Toshiharu Yasaka

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.

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.

Prolonged effects of polyriboinosinic:polyribocytidylic acid on spontaneous running wheel activity and brain interferon-α mRNA in rats: A model for immunologically induced fatigue

Following 2 weeks acclimation to the running wheel in the home cages, an i.p. injection of a synthetic double-stranded RNA, polyriboinosinic:polyribocytidylic acid (poly I:C, 3 mg/kg), was performed to produce the immunologically induced fatigue in rats. The daily amounts of spontaneous running wheel activity decreased to about 40-60% of the preinjection level until day 9 with normal circadian rhythm, then gradually returned to the baseline level by day 14. Rats given a heat exposure (36 degrees C for 1 h) for the consecutive 3 days showed an increase in activity except for the first day. In the open field test, the total moving distance and the number of rearing of the poly I:C-injected rats decreased on day 1, but they were not different from the saline-injected group on day 7, suggesting that the poly I:C-induced fatigue on day 7 was not due to the peripheral problems such as muscle/joint pain, but involved the CNS. Quantitative analysis of mRNA levels using a real-time capillary reverse transcriptase-polymerase chain reaction (RT-PCR) method revealed that interferon-alpha (IFN-alpha) mRNA contents in the cortex, hippocampus, hypothalamic medial preoptic, paraventricular, and ventromedial nuclei were higher in the poly I:C group than those in the saline and heat-exposed groups on day 7, although the amount of interleukin-1 beta mRNA showed no differences. Serum adrenocorticotropic hormone and catecholamine levels were not significantly different between groups. The present results indicate that the prolonged fatigue induced by poly I:C, which is evaluated by the spontaneous running wheel activity, can be used as an animal model for the immunologically induced fatigue associated with viral infection, and suggest that brain IFN-alpha may play a role in this model.

Organization of Intralaminar and Translaminar Neuronal Connectivity in the Superficial Spinal Dorsal Horn

The spinal dorsal horn exhibits a high degree of intrinsic connectivity that is critical to its role in the processing of nociceptive information. To examine the spatial organization of this intrinsic connectivity, we used laser-scanning photostimulation in parasagittal and transverse slices of lumbar spinal cord to stimulate presynaptic neurons by glutamate uncaging, and mapped the location of sites that provide excitatory and inhibitory synaptic input to neurons of the superficial laminae. Excitatory interneuronal connectivity within lamina II exhibited a pronounced sagittal orientation, in keeping with the somatotopic organization present in the pattern of primary afferent projections. Excitatory inputs to all classes of lamina II neurons arose from a wider rostrocaudal area than inhibitory inputs, whereas both excitatory and inhibitory input zones were restricted mediolaterally. Lamina I–II neurons exhibited cell type-specific patterns in the laminar distribution of their excitatory inputs that were related to their dorsoventral dendritic expanse. All cell types received excitatory input predominantly from positions ventral to that of their soma, but in lamina I neurons and lamina II vertical cells this ventral displacement of the excitatory input zone was greater than in the other cell types, resulting in a more pronounced translaminar input pattern. A previously unknown excitatory input to the superficial dorsal horn from lamina III–IV was identified in a subset of the vertical cell population. These results reveal a specific three-dimensional organization in the local patterns of excitatory and inhibitory connectivity that has implications for the processing of information related to both somatotopy and sensory modality.

Electrophysiological mapping of the nociceptive inputs to the substantia gelatinosa in rat horizontal spinal cord slices.

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 Aδ‐ and C‐afferent‐mediated excitatory postsynaptic currents (EPSCs) in more than 70% of the neurones tested at the L5 level. Both Aδ‐ 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 Aδ‐afferent EPSCs (< 20%). On the other hand, the Aδ‐ 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 Aδ‐ and C‐afferent‐mediated EPSPs, some of which initiated action potentials (APs). Most of the Aδ‐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 Aδ 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.