Real-time fluorescence microscopic imaging of nitric oxide production in rat brain neuronal cells

S126

~&I-EVEFI_LJORE~CEN~E MICROSCOPIC

195

IMAGING OF NKIRIC OXIDE PRODUCTION

lNRATBRAlNNEUR0NALCELI-S.

NORIO TAKATA’, TETSUYA KJMOTO’,

TAIKI TAKAHASHI’, HIROTATSU

KOJIMA’, TETSUO NAGAN@ and SUGlJRlJ

KAWATO’ ‘Dep. ofBiophysics, Graduate School of Arts and Sciences, Univ. of Toho at Komaba Megur~_lol, Tokyo I53-004I %&duate School ofPharmac&cal

Sciences, Univ. ofTokyo, Bmlk@u

Tokyo 113-0033

We achieved a real-time imaging of nitric oxide (NO) production in rat cerebellar granule neurons using a novel fluorescerd NO probe DAF-2. NO was produced by neuronal nitric oxide synthase (nNOS) 6om Garginine in some kind of ntwrons of rat central newous system. This process is regulated by a Ca2+-calmodulin-dependent signaling system. For fluorescence NO imaging we loaded DAF-2 diacetate, the membrane-permeable form, into granule neurons derived horn brains of post natal 4 days rats, and then the NO production was monitored as an increase of fluorescence ( 2 5 1Onm) using a video fluorescence microscope (ex : 470-49onm). Upon supplement of Larginine the rapid NO procluction was observed in the neurons We observed the increase of NO prcduction by both an application of Ca2’ionophore ionomycin and stimulation with L-me induced ca2’ signaling. These stimulation effects on the NO production were almost completely-suppressed

which

by LEMMA

and

trifluoperazine which were specific inhibitors of nNOS and cahnodulin, respectively. We will also report the NO production in hippocampal neumns.

196 TETSUYA

EFFECTS OF ANTISENSE OLIGODEOXYNUCLEOTIDES FOR TARGETING TACHYKININ RECEPTORS ON THE EXPRESSIONS OF C-FOS AND ZIFi268 IN THE RAT SPINAL DORSAL HORN. IKEDA I, TADASHI

NAKAMURA2,

RYUJI TERAYAMA3

‘Division of Biology, 2Department of Anesthesiology and 3Departmcnt Medical College, Kiyotake-cho, Miyazaki 889-1692, Japan,

AND TOSHIKAZU

NISHIMORI1.

of Oral and Masillofacial

Surgery,

Miyazaki

Peripheral noxious stimulation induces the espression of transcriptional regulatory protetns, c-Fos and Zifi368, tn the rat spinal dorsal horn. In the present study, for examination of the role of the three tachykinin receptors NK-1, NK-2 and NK-3 on the expression of c-Fos and Zifi268 induced by noxtous stimulation, wc used phosphorothioate anttsense These antisenses were admunstered to the rat oligodeosynucleotides for targeting the three tachykmm receptors. subarachnoid space of lumber spinal cord through catheter 3 to 7 times each 24 hour. The rats were perfused wtth 4% parafotmaldehy,de after 2 hours of subcutaneous injection of 5% formalin as pain stimulant and 50 urn sections of the lumber spinal cord were immunostained. The corrcspondmg control groups were administered sense and nonsense oligodeoxynucleottdes. Quantttative analyses rev,ealed that pretreatments with antisense oligodeoxynucleotides to NK-I and NK-2 receptors decreased c-Fos-like immunoreacti\.tty in the spinal dorsal horn, but did not change Zif/268-like immunoreactivity. On the other hand, pretreatment with antisense to NK-3 did not show any effect on expressions of both c-Fos and Zif/268-like proteins. These results indicate that NK-1 and NK-‘2 receptors but not NK-3 receptor play an important role in the expresstons of c-Fos protein following noxtous sttmulation.

197 MASARU

DIFFERENTIALCALCIUM-SIGNALING CEREBELLAR CRANULE CELLS YUKIMINE,

AKIKO TABUCHI

PATHWAYS

AND MASAAKI

TO BDNF AND C-FOS GENES IN MOUSE

TSUDA

Dept. of Biological Chemistry. Faculty of Pharmaceutical Scicnccs. Toyama Medical and Pharmaceutical University. Sugitani 2630, Toyama In cultured mouse ccrcbcllar granule cells (CGCs). stimulation of L-type volt+-depcndcnt calcium channels (L-VDCCs) or NMDA rcccptors (NMDA-R) Icads to an activation of BDNF and c-fi~., gcnc cxprcssion through Ca” influx into CC;&. Howcvcr, it is not certain whcthcr the calcium signals cvokcd via thcsc diffcrcnt calcium channels could he transfcrrcd to gcnc cxprcssion through diffcrcnt or same calcium signaling pathways. Focusing on this point, WC investigated the changes in BDNF and c-fia mRNA cxprcssions in CGCs stimulated by NMDA or mcmhranc depolarization using Northern blot hybridization. When the non-dcpolarizcd CGCs wcrc stimulated with 100 uM NMDA, significant dccreasc in the C;i’t influx into CGCs was dctcctcd in the prcscncc of nicardipinc, indicating that the Ca” influx through L-VDCCs as ucll as through NMDA-R can bc induced by stimulation of NMDA-R. Thcrcforc. WC cxamincd the BDNF and c-fo.~ gcnc cxprcssion in CGCs stimulated by NMDA in the prcscncc of nicardipinc. As the results. w*c found that the c-f0.s induction was induced by both the Ca” influxes through L-VDCCs and NMDA-R hut the BDNF gcnc cxprc\sion was activated onI> by the Ca” influx through L-VDCCo, suggesting a prcscncc of diffcrcnt signaling pathways for trunfcrring the Ca” signals cvokcd via L-VDCCs and NMDA-R to gcnc cxprcssion in nucleus.