Masafumi Kosugi

Activation of TRPA1 Channel Facilitates Excitatory Synaptic Transmission in Substantia Gelatinosa Neurons of the Adult Rat Spinal Cord

TRPA1 is expressed in primary sensory neurons and hair cells, and it is proposed to be activated by cold stimuli, mechanical stimuli, or pungent ingredients. However, its role in regulating synaptic transmission has never been documented yet. In the present study, we examined whether activation of the TRPA1 channels affects synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. A chief ingredient of mustard oil, allyl isothiocyanate (AITC), superfused for 2 min markedly increased the frequency and amplitude of spontaneous EPSCs (sEPSCs), which was accompanied by an inward current. Similar actions were produced by cinnamaldehyde and allicin. The AITC-induced increases in sEPSC frequency and amplitude were resistant to tetrodotoxin (TTX) and La3+, whereas being significantly reduced in extent in a Ca2+-free bath solution. In the presence of glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activities. The AITC-induced increases in sEPSC frequency and amplitude were reduced by ruthenium red, whereas being unaffected by capsazepine. AITC also increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents; this AITC action was abolished in the presence of TTX or glutamate receptor antagonists. These results indicate that TRPA1 appears to be localized not only at presynaptic terminals on SG neurons to enhance glutamate release, but also in terminals of primary afferents innervating onto spinal inhibitory interneurons, which make synapses with SG neurons. This central modulation of sensory signals may be associated with physiological and pathological pain sensations.

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.

Separate Inhibitory and Excitatory Components Underlying Receptive Field Organization in Superficial Medullary Dorsal Horn Neurons

Extracellular recording has shown that dorsal horn neurons can have an inhibitory surround outside their excitatory receptive field, but cannot reveal inhibitory inputs within the excitatory field, or show the underlying excitatory and inhibitory synaptic inputs that determine net output. To study the underlying components of receptive field organization, in vivo patch-clamp recording was used to compare the size and distribution of subthreshold, suprathreshold, and inhibitory fields, in neurons in the mouse superficial medullary dorsal horn that were characterized by their responses to noxious and innocuous mechanical facial stimulation. Subthreshold excitatory fields typically extended some distance beyond the borders of the suprathreshold field, and also commonly exhibited broader stimulus selectivity, in that the majority of nociceptive-specific neurons exhibited subthreshold responses to brush. Separate voltage-clamp recording of excitatory and inhibitory inputs using different holding potentials revealed that inhibition could be evoked from both within and outside the excitatory field. In nociceptive neurons, inhibition tended to be maximal at the excitatory receptive field center, and was usually greater for pinch than brush, although the selectivity for pinch versus brush was not as great as with excitatory responses. Based on current data on dorsal horn organization, we propose that the localized peak of inhibition at the excitatory field center could be mediated by local interneurons, while the more widespread surrounding inhibition may depend on supraspinal circuitry.

Subpopulation-specific patterns of intrinsic connectivity in mouse superficial dorsal horn as revealed by laser scanning photostimulation

•  Sensory neurons that detect painful and non‐painful stimulation of body tissues have axons that project to the dorsal horn of the spinal cord, where their terminations are partially segregated into superficial (I–II) and deep (III–IV) dorsal horn laminae, respectively. •  The dorsal horn contains many excitatory and inhibitory interneurons whose axons synapse on other dorsal horn neurons to enhance or suppress sensory transmission. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of synaptic connections between dorsal horn interneurons, in an in vitro‘slice’ preparation of the mouse lumbar spinal cord. •  Some neurons in superficial layers of the dorsal horn have long dendrites that extend ventrally into deeper layers of the dorsal horn, and these neurons can receive excitatory or inhibitory synaptic input from neurons in the deeper layers. •  These interlaminar connections may be involved in interactions between transmission of signals underlying painful versus non‐painful sensations.

Transoral Carotid Ultrasonography Is Useful for Detection and Follow-Up of Extracranial Internal Carotid Artery Dissecting Aneurysm

Case Report A 62-year-old man having a history of lacunar infarction in the left putamen 4 years previously was referred to a regional hospital with severe headache of sudden onset (day 0). He happened to have had brain magnetic resonance angiography 10 months before, which showed no abnormalities. He had a history of no cardiovascular diseases, hypertension, hyperlipidemia and cervical and cranial trauma. There was no family history of cardiovascular diseases. He was almost normal on general physical and neurological examinations. Although diffusion-weighted magnetic resonance imaging showed no acute ischemic lesions in the brain, magnetic resonance angiography showed the complete disappearance of the right internal carotid artery (ICA). On carotid angiography (CAG), luminal irregularity, severe stenosis and aneurysmal form were found at the C 1 vertebral level of the right ICA. He was transferred to our hospital, and conventional carotid ultrasonography was performed on day 29, which, however, did not find any difference in the diameter and the end-diastolic flow velocities between bilateral common carotid arteries (right 30.3 cm/s, left 31.7 cm/s). The right CAG performed on day 32 showed the persistence of luminal stenosis and aneurysm-like dilatation, with mild improvement of the stenosis ( fig. 1 a). On the same day, TOCU was first performed after informed consent. We used Sonosite Titan version C 2.2 (Sonosite Inc., Bothell, Wash., USA) equipped with an 8to 5-MHz convex array transducer (C8/8–5 MHz) [2] . The transducer was originally designed for prostate imaging and was equipped with a color power Doppler imaging system. The probe tip was painted with echo jelly, covered with a clean cover and softly attached to the posterolateral pharyngeal wall. The attachment of the probe tip to the pharyngeal wall was confirmed by roentgenograms ( fig. 1 b). TOCU clearly demonstrated the dilatation of the right ICA (diameters: right, 8.2 mm; left, 4.0 mm) with narrowing of the true lumen (3.3 mm; fig. 1 c). The aneurysm sac observed on CAG was shown as a protrusion toward the ventral side (the whole length, 2.5 mm; intimal tear length, 7.3 mm). In the false lumen, the isoechoic area indicating intramural hematoma was present around the aneurysm sac, of which thickness was 2.6 mm from the brim of the aneurysm sac to the arterial adventitia. A severe stenotic flow pattern was observed at the distal part of the aneurysm sac (peak systolic flow velocity: right, 232.6 cm/s; left, 120 cm/s). The peak systolic flow velocity ratio (right ICA/ left ICA) was increased to 1.98. On magnetic resonance angiography performed on the same day, the size of the ICA was larger on the right and axial source imaging of time of flight depicted the double lumen in the right ICA: one lumen was hyperintense, indicating blood flow, whereas the other was isointense, indicating intramural hematoma in the false lumen ( fig. 1 d). The false lumen protruded toward the pharyngeal wall. All these findings suggested a diagnosis of spontaneous EICAD. He was treated with long-term antiplatelet (aspirin) therapy. TOCU was performed once a month thereafter, which confirmed the gradual improvement of the narrowing of the true lumen and the decrease in size of both intramural hematoma and aneurysm sac. The peak systolic f low velocity at the distal portion of the right ICA also decreased to that of the left ICA on day 118 (right 92.6 cm/s; left 97.4 cm/s; f low velocity ratio 0.95). On day 245, the protrusion and intimal tear of the aneurysm sac were found to decrease to 1.7 and 5.8 mm, respectively ( fig.1 e). Intramural hematoma, which had been seen at the ventral side of the affected ICA on the first TOCU, was seen at its dorsal side. At the follow-up after 10 months, the patient displayed neither ischemic events nor iatrogenic adverse effects, but mild headache still remained.

Three‐dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III–IV of the spinal dorsal horn

•  Axons of sensory neurons that detect painful and non‐painful stimulation of body tissues project centrally to the dorsal horn of the spinal cord, where they are partially segregated in the superficial and deep laminae, respectively. •  Interneuronal connections between superficial and deep laminae could potentially modulate sensory transmission and contribute to alterations that occur under conditions of pain hypersensitivity. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of local interneuronal synaptic connections to laminae III–IV neurons, combined with intracellular staining for morphological analysis, in an in vitro‘slice’ preparation of the rat lumbar spinal cord. •  Synaptic input from superficial laminae (I–II) was received by laminae III–IV neurons with long dorsal dendrites, supporting the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and, more generally, that local connectivity is governed by rules that are specific to the laminar position and morphology of the postsynaptic neuron.

Four mutations of the spastin gene in Japanese families with spastic paraplegia

AbstractHereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower limbs. HSP is caused by failure of development or selective degeneration of the corticospinal tracts, which contain the longest axons in humans. The most common form of HSP is caused by mutations of the spastin gene (SPAST), located on chromosome 2p21-p22, which encodes spastin, one of the ATPases associated with diverse cellular activities (AAA). In this study, we detected four causative mutations of SPAST among 14 unrelated patients with spastic paraplegia. Two missense mutations (1447A→G, 1207C→G) and two deletion mutations (1465delT, 1475-1476delAA) were located in the AAA cassette region. Three of these four mutations were novel. Previous reports and our results suggest that the frequency of SPAST mutations is higher among Japanese patients with autosomal dominant HSP, although SPAST mutations are also observed in patients with sporadic spastic paraplegia.

Presynaptic TRPA1 activation enhances glutamate release onto substantia gelatinosa neurons of the adult rat spinal cord

In order to clarify the neuronal mechanisms of abnormal pain sensation in the face reinnervated by the regenerated inferior alveolar nerve (IAN), the nocifensive behavioral and single neuronal activity were precisely analyzed in rats with IAN transection. The escape thresholds to mechanical stimulation of the mental skin was significantly higher at 3 days and lower at 14 days after IAN transection, and returned to the preoperative level at 60 days after that. The population of WDR neurons with background activity was significantly higher in rats 14 and 60 days after IAN transection compared with that of naive. Mechanical evoked responses were significantly larger in WDR and LTM neurons 14 days after IAN transection compare with naive rats, but not in WDR neurons at 60 days after transection. The present findings suggest that the regenerated IAN may have an ability to generate abnormal firing reflecting in the abnormal neuronal discharge in Vc neurons.