$170

32 Tissue culture

DEVELOPMENT OF GABAERGIC AND CHOLINERGIC NEURONS IN PRIMARY CULTURE

SEITARO OHKUMA*, MASAAKI HIROUCHI*, SHOICHI TOMONO* and TSUNEICHI HASHIMOTO*, Department of Pharma- cology, Kyoto Prefectural University of Mddlcine, Kamikyo-Zu, Kyoto 602, Japan

Developmental patterns of GABAergic and cholinergic neurons in the brain were studied using primary cultured cerebral cortical neurons prepared from lS-day-old mouse fetuses of STD:ddy strain

these cells possessed typical features of neuronal cells. In addition, the immunohistochemical study using antibody to gllal fibrillary acidic protein, a marker for astroglia, showed that the cent tamination of astrogllas was negligible. In the cultured cerebral cortical neurons, it was found

mental patterns observed in neuronal cells in vitro coincided with those found in cerebral cortices obtained from age-matched mice..Furthermore, immunohistochemical studies using anti-GAD antibody

bodies and neuronal processes of cultured cells and its intensity increased in parallel with the development of neuronal cells. Activities of choline acetyltransferase and acetylcholinesterase,

other band, bhe contents of taurine and its intermediates in taurine biosynthesis such as cystelne sulfinic acid and cysteic acid showed a progressive reduction in conjunction with the development of cultured neurons. In contrast, the enzyme activities of cysteine sulflnic acid decarboxylase and cysteine elevation with the development of cerebral cortical neurons in vitro. These developmental patterns in the taurlne biosynthesizlng system were essentially identical withthose found in the brain of age-matched mice culture possess a similar capacity to synthesize GABA, acetylcholine and taurine as developing neurons in vivo. The present results also suggest that primary cultured neurons may be a suitable experimental model not only for analyzing the developmental pattern of neurotransmitter systems but also for investigating the effect of various centrally acting drhgs on the development and/or maintenance of neuronal cells.

RITSUKO KATOH-SEMBA, LAURA FACCI*, STEPHEN D. SKAPER*, and SILVIO VARON*

Department of Biology, University of California, San Diego, La Jolla, CA 92093,

U.S.A.

Our previous studies have shown the ability of gangliosides to inhibit the

cAMP-associated conversion of cultured astroglial cells from a flat, epithelioid

to a stellate (star-shaped) morphology. We have now investigated the capability

of gangliosides to stimulate DNA synthesis and to increase cel~ numbers in

similar secondary cultures of newborn rat astroglial cells. In the standard

culture, GMI ganglioside at a concentration of 6 x 10-SM enhanced the

incorporation of 3H-thymidine into DNA with a peak within 24 h, and increased

the cell number to twice as many after 48 h. The ganglios[de-induced

proliferative response occurred with GDIa, GDI b and ST1b, GTI b being the most

potent at I0-5M, while aslalo GMI and sialic acid were without effect. Serum

also produced the same behaviour as GMI. O~ur present results show that the

introduction of GMI to the astroglial cell culture caused a burst of

proliferative activity, which was identical to that elicited by serum.

GLUTAMATE-SENSITIVE, NEUROBLASTOMA CELL LINES SELECTED AND ESTABLISHED IN A CHEMICALLY DEFINED MEDIUM

JUN FUKUDA, KOUICHI ITO*, MADOKA FUKUMOTO*, TOSHIHIKO AOSAKI* and KAZUKO KEINO*, Department of Physiology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo I13, Japan

The present study aimed to establish neuroblastoma cell lines which were rich in glutamate- receptor molecules in plasma membranes. Twenty species of cell l ines (11 were original neuroblastoma cell lines, 6 were modified neuroblastoma cell lines developed in our laboratory, 3 were non-neuroblastoma cell l ines) were selected in a completely chemically defined medium. Glutamate sensitivity was tested on twelve species of neuroblastoma cell lines which exhibited a good capability to proliferate in the medium. An ultra-low density culture (2 t~ 20 cells/mm in plastic dishes of 35 .mm in diameter, Lux, U.S.A.) of the neuroblastoma cells was made in a chemically defined medium containing either 100 uML-glutamate, kainate, N-methyl-D-aspartic acid (NMDA) or quiscalate, respectively, and, the survival fraction and neurite growth were examined under the phase contrast microscope. These ce|l lines were classif ied into several groups as follows: one species of the cell lines (NIB-SF) prol i ferated in the presence of the glutamate analogues. NB41A3 was unaffected by the glutamate analogues. Neurite growth from IMR32 was suppressed. Survival fractions of 4 species of neuroblastoma (N2A-SF, Neuro 2A, NI15-SF and S20Y- SF) were reduced by the glutamate analogues and were thus considered to have glutamate receptors in the plasma membranes; specificity to the glutamate analogues was varied among species of the neuroblastoma cell lines. Two new species of neuroblastoma cell l ines were developed in our laboratory; one of which was resistant to glutamate(1 mM) and the other was resistant to Kainate (! mM). The glutamate-resistant cell line (N2A-SFGR) exhibited no reduction of proliferation in the presence of any one of the glutamate analogues in the culture medium. The kainate resistant cell line (N2A-$FKR) was, by contrast, sensitive to NMDA (reduction in survival fraction) but not to the other glutamate analogues. These observations suggest that the sites responsible to She kainate sensitivity differ from those to the NMDA sensitivity on the glutamate receptors of the neuroblastoma cells. In conclusion, I) several species of cell l ines which were speci f ical ly sensi . . . . . . . . . . . . . . . . . ci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cell lines. 2) N2A-SFKR and Neuro 2A were the most sensitive to NMDA, 3) NIIS-SF was the most sensitive to kainate and 4) S20Y-SF was the most sensitive to quiscalate, respectively.

VOLTAGE-GATED AND SYNAPTIC IONIC CURRENTS IN CULTURED PURKINJE CELLS

TOMOO HIRANO e, YOSHIHIRO KUBO', MICHAEL M. WU'0 and HARUNORI OHMORI Dept. of Neurobiol.,

Brain Hongo 7-3-i, Bunkyo-ku, Tokyo I~3, Japan

Ionic currents of rat PurklnJe cells were studied in dissociated cell culture. PurkinJe

cells were identified with a monoelonal antibody Leu-4, which in the cerebellum is known to

bind PurkinJe cells specifically. LarEe, round cells with a diameter of about 25 ~m were

always stained, therefore such neurone~ were identified as PurkinJe cells. The PurkinJe

cell soma was whole cell clamped and ionic currents were analyzed. With intracellular K

solution, early and late inward currents and outward currents were recorded. Outward

currents disappeared when intracellular Cs was used. Early inward currents disappeered

when extracellular Na was replaced with tetramethylammonium or when 5xlO -5 g/ml

tetrodotoxin was added. Late inward currents disappeared when ex~racellular Ca was replaced

with ME or when 2 mM Cd was added. Thus, early Na and late Ca inward currents and g

outward currents were demonstrated in PurMinJe cells. We also recorded spontaneous

excitatory synaptie currents in PurkinJe cells co-cultured with granule cells. The

synaptlc currents reversed direction at a membrane potential of 2.$ mV, similar to currents

induced by L-glutamate. Therefore the action of L-glutamate as a granule cell to PurkinJe

cell neurotransmltter is further suggested by this experiment.

ELECTROPHYSIOLOGIC SCREENING OF MONOCLONAL ANTIBODIES TO PC-12 CELL SURFACE

ANTIGENS

KAZUO KOBAYASHI*,YOSHIKO OHGUCHI* and YOICHIRO KURODA, Department of Neurochemistry,

Tokyo Metropolitan Institute for Neurosciences, Fuchu-shi, Tokyo 183, Japan

A library of monoclonal antibodies to cell surface antigens of rat pheochromo-

cytoma (PC-12) cells was examined for their abilities to change synaptic functions

in in vitro formed synapses. Mice were immunized by intact PC-12 cells. Supernatant

from the hybrid cell culture was first screened by enzyme-linked immunosorbent

assay (ELISA) using the fixed cells. ELISA-positive bybrid cell lines were cloned

and their antibody fractions were obtained. More than 70% of the antibodies

appeared to recognize the cell surface antigens. Only two clones secreted IgG.

In the second step, the antibody library was screened to see its effect on synaptic

transmission. In vitro synapses were formed between neurons which differentiated

from rat chromaffin cells by the method of OGAWA et al. (Nature, 307, 66, 1984).

Synaptic interaction was observed by impaling a pair of cells with microelectrodes.

Intracellular stimulation of cell A elicited an excitatory postsynaptic potential

(e.p.s.p.) in cell B which was shown to be cholinergic. After checking the

stability of the e.p.s.p., monoclonal antibody solution was added to the medium

at a known concentration. Among the monoclonal antibodies tested, only one

(PCH-32-20) depressed the synaptic transmission within 30 s after addition at

5 ug/ ml.