Katsuhiro Nagatomo

Singular localization of sodium channel β4 subunit in unmyelinated fibres and its role in the striatum

Voltage-gated Na(+) channel β-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. β-subunits including β4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse β4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report β4 dimer expression in the mouse brain, with high levels of β4 dimers in the striatal projection fascicles, suggesting a specific role of β4 in those fibres. Scn4b-deficient mice show a resurgent Na(+) current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of β4 in the striatum.

Auto-oxidation Products of Epigallocatechin Gallate Activate TRPA1 and TRPV1 in Sensory Neurons

The sensation of astringency is elicited by catechins and their polymers in wine and tea. It has been considered that catechins in green tea are unstable and auto-oxidized to induce more astringent taste. Here, we examined how mammalian transient receptor potential V1 (TRPV1) and TRPA1, which are nociceptive sensors, are activated by green tea catechins during the auto-oxidation process. Neither TRPV1 nor TRPA1 could be activated by any of the freshly prepared catechin. When one of the major catechin, epigallocatechin gallate (EGCG), was preincubated for 3h in Hanks balanced salt solution, it significantly activated both TRP channels expressed in HEK293 cells. Even after incubation, other catechins showed much less effects. Results suggest that only oxidative products of EGCG activate both TRPV1 and TRPA1. Dorsal root ganglion (DRG) sensory neurons were also activated by the incubated EGCG through TRPV1 and TRPA1 channels. Liquid chromatography-mass spectrometry revealed that theasinensins A and D are formed during incubation of EGCG. We found that purified theasinensin A activates both TRPV1 and TRPA1, and that it stimulates DRG neurons through TRPV1 and TRPA1 channels. Results suggested a possibility that TRPV1 and TRPA1 channels are involved in the sense of astringent taste of green tea.

Syntheses of 2-NBDG analogues for monitoring stereoselective uptake of D-glucose.

2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.

Uptake of a fluorescent L-glucose derivative 2-NBDLG into three-dimensionally accumulating insulinoma cells in a phloretin-sensitive manner.

Of two stereoisomers of glucose, only d- and not l-glucose is abundantly found in nature, being utilized as an essential fuel by most organisms. The uptake of d-glucose into mammalian cells occurs through glucose transporters such as GLUTs, and this process has been effectively monitored by a fluorescent d-glucose derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) at the single cell level. However, since fluorescence is an arbitrary measure, we have developed a fluorescent analog of l-glucose 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-l-glucose (2-NBDLG), as a negative control substrate for more accurately identifying the stereoselectivity of the uptake. Interestingly, a small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (≳10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage (≲6 DIV). The 2-NBDLG uptake was persistently observed in the presence of a GLUT inhibitor cytochalasin B. Neither d- nor l-glucose in 50 mM abolished the uptake. No significant inhibition was detected by inactivating sodium/glucose cotransporters (SGLTs) with Na+-free condition. To our surprise, the 2-NBDLG uptake was totally inhibited by phloretin, a broad spectrum inhibitor against transporters/channels including GLUTs and aquaporins. From these, a question might be raised if non-GLUT/non-SGLT pathways participate in the 2-NBDLG uptake into spheroid-forming MIN6 insulinoma. It might also be worthwhile investigating whether 2-NBDLG can be used as a functional probe for detecting cancer, since the nuclear heterogeneity is among critical features of malignancy. Electronic supplementary material The online version of this article (doi:10.1007/s13577-015-0125-3) contains supplementary material, which is available to authorized users.

Dopamine D1 Receptor Immunoreactivity on Fine Processes of GFAP-Positive Astrocytes in the Substantia Nigra Pars Reticulata of Adult Mouse

Substantia nigra pars reticulata (SNr), the major output nucleus of the basal ganglia, receives dopamine from dendrites extending from dopaminergic neurons of the adjacent nucleus pars compacta (SNc), which is known for its selective degeneration in Parkinsons disease. As a recipient for dendritically released dopamine, the dopamine D1 receptor (D1R) is a primary candidate due to its very dense immunoreactivity in the SNr. However, the precise location of D1R remains unclear at the cellular level in the SNr except for that reported on axons/axon terminals of presumably striatal GABAergic neurons. To address this, we used D1R promotor-controlled, mVenus-expressing transgenic mice. When cells were acutely dissociated from SNr of mouse brain, prominent mVenus fluorescence was detected in fine processes of glia-like cells, but no such fluorescence was detected from neurons in the same preparation, except for the synaptic bouton-like structure on the neurons. Double immunolabeling of SNr cells dissociated from adult wild-type mice brain further revealed marked D1R immunoreactivity in the processes of glial fibrillary acidic protein (GFAP)-positive astrocytes. Such D1R imunoreactivity was significantly stronger in the SNr astrocytes than that in those of the visual cortex in the same preparation. Interestingly, GFAP-positive astrocytes dissociated from the striatum demonstrated D1R immunoreactivity, either remarkable or minimal, similarly to that shown in neurons in this nucleus. In contrast, in the SNr and visual cortex, only weak D1R immunoreactivity was detected in the neurons tested. These results suggest that the SNr astrocyte may be a candidate recipient for dendritically released dopamine. Further study is required to fully elucidate the physiological roles of divergent dopamine receptor immunoreactivity profiles in GFAP-positive astrocytes.

A quantification of stereoselective and non-stereoselective uptake of D-glucose into glial cells

P4-b01 Postnatal changes of Cl− homeostasis in respiration-related rhythmic activity in mouse hypoglossal nucleus Akihito Okabe 1 , Akiko Arata 1, Chigusa Shimizu-Okabe 2, Chitoshi Takayama 3, Shiro Konishi 4, Atsuo Fukuda 5 1 Div. of Physiome, Dept. of Physiol., Hyogo Col. of Med., Nisinomiya, Japan 2 Facul. Pharmaceu. Sci. at Kagawa, Tokushima Bunri Univ 3 Dept. Mol. Anat., Sch. Med., Univ. of the Ryukyus 4 Dept. Neurophysiol., Kagawa Sch. Pharm. Sci., Tokushima Bunri Univ 5 Dept. Physiol., Hamamatsu Univ. Sch. Med Developmental changes in the role of Cl− cotransporters associated with the respiration-related rhythmic activity (RRA) in the medulla have not been well understood. The concentration of intracellular chloride ([Cl−]i) is considered to be determined by the balance of chloride co-transporters, KCC2 (K+-Cl− cotransporter-extrusion system) and NKCC1 (Na+, K+-2Cl− cotransporteraccumulation system). Here, we examined how the Cl− cotransporters contribute to the RRA during development in the hypoglossal motor nucleus where inspiratory neurons reside. We recorded the RRA extracellulary in 700m-thick-medullary slice preparation obtained from postnatal day (P) 0–7 mice. Soaking slices with 8-mM-K+-containing artificial cerebrospinal fluid (ACSF) induced the RRA. Under these conditions, application of either 100 M GABA or a KCC2 blocker (1 mM furosemide or 50 M DIOA) increased the frequency of the RRA in P0-7 slices. A NKCC1 blocker (10 M bumetanide) also caused the increase of RRA frequency in P0-7 mice. The [Cl−]i of inspiratory neurons thus appeared to be high enough to lead to depolarization in response to GABAA receptor activation. The immunoreactivity of KCC2 in the hypoglossal nucleus was higher at P0 than at P7. In contrast, there were no significant differences of the KCC2 immunoreactivity in the vagus nerve nucleus between P0 and P7. Thus, the intracellular Cl− concentration of the motor nucleus in the medulla appears to keep high levels in a week after birth. Research fund: Supported by KAKENHI (21791046).

An analysis of glucose uptake into neurons and astrocytes using 2-NBDG combined with newly synthesized fluorescent L-glucose derivatives

P4-b01 Postnatal changes of Cl− homeostasis in respiration-related rhythmic activity in mouse hypoglossal nucleus Akihito Okabe 1 , Akiko Arata 1, Chigusa Shimizu-Okabe 2, Chitoshi Takayama 3, Shiro Konishi 4, Atsuo Fukuda 5 1 Div. of Physiome, Dept. of Physiol., Hyogo Col. of Med., Nisinomiya, Japan 2 Facul. Pharmaceu. Sci. at Kagawa, Tokushima Bunri Univ 3 Dept. Mol. Anat., Sch. Med., Univ. of the Ryukyus 4 Dept. Neurophysiol., Kagawa Sch. Pharm. Sci., Tokushima Bunri Univ 5 Dept. Physiol., Hamamatsu Univ. Sch. Med Developmental changes in the role of Cl− cotransporters associated with the respiration-related rhythmic activity (RRA) in the medulla have not been well understood. The concentration of intracellular chloride ([Cl−]i) is considered to be determined by the balance of chloride co-transporters, KCC2 (K+-Cl− cotransporter-extrusion system) and NKCC1 (Na+, K+-2Cl− cotransporteraccumulation system). Here, we examined how the Cl− cotransporters contribute to the RRA during development in the hypoglossal motor nucleus where inspiratory neurons reside. We recorded the RRA extracellulary in 700m-thick-medullary slice preparation obtained from postnatal day (P) 0–7 mice. Soaking slices with 8-mM-K+-containing artificial cerebrospinal fluid (ACSF) induced the RRA. Under these conditions, application of either 100 M GABA or a KCC2 blocker (1 mM furosemide or 50 M DIOA) increased the frequency of the RRA in P0-7 slices. A NKCC1 blocker (10 M bumetanide) also caused the increase of RRA frequency in P0-7 mice. The [Cl−]i of inspiratory neurons thus appeared to be high enough to lead to depolarization in response to GABAA receptor activation. The immunoreactivity of KCC2 in the hypoglossal nucleus was higher at P0 than at P7. In contrast, there were no significant differences of the KCC2 immunoreactivity in the vagus nerve nucleus between P0 and P7. Thus, the intracellular Cl− concentration of the motor nucleus in the medulla appears to keep high levels in a week after birth. Research fund: Supported by KAKENHI (21791046).

The expression of the dopamine receptors in neurons and astrocytes of the substantia nigra pars reticulata by single-cell real time RT-PCR

P1-c14 The expression of the dopamine receptors in neurons and astrocytes of the substantia nigra pars reticulata by single-cell real time RT-PCR Katsuhiro Nagatomo 1,2 , Sechiko Suga 2,3, Koji Shibasaki 4, Nanoko Watanabe 5, Satoshi Kodama 1, Hongjie Yuan 6, Nobuya Inagaki 7, Yoshio Yamamoto 5, Makoto Tominaga 8, Katsuya Yamada 1,2 1 Dept. Physiol., Hirosaki Univ. Grad. Sch. Med., Hirosaki, Japan 2 PAR, JST, Kawaguchi, Japan 3 Hirosaki Univ. Health & Welfare, Hirosaki, Japan 4 Dept. Mol. Cellular Neurobiology, Gunma Univ. Grad. Sch. Med., Maebashi, Japan 5 Lab. Vet. Biochem. & Cell Biol., Dept. Vet. Sci., Fac. Agr., Iwate Univ, Morioka, Japan 6 Dept. Phrmacol., Emory Univ. Sch. Med., Atlanta, USA 7 Dept Diabet. & Clin. Nutr., Kyoto Univ. Grad. Sch. Med., Kyoto, Japan 8 Sect. Cell Signal., Okazaki Inst. Integrative Biosci., Okazaki, Japan