diabetic genetic controls Long Evans Tokusima Otsuka (LETO) rats, were used. The animals were fed with standard laboratory chow and divided into the following four groups: Group O-sucrose (n = 20) – OLETF rats were loaded for 2 months with 30% sucrose in the drinking water (sucrose period). Then the rats were put again on pure tap water (washout period) until the end of the experiment. Group O-control (n = 9) – on tap water in the course of the whole experiment. Group L-sucrose (n = 16) and group L-control (n = 9) – age-matched LETO rats had the same schedule like the groups O-sucrose and O-control, respectively. Sensitivity to noxious thermal stimuli was assessed by the latency time during tail-flick test. The beam was focused on the ventral surface, 5 cm from the tip of the tail. Results. At baseline, at the end of the sucrose period, and 2 moths later (during the washout period), respectively, the groups presented with following latencies (means ± SD): O-sucrose = 6.9 ± 1.4; 7.5 ± 0.9 and 8.4 ± 0.9 (p < 0.5 vs.baseline and vs. O-control); O-control = 7.6 ± 0.6; 7.1 ± 0.7 and 7.2 ± 1.0 (p < 0.5 vs. Lcontrol); L-sucrose = 7.6 ± 1.0; 6.2 ± 1.0 and 5.8 ± 1.4 (p < 0.5 vs. baseline); L-control = 8.4 ± 1.4; 6.0 ± 1.4 and 6.0 ± 0.8 (p < 0.5 vs. baseline). Conclusion. Sucrose loading decreases pain sensitivity, measured with tail-flick test in diabetic, but not in non-diabetic rats.