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344 Characterization of nmda-induced changes in intracellular pH of rat hippocampal slices
S62 342
IMMUNOHISTOCHEMICAL LOCALIZATION OF INSULIN-LIKE GROWTH FACTOR II RECEPTORS IN MOUSE BRAIN-IN RELATIONSHIP TO CHOLINE ACETYLTRANSFERASE IMMUNOREACTMTY. YOSHIHIRO KONISHI. SHIGEKO FUSHIMI, DE-HUA CHUI ’ , KEIKICHI TAKAHASHI ’ , TAKESHI TABIRA * 1 TERUO SHIRABE. Dent. of Neuronathol., Kawasaki Med. Sch., Kurashiki. 701-01, Janan. Dent. of Demvelinat. Dis. and -Nat?. Inst. of Nenrosci., NCNP, Kodaira, 187, Janan We previously reported that insulin-like growth factor IRIGFII) is one of the neurotrophic factors for murine central cholinergic neurons in vitro (Brain Res. 64953, 1994). Although at that report we showed IGFII immunoreactivity in primary neurons including cholinorgic ones cultured from the septal regions of embryonic mouse brain, herein we demonstrate localization of IGFII immnuoreactivity in the coronal sections of adult mouse brain to clarify the target cells of IGFII. The immtmohistochemical study was performed with indirect immtmoperoxidase technique and a rabbit IGFII/mamrose-6-phosphate receptor antibody was provided by Dr. M. Himono (Kyushu Univ., Fukuoka, Japan). This antibody labeled the cell bodies in many neurons throughout the btain, but did not those in astmcytes. The result is consistent with the autoradiographic localization reported by Lesniak et al. (Endocrinol. 123:2089, 1988). Furthermore, the localization of IGFII immtmoreactivity in neurons was confirmed with electron immtmohistochemistry. To demonstrate that choline&z neurons are also involved in the target cells of IGFII, we performed double immtmofluorescence staining with IGFII receptor antibody and choline acetybtanferase (ChAT) antibody (provided by Dr. B. Wainer, Emory Univ., Atlanta, GA, USA). The staining was also imaged by laser scanning confocal microscopy. The immnnoreactive IGFII receptors were also localized in cholinergic neurons in the medial septum and nucleus diagonal band of Broca. IGFII is thought to directly act on cholinergic neurons via its receptors to elevate ChAT activity.
343
MAPPING THE GLUTAMATE RECEPTOR DISTRIBUTION BY LASER PHOTOLYSIS OF CAGED L-GLUTAMATE IN CULTURED EMBRYONIC DROSOPHILA MYOTUBES. HIROYUKI KOSHIMOTO’. MINORU SAITOE2. MASAHIKO HIRANO’. AKIHIKO WATANABE’ AND YOSHIAKI KIDOKOR02ti1 Laboratorv of Molecular Bioohotonics. Hiraauchi. Hamakita 434 and nlnstitute for Behavioral Sciences. Gunma Universitv School of Medicine, Showa-machi. Maebashi 371. Japm Laser photostimulation provides a sufficiently rapid change in the concentration of Lglutamate at a restricted surface area of a cell pre-equilibrated with pharmacologically inert ‘caged’ L-glutamate. In the present study we examined the distribution of functional glutamate receptors which show Ca2+ permeability in cultured single embryonic Drosophila myotubes by laser spot (slum in diameter) photostimulation using caged L-glutamate in conjunction with measurement of [Caz+]i changes using fluo-3. [Cas+]i showed large increases when laser spots were located close to some of myotube nuclei. This indicates that functional glutamate receptors form ‘hot spots’ in the surface membrane at some of muscle nuclei. This finding is in accord with our previous finding that DGIuR-II, a Drosophila glutamate receptor subunit, immunoreactivity was associated in some of nuclei of myotubes in culture as well as in vivo.
344
CHARACTBRIZATION SLICES
OF NMDA-INDUCED
NAOSI-B FIJJIWARA.
CHANGE8
RBN-WI
IN INTRACBLLULAR
pH OF RAT HIPPOCAMPAL
ZHAN. KOKI SHIMOJI,
Dept. of Anesth., Niigata Univ. Sch. of Med., l-757 Niigata 95 1, Japan. NMDA-induced
changes in intracellular
fluorescence in 5% CO2/25 mM HC03-buffering
pH (PI+) of the CA1 cell layer of hippocampal slice were observed using BCECF solution at 37’C.
initial transient alkabne shift was followed by a long-lasting NMDA-induced
increase in intracellular
Exposure of NMDA
induced a biphasic change in pHi: an
acid shift. Ca2+-free solution with 1mM EGTA (0 Ca), eliminating
Ca2+ suppressed the intracellular
alkaline shift by 80% but partially (11%) reduced the
acid shift. Substitution of glucose with equivalent amount of pyruvate (in mM) profoundly reduced the NMDA-induced yielding augmentation of the alkaline shift. 0 Ca plus glucose-replacement
acid shift,
(with pyntvate) or addition of NaF, as an inhibitor
glycolysis, profoundly inhibited both of the alkaline and acid shifts. The results indicate that the NMDA-induced shift of pI$ depends on calcium influx andthe following
an
of
initial alkaline
acid shift is caused mainly by increase in the glycolytic acid production.
The NMDA receptor channels are known to be inactive at low pH. Thus, the increase in tbe acid production following activation of the NMDA receptors may serve an self-regulating mechanism to prevent overactivity of the NMDA receptors.
IMMUNOHISTOCHEMICAL LOCALIZATION OF INSULIN-LIKE GROWTH FACTOR II RECEPTORS IN MOUSE BRAIN-IN RELATIONSHIP TO CHOLINE ACETYLTRANSFERASE IMMUNOREACTMTY. YOSHIHIRO KONISHI. SHIGEKO FUSHIMI, DE-HUA CHUI ’ , KEIKICHI TAKAHASHI ’ , TAKESHI TABIRA * 1 TERUO SHIRABE. Dent. of Neuronathol., Kawasaki Med. Sch., Kurashiki. 701-01, Janan. Dent. of Demvelinat. Dis. and -Nat?. Inst. of Nenrosci., NCNP, Kodaira, 187, Janan We previously reported that insulin-like growth factor IRIGFII) is one of the neurotrophic factors for murine central cholinergic neurons in vitro (Brain Res. 64953, 1994). Although at that report we showed IGFII immunoreactivity in primary neurons including cholinorgic ones cultured from the septal regions of embryonic mouse brain, herein we demonstrate localization of IGFII immnuoreactivity in the coronal sections of adult mouse brain to clarify the target cells of IGFII. The immtmohistochemical study was performed with indirect immtmoperoxidase technique and a rabbit IGFII/mamrose-6-phosphate receptor antibody was provided by Dr. M. Himono (Kyushu Univ., Fukuoka, Japan). This antibody labeled the cell bodies in many neurons throughout the btain, but did not those in astmcytes. The result is consistent with the autoradiographic localization reported by Lesniak et al. (Endocrinol. 123:2089, 1988). Furthermore, the localization of IGFII immtmoreactivity in neurons was confirmed with electron immtmohistochemistry. To demonstrate that choline&z neurons are also involved in the target cells of IGFII, we performed double immtmofluorescence staining with IGFII receptor antibody and choline acetybtanferase (ChAT) antibody (provided by Dr. B. Wainer, Emory Univ., Atlanta, GA, USA). The staining was also imaged by laser scanning confocal microscopy. The immnnoreactive IGFII receptors were also localized in cholinergic neurons in the medial septum and nucleus diagonal band of Broca. IGFII is thought to directly act on cholinergic neurons via its receptors to elevate ChAT activity.
343
MAPPING THE GLUTAMATE RECEPTOR DISTRIBUTION BY LASER PHOTOLYSIS OF CAGED L-GLUTAMATE IN CULTURED EMBRYONIC DROSOPHILA MYOTUBES. HIROYUKI KOSHIMOTO’. MINORU SAITOE2. MASAHIKO HIRANO’. AKIHIKO WATANABE’ AND YOSHIAKI KIDOKOR02ti1 Laboratorv of Molecular Bioohotonics. Hiraauchi. Hamakita 434 and nlnstitute for Behavioral Sciences. Gunma Universitv School of Medicine, Showa-machi. Maebashi 371. Japm Laser photostimulation provides a sufficiently rapid change in the concentration of Lglutamate at a restricted surface area of a cell pre-equilibrated with pharmacologically inert ‘caged’ L-glutamate. In the present study we examined the distribution of functional glutamate receptors which show Ca2+ permeability in cultured single embryonic Drosophila myotubes by laser spot (slum in diameter) photostimulation using caged L-glutamate in conjunction with measurement of [Caz+]i changes using fluo-3. [Cas+]i showed large increases when laser spots were located close to some of myotube nuclei. This indicates that functional glutamate receptors form ‘hot spots’ in the surface membrane at some of muscle nuclei. This finding is in accord with our previous finding that DGIuR-II, a Drosophila glutamate receptor subunit, immunoreactivity was associated in some of nuclei of myotubes in culture as well as in vivo.
344
CHARACTBRIZATION SLICES
OF NMDA-INDUCED
NAOSI-B FIJJIWARA.
CHANGE8
RBN-WI
IN INTRACBLLULAR
pH OF RAT HIPPOCAMPAL
ZHAN. KOKI SHIMOJI,
Dept. of Anesth., Niigata Univ. Sch. of Med., l-757 Niigata 95 1, Japan. NMDA-induced
changes in intracellular
fluorescence in 5% CO2/25 mM HC03-buffering
pH (PI+) of the CA1 cell layer of hippocampal slice were observed using BCECF solution at 37’C.
initial transient alkabne shift was followed by a long-lasting NMDA-induced
increase in intracellular
Exposure of NMDA
induced a biphasic change in pHi: an
acid shift. Ca2+-free solution with 1mM EGTA (0 Ca), eliminating
Ca2+ suppressed the intracellular
alkaline shift by 80% but partially (11%) reduced the
acid shift. Substitution of glucose with equivalent amount of pyruvate (in mM) profoundly reduced the NMDA-induced yielding augmentation of the alkaline shift. 0 Ca plus glucose-replacement
acid shift,
(with pyntvate) or addition of NaF, as an inhibitor
glycolysis, profoundly inhibited both of the alkaline and acid shifts. The results indicate that the NMDA-induced shift of pI$ depends on calcium influx andthe following
an
of
initial alkaline
acid shift is caused mainly by increase in the glycolytic acid production.
The NMDA receptor channels are known to be inactive at low pH. Thus, the increase in tbe acid production following activation of the NMDA receptors may serve an self-regulating mechanism to prevent overactivity of the NMDA receptors.