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Intracellular recording from hypothalamic neurosecretory cells in tissue culture (clone HT9-C7)
Neuroscience Letters, 4 (1977) 257--262 © Elsevier/North-Holland Scientific Publis~hem Ltd.
257
INTRACELLULAR RECORDING FROM HYPOTHALA.MIC NEURO. SECRETORY CELLS IN TIS~SUE CULTURE (CLONE HT9-C7)
B. BIOULAC*, B DUFY, F. DE VITRY, H. FLEURY, A. TIXIER-VIDAL and J.D. VINCENT Laboratoire de Neurophysiologie, Universite de Bordeaux H, et (H.F.) Laboratoire de Virology (Pr du Poaquier), Universite de Bordeaux II, 33 Bordea~x et (F. de V. et A.T.-V.) Groupe de Neuroendocrinologie Cellulaire, Laboratoire de Physiologie Cellulaire, Coll~ge de France, Paris (France)
(Received January 5th, 1977) (Accepted January 28th, 1977)
SUMMARY Electrophysiological and pharmacological studies have been performed on a clone of mouse hypothalamie neuroseeretory cells synthesizing neurophysin and vasopressin (HTg-c7). These neurons p ~ s e d low resting membrane potential {RMP) and weak membrane resistance (MR). They did not exhibit spontaneous activity. Electrical stimulation or microiontophoretic apphcation oi putative neurotransmi~ters did not induce action potentials. Nevetheless, dopamine and 7-aminobutyric acid appeared to exert a slight hyperpol~zing effect on RMP. Radioimmunoassays, carried out on the culture rnedi am after electrical stimulation, did not reveal any measurable quantities o ~vasopressin. However, an electron microscopic analysis of the cytoplasmic processes of these cells did not reveal axonal outgrowth. It can be assumed that the weak electrophysiological and pharmacological properties of these neurons have to be related to their weak morphological differentiation. Two hypotheses might account for the absence of most characteristics cf in situ magnocellular hypothalamic neurons in HTg-C7 cells, the lack of pituicytes, the target cells for the axon terminal of magnocellular neurons, and the SV40 transformation itself which may impede neuronal maturation. The complexity of the hypothalamus imposes numerous limitations on eleetrophysiological and pharmacological studies at the cellular level. The availability of a elonal neurosecretory celI line offers a unique approach to the study of electrophysiological properties and pharmacological regulations of magnoceUularneurons. * Reprint requests should be sent to Dr. B. Biouiac at: Laboratoire de Neurophysiologie, Universit~ de Bordeaux H, 146 rue L ~ Saignat, 33076 Bordeaux, France.
258 An hypothalemic continuous cell line was o~.-tamedby simian ~ 40 (SV40) ~ansformation of primary c u l t ~ s of di~ociated mouse fetal hypothalan~¢ cell [ 17,18]. One clone from this cel~ line has been previously shown to p o ~ some ultr~tructuml features, immunological p r o p e ~ and syn~he~zing cepacities of the magnocellu_ler hypotb~i~mjc neurons which secrete vasopr'~sin and neurophysin [17,19]. The present paper reports on the electrophysiological characteristics of these cells. Cell c~ltures. Cont~uous cell lines were grown according to de Vitry et al. [18] with the follo-~ing modifications of the c~Jlture medium: Ham F 10 plus 10% fetal calf serum (GIBCO)~ 1.5% of 200 mM L-glutamine, 1% HEPES 1 M with 2.5% NaPI2CO3 and gentamycin (10 ~g/ml). Electrophyaiological studies. Just before the onset of recording sessions the culture medium was removed and replaced by phosphate-buffered saline (PBS) (NaCI 136 raM, KC1 2.65 mM CaCI2 0.68 mM, MgCI2 0.49 mM, Merieux). To maintain a stable temperature (3~.8--370C) the culture dishes were placed on a heated stage mounted on an inverted microscope. The constancy of pH (7.3) was regularly checked throughout the duration of the experiments. Intracellutar measurements were performed vi~ glass microelectrodes of 10--40 M f2 resistance filled with 3 M KCI (diameter 0.2--0.4 ~m). Penetrations were carried out with the help of a micromanipulator. In some cases, to deliver direct extracellular electrical shocks, a stimulating electrode was located near the recorded neuron. Membrane impedance was measured by the voltage change on p ~ i n g current pulses of either 20 msec or 1 msec duration. The 20 msec p ~ gave the sum of the elect,rode and membra~le impedance, whereas the I msec pulses gave the electrode impedance alone. Membrane impedance was obtained by subtraction [11]. During certain intracellular recordings, putative neurotransmitter~ were extracellularly applied by mJcroiontophoresis (Figs. 1 and 2). For this purpose, multibarrelled glass microelectrode~ were used and filled with L-glutamate sodium (Glu) (200 raM, pH 4.5, Geigy); acetyicholine chloride (ACh) (1 M, pH 4.0, British Drug Houses), 7-aminobutyric acid (GABA) (1 M, pH 4.3, Calbiochem) and dopamine hydrochloride (DA) (0.8 M, pH 3.7, Sigma). Finally, in some experiments, massive electrical stimulations (5--15 Hz, 10--20 V) were realized with the help of special grids on which cells were grown. Stimulations were delivered over short time periods (5--10 min) and immediately after each stimulation sequence fractions of medium (5 ml) were collected to estimate the ADH release by radioimmunoassay (sensitivity of the method: 5 pg) [6]. Resting membrane potentials (RMP) and membrane resistance (MR) were measured in many neurons. The mean valae for RMP was 11.3 mV .+-2.8 S.D. (n -- 234) and for MR 0.72 M ~ -+ 0.67 S.D. (n = 82). Neither spontaneous nor electrically evoked action potentials were recorded from any of these cells (Fig. ~) Iontophoretic applicatiol~.s were performed upon 97 neurons. High currents (250--400 nA) of neurotransmitter were injected over prolonged
259
Figs. 1 and 2. HTg-C7 cells. Phase contrast observation (x 280) during electropbysiological recording. 1 : round cell. 2: cell with cytoplasmic processes. Note in both cases the absence of axonal differentiation; r, microelectrode for intracellular recording; m, multibarrelled micropipette for iontophoretic applications.
periods (2--8 min). In no case could an action potential be elicited. Application of Glu and ACh did iaot induce any modifications of RMP. Nevertheless, as shown in Fig. 3, injections of GABA and DA produced a weak, reproducible and durable hyperpolarization. Radioimmunoassays performed on medium fractions collected after electrical stimulations revealed that there was no significant release of vasopressin. The C7 cells have a very |ow RMP and a weak MR, they do not display spontaneous activity, they do not respond to electrical stimulation, although the iontophoretic studies reveal that their membrane is slightly sensitive to GABA and DA. Finally, they are not able to release measurable quantities of vasopre~in, upon electrical stimulation. In comparison with hypothalamic neurosecretory cells in situ which exhibit RMPs ranging from 40 to 80 mV, and specific firing patterns either spontaneously or induced by electrical stimulation or applications of putative neurotransmitters [1,9,10,15], the C7 ceils do not appear to possess the electrophysiological characteristics of magnocellular neurosecretory units. Nervous tissue cultures have been widely used to study, in a simplified situation, some morphological and physiological aspects of the central nervous system. Three methods have permitted such approaches: explant culture, dissociated nervous cells in culO.lre, and continuous cell line [3--5,12, 13]. Thus electrical recordings carried out on explant cultures as well ~ on dissociated cells indicate that many neurons in these conditions display substantial RMPs, spontar, eous and induced activity, and chemosensitivity. These findings should be considered as a reflection of the relatively high
GA~ 25o~A
DA 250 ~.~
ffl
OLU 250 n~
.4~h 250n~
T~4~
Fig. 3. Microiontophoretic applications during intracellular recording of three C7 cells. The two lower traces correspond to the same neuron. Bars indicate periods of neurotransmitter applications.
degree of morphological differentiation of these neurons. For instance, Geller [7] has demonstrated that neurons in explants of neona/al rats' tubers] hypothalamus display spontaneous firing patterns similar to those of their in situ counterparts. The only continuous nervous cell line on which extensive electrophysiological and pharmacological studies have been performed are clones of the C1300 mouse tumor. Depending on the clone, they may exhibit high ~MPs, spontaneous activity and firing elicited by electrical or chemical stimulation [2,5,8,13,15]. Occasionally, in one clone, electrotonic coupling between adjacent neurons has even been observed [8,14]. Although there is no clear-cut correlation between neurite outgrowth, induction of neuro~ransmitter enzymes and occurrence of elech'ical excitability, one can say that, in a given clone, excitability increases vrith increas-
261
ing morphological differentiation of the culture [8,12,13]. In the case of C7 cells the weak electrical properties can be correlated with the lack of axonal differentiation as revealed by the electron microscopic analysis of cytoplasmic processes [ 16]. Furthermore, the invariant RMP in response to ACh applications is in good agreement with the fact that these cells were found, by Merlie and de Vitry (1976, unpublished results), to be unable to bind labeled a-bungarotoxin (3H or ~2sI) whether the binding test was performed on intact cells or on solubilized receptors. On the other hand, the hyperpolarizing effects of GABA and DA suggest the presence of membrane receptors to these two neurotransmitters. Finally, the fact that there is no measurable-~mount of vasopressin in the medium after electrical stimulation agrees with the unsuccessful attempts, by de Vitry [20] to induce vasopressin release from 2-106 cells in 3 ml of medium by various agents (cooling, high potassium concentration, and carbachol at 10 -s M). Two hypotheses might account for the absence of most characteristics of fully differentiated magnocellular neurons in SV40 transformed neurosecretory cell lines: (1) the lack of pituicytes, the specific target cells for the axon terminals of magnocellular neurons, and (2) the SV40 transformation itself, which might have arrested the maturation of neurosecretory cells at the time of penetration of the virus [17]. ACKNOWLEDGEMENTS
We are grateful to Prof. J. MacKenzie for reading this manuscript. We express our thanks to Mr. F. Rodriquez and Mr. G. Labayle for their skillful technical assistance. This work was supported by Grants from CNRS (ERA-493 and ATP 2110). REFERENCES 1 Arnauld, E., Vincent, J.D. and Dreifuss, J.J., Firing patterns of hypothalamic supraoptic neurons during water deprivation in monkeys, Science 185 (1974) 535--537. 2 Augtmti-Tocco, G. and Sato, G.H., Establishment of functional clonal lines of neurons from motme neuroblastoma, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 311--315. 3 Crain, S.M., Microelectrode recording in brain tissue cultures. In R.F. Thompson and M.M. Patterson (Eds.), Methods in Physiological Psychology, Bioelectric Techniques; Cellular Processes and Brain Potentials, Vol. 1, Academic Press, New York, 1973, pp. 39--75. 4 Crain, S.M., Tissue culture models of developing brain functions In G. Gottlieb (Ed.), Studies on the Development of Behavior and the Nervous Sys%m, A~pect~ of Neurogenesis, Vol. 2, Academic Pres~, New York, 1974, pp. 69--114. 5 Crain, S.M., Netuophysiologie Studies in Ti~ue Culture, Raven Pres~, New York, 1976, 280 pp. 6 Czernichow, P., Merkelbach, U. and Valloton, M.C., Radioimmunoassay of (8-arginine)~vasopre~in,Acta endocr. (Kbh.), 80 (1975) 444--452. 7 Geller,H.M., Phasic discharge of neurons in long-~rrn culture8 of funeral hypotha|amus, Brain Res., 93 (1975) 511--515.
262
8 Oilier, E.L., Breakfieid, X.O., Christian, CJN., Neslem, E ~ - and Nelson, P.G., Expression of neuronal cbersct~ristlcs in culture: some pros and cons of primary cultures and continuons cell lines, In M. 8antini (Ed.), l ' ~ e s in Neurobioloty, Golgi Centennial Bymposium, Raven Press, New York, 1975, pp. 603-623. 9 Kandel, E.R., Electrical properties of hypothalamie meuxoezldo~im~ cells, J. ge~ Physiol., 47 (1964) 691-719. 10 Koizumi, K. and YamMhita, I-L, Studies of autidromiceg¥ identified ~ r y celb of the h ~ u s by intraceUular and extracellular recordings, J. P h i a l . i,Ixmd.), 221 (1972) 683--705. 11 Mentor Operating Menual, Intracellular Probe System (Model N~50). Mentor Corporation, Minneapolis, 1969, Revised 1973, 4--5. 12 Nelson, P.G., Eh~-trophy~ologiesl studi~ of normal and neopia~c cells in tissue culture. In G. Sato (Ed.) Tissue Culture of the Nervous System, Current Topics in Neurobiology, VoL 1, Plenum Press, New York, 1973, pp. 135-159. 13 Nelson, P.G., Nerve and muscle ceils in culture, Physiol. Rev., 55 (1975) 1--61. 14 Nelson, P., Christian, C. and Nirenberg, M., Synapse formation between elonal neurob ~ X giioma cells and striated muscle celk, Proc. nat. Acad. SCi. (Wash.), 73 (1976) 123--127. 15 Nt¢oll, R.A. and Barker, J.L., The pharmaeolo3y ot recurrent inhibition in the supraoptic neuroseeretogy system, Brain Rce, 35 (1971) 501--511. 16 Schubert, D., Harris, A.J., Heinemann, $., Kidokogo, Y., Patrick, J. and Steint:aeh, J.H., Differentiation and interaction of elonal cell lines of nerve and muscle. In G. 8ato (gal.), Tissue Culture of the Nervous System,Current Topics in Neurobiology, Voi. 1, Plenum Prom, New York, 1973, pp. 55--86. 17 Tixier-Vidsl, A. end de Vitry, F., Ultrutntetural and eytoehemieal features of SV40 transformed hypothalamie cell lines, Cell. Tissue Res., 171 (1976) 3 H 9 . 18 Vitry, F. de, Caroler, M., Cze~ichow, P., Bends, Ph., Cohen, Po and TixlePVidal, A., Establishment of a clone of mouse hypothalamte neurc~eeretory cells synthesizing neurophysin and vuopremin, Proe. nat. Aead. 8el. (Wsdt.), 71 (1974) 3575-3579. 19 Vttry, F. de, Caroler, M., Czemichow, P., Bends, Ph., Cohen, P. and TixkHr-Vldsl,A., Characterization of a clonal strain of mouse hypothalimle cells synthesizing neurophysin and vssopressin. Ann. N.Y. Aead. 8eL, 246 (1975) 64--79. 20 Vitry, F. ce, Attempts to induce ADH release from a clone of mouse hypothalamic neurosecv¢ ~ory cells, to be published.
257
INTRACELLULAR RECORDING FROM HYPOTHALA.MIC NEURO. SECRETORY CELLS IN TIS~SUE CULTURE (CLONE HT9-C7)
B. BIOULAC*, B DUFY, F. DE VITRY, H. FLEURY, A. TIXIER-VIDAL and J.D. VINCENT Laboratoire de Neurophysiologie, Universite de Bordeaux H, et (H.F.) Laboratoire de Virology (Pr du Poaquier), Universite de Bordeaux II, 33 Bordea~x et (F. de V. et A.T.-V.) Groupe de Neuroendocrinologie Cellulaire, Laboratoire de Physiologie Cellulaire, Coll~ge de France, Paris (France)
(Received January 5th, 1977) (Accepted January 28th, 1977)
SUMMARY Electrophysiological and pharmacological studies have been performed on a clone of mouse hypothalamie neuroseeretory cells synthesizing neurophysin and vasopressin (HTg-c7). These neurons p ~ s e d low resting membrane potential {RMP) and weak membrane resistance (MR). They did not exhibit spontaneous activity. Electrical stimulation or microiontophoretic apphcation oi putative neurotransmi~ters did not induce action potentials. Nevetheless, dopamine and 7-aminobutyric acid appeared to exert a slight hyperpol~zing effect on RMP. Radioimmunoassays, carried out on the culture rnedi am after electrical stimulation, did not reveal any measurable quantities o ~vasopressin. However, an electron microscopic analysis of the cytoplasmic processes of these cells did not reveal axonal outgrowth. It can be assumed that the weak electrophysiological and pharmacological properties of these neurons have to be related to their weak morphological differentiation. Two hypotheses might account for the absence of most characteristics cf in situ magnocellular hypothalamic neurons in HTg-C7 cells, the lack of pituicytes, the target cells for the axon terminal of magnocellular neurons, and the SV40 transformation itself which may impede neuronal maturation. The complexity of the hypothalamus imposes numerous limitations on eleetrophysiological and pharmacological studies at the cellular level. The availability of a elonal neurosecretory celI line offers a unique approach to the study of electrophysiological properties and pharmacological regulations of magnoceUularneurons. * Reprint requests should be sent to Dr. B. Biouiac at: Laboratoire de Neurophysiologie, Universit~ de Bordeaux H, 146 rue L ~ Saignat, 33076 Bordeaux, France.
258 An hypothalemic continuous cell line was o~.-tamedby simian ~ 40 (SV40) ~ansformation of primary c u l t ~ s of di~ociated mouse fetal hypothalan~¢ cell [ 17,18]. One clone from this cel~ line has been previously shown to p o ~ some ultr~tructuml features, immunological p r o p e ~ and syn~he~zing cepacities of the magnocellu_ler hypotb~i~mjc neurons which secrete vasopr'~sin and neurophysin [17,19]. The present paper reports on the electrophysiological characteristics of these cells. Cell c~ltures. Cont~uous cell lines were grown according to de Vitry et al. [18] with the follo-~ing modifications of the c~Jlture medium: Ham F 10 plus 10% fetal calf serum (GIBCO)~ 1.5% of 200 mM L-glutamine, 1% HEPES 1 M with 2.5% NaPI2CO3 and gentamycin (10 ~g/ml). Electrophyaiological studies. Just before the onset of recording sessions the culture medium was removed and replaced by phosphate-buffered saline (PBS) (NaCI 136 raM, KC1 2.65 mM CaCI2 0.68 mM, MgCI2 0.49 mM, Merieux). To maintain a stable temperature (3~.8--370C) the culture dishes were placed on a heated stage mounted on an inverted microscope. The constancy of pH (7.3) was regularly checked throughout the duration of the experiments. Intracellutar measurements were performed vi~ glass microelectrodes of 10--40 M f2 resistance filled with 3 M KCI (diameter 0.2--0.4 ~m). Penetrations were carried out with the help of a micromanipulator. In some cases, to deliver direct extracellular electrical shocks, a stimulating electrode was located near the recorded neuron. Membrane impedance was measured by the voltage change on p ~ i n g current pulses of either 20 msec or 1 msec duration. The 20 msec p ~ gave the sum of the elect,rode and membra~le impedance, whereas the I msec pulses gave the electrode impedance alone. Membrane impedance was obtained by subtraction [11]. During certain intracellular recordings, putative neurotransmitter~ were extracellularly applied by mJcroiontophoresis (Figs. 1 and 2). For this purpose, multibarrelled glass microelectrode~ were used and filled with L-glutamate sodium (Glu) (200 raM, pH 4.5, Geigy); acetyicholine chloride (ACh) (1 M, pH 4.0, British Drug Houses), 7-aminobutyric acid (GABA) (1 M, pH 4.3, Calbiochem) and dopamine hydrochloride (DA) (0.8 M, pH 3.7, Sigma). Finally, in some experiments, massive electrical stimulations (5--15 Hz, 10--20 V) were realized with the help of special grids on which cells were grown. Stimulations were delivered over short time periods (5--10 min) and immediately after each stimulation sequence fractions of medium (5 ml) were collected to estimate the ADH release by radioimmunoassay (sensitivity of the method: 5 pg) [6]. Resting membrane potentials (RMP) and membrane resistance (MR) were measured in many neurons. The mean valae for RMP was 11.3 mV .+-2.8 S.D. (n -- 234) and for MR 0.72 M ~ -+ 0.67 S.D. (n = 82). Neither spontaneous nor electrically evoked action potentials were recorded from any of these cells (Fig. ~) Iontophoretic applicatiol~.s were performed upon 97 neurons. High currents (250--400 nA) of neurotransmitter were injected over prolonged
259
Figs. 1 and 2. HTg-C7 cells. Phase contrast observation (x 280) during electropbysiological recording. 1 : round cell. 2: cell with cytoplasmic processes. Note in both cases the absence of axonal differentiation; r, microelectrode for intracellular recording; m, multibarrelled micropipette for iontophoretic applications.
periods (2--8 min). In no case could an action potential be elicited. Application of Glu and ACh did iaot induce any modifications of RMP. Nevertheless, as shown in Fig. 3, injections of GABA and DA produced a weak, reproducible and durable hyperpolarization. Radioimmunoassays performed on medium fractions collected after electrical stimulations revealed that there was no significant release of vasopressin. The C7 cells have a very |ow RMP and a weak MR, they do not display spontaneous activity, they do not respond to electrical stimulation, although the iontophoretic studies reveal that their membrane is slightly sensitive to GABA and DA. Finally, they are not able to release measurable quantities of vasopre~in, upon electrical stimulation. In comparison with hypothalamic neurosecretory cells in situ which exhibit RMPs ranging from 40 to 80 mV, and specific firing patterns either spontaneously or induced by electrical stimulation or applications of putative neurotransmitters [1,9,10,15], the C7 ceils do not appear to possess the electrophysiological characteristics of magnocellular neurosecretory units. Nervous tissue cultures have been widely used to study, in a simplified situation, some morphological and physiological aspects of the central nervous system. Three methods have permitted such approaches: explant culture, dissociated nervous cells in culO.lre, and continuous cell line [3--5,12, 13]. Thus electrical recordings carried out on explant cultures as well ~ on dissociated cells indicate that many neurons in these conditions display substantial RMPs, spontar, eous and induced activity, and chemosensitivity. These findings should be considered as a reflection of the relatively high
GA~ 25o~A
DA 250 ~.~
ffl
OLU 250 n~
.4~h 250n~
T~4~
Fig. 3. Microiontophoretic applications during intracellular recording of three C7 cells. The two lower traces correspond to the same neuron. Bars indicate periods of neurotransmitter applications.
degree of morphological differentiation of these neurons. For instance, Geller [7] has demonstrated that neurons in explants of neona/al rats' tubers] hypothalamus display spontaneous firing patterns similar to those of their in situ counterparts. The only continuous nervous cell line on which extensive electrophysiological and pharmacological studies have been performed are clones of the C1300 mouse tumor. Depending on the clone, they may exhibit high ~MPs, spontaneous activity and firing elicited by electrical or chemical stimulation [2,5,8,13,15]. Occasionally, in one clone, electrotonic coupling between adjacent neurons has even been observed [8,14]. Although there is no clear-cut correlation between neurite outgrowth, induction of neuro~ransmitter enzymes and occurrence of elech'ical excitability, one can say that, in a given clone, excitability increases vrith increas-
261
ing morphological differentiation of the culture [8,12,13]. In the case of C7 cells the weak electrical properties can be correlated with the lack of axonal differentiation as revealed by the electron microscopic analysis of cytoplasmic processes [ 16]. Furthermore, the invariant RMP in response to ACh applications is in good agreement with the fact that these cells were found, by Merlie and de Vitry (1976, unpublished results), to be unable to bind labeled a-bungarotoxin (3H or ~2sI) whether the binding test was performed on intact cells or on solubilized receptors. On the other hand, the hyperpolarizing effects of GABA and DA suggest the presence of membrane receptors to these two neurotransmitters. Finally, the fact that there is no measurable-~mount of vasopressin in the medium after electrical stimulation agrees with the unsuccessful attempts, by de Vitry [20] to induce vasopressin release from 2-106 cells in 3 ml of medium by various agents (cooling, high potassium concentration, and carbachol at 10 -s M). Two hypotheses might account for the absence of most characteristics of fully differentiated magnocellular neurons in SV40 transformed neurosecretory cell lines: (1) the lack of pituicytes, the specific target cells for the axon terminals of magnocellular neurons, and (2) the SV40 transformation itself, which might have arrested the maturation of neurosecretory cells at the time of penetration of the virus [17]. ACKNOWLEDGEMENTS
We are grateful to Prof. J. MacKenzie for reading this manuscript. We express our thanks to Mr. F. Rodriquez and Mr. G. Labayle for their skillful technical assistance. This work was supported by Grants from CNRS (ERA-493 and ATP 2110). REFERENCES 1 Arnauld, E., Vincent, J.D. and Dreifuss, J.J., Firing patterns of hypothalamic supraoptic neurons during water deprivation in monkeys, Science 185 (1974) 535--537. 2 Augtmti-Tocco, G. and Sato, G.H., Establishment of functional clonal lines of neurons from motme neuroblastoma, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 311--315. 3 Crain, S.M., Microelectrode recording in brain tissue cultures. In R.F. Thompson and M.M. Patterson (Eds.), Methods in Physiological Psychology, Bioelectric Techniques; Cellular Processes and Brain Potentials, Vol. 1, Academic Press, New York, 1973, pp. 39--75. 4 Crain, S.M., Tissue culture models of developing brain functions In G. Gottlieb (Ed.), Studies on the Development of Behavior and the Nervous Sys%m, A~pect~ of Neurogenesis, Vol. 2, Academic Pres~, New York, 1974, pp. 69--114. 5 Crain, S.M., Netuophysiologie Studies in Ti~ue Culture, Raven Pres~, New York, 1976, 280 pp. 6 Czernichow, P., Merkelbach, U. and Valloton, M.C., Radioimmunoassay of (8-arginine)~vasopre~in,Acta endocr. (Kbh.), 80 (1975) 444--452. 7 Geller,H.M., Phasic discharge of neurons in long-~rrn culture8 of funeral hypotha|amus, Brain Res., 93 (1975) 511--515.
262
8 Oilier, E.L., Breakfieid, X.O., Christian, CJN., Neslem, E ~ - and Nelson, P.G., Expression of neuronal cbersct~ristlcs in culture: some pros and cons of primary cultures and continuons cell lines, In M. 8antini (Ed.), l ' ~ e s in Neurobioloty, Golgi Centennial Bymposium, Raven Press, New York, 1975, pp. 603-623. 9 Kandel, E.R., Electrical properties of hypothalamie meuxoezldo~im~ cells, J. ge~ Physiol., 47 (1964) 691-719. 10 Koizumi, K. and YamMhita, I-L, Studies of autidromiceg¥ identified ~ r y celb of the h ~ u s by intraceUular and extracellular recordings, J. P h i a l . i,Ixmd.), 221 (1972) 683--705. 11 Mentor Operating Menual, Intracellular Probe System (Model N~50). Mentor Corporation, Minneapolis, 1969, Revised 1973, 4--5. 12 Nelson, P.G., Eh~-trophy~ologiesl studi~ of normal and neopia~c cells in tissue culture. In G. Sato (Ed.) Tissue Culture of the Nervous System, Current Topics in Neurobiology, VoL 1, Plenum Press, New York, 1973, pp. 135-159. 13 Nelson, P.G., Nerve and muscle ceils in culture, Physiol. Rev., 55 (1975) 1--61. 14 Nelson, P., Christian, C. and Nirenberg, M., Synapse formation between elonal neurob ~ X giioma cells and striated muscle celk, Proc. nat. Acad. SCi. (Wash.), 73 (1976) 123--127. 15 Nt¢oll, R.A. and Barker, J.L., The pharmaeolo3y ot recurrent inhibition in the supraoptic neuroseeretogy system, Brain Rce, 35 (1971) 501--511. 16 Schubert, D., Harris, A.J., Heinemann, $., Kidokogo, Y., Patrick, J. and Steint:aeh, J.H., Differentiation and interaction of elonal cell lines of nerve and muscle. In G. 8ato (gal.), Tissue Culture of the Nervous System,Current Topics in Neurobiology, Voi. 1, Plenum Prom, New York, 1973, pp. 55--86. 17 Tixier-Vidsl, A. end de Vitry, F., Ultrutntetural and eytoehemieal features of SV40 transformed hypothalamie cell lines, Cell. Tissue Res., 171 (1976) 3 H 9 . 18 Vitry, F. de, Caroler, M., Cze~ichow, P., Bends, Ph., Cohen, Po and TixlePVidal, A., Establishment of a clone of mouse hypothalamte neurc~eeretory cells synthesizing neurophysin and vuopremin, Proe. nat. Aead. 8el. (Wsdt.), 71 (1974) 3575-3579. 19 Vttry, F. de, Caroler, M., Czemichow, P., Bends, Ph., Cohen, P. and TixkHr-Vldsl,A., Characterization of a clonal strain of mouse hypothalimle cells synthesizing neurophysin and vssopressin. Ann. N.Y. Aead. 8eL, 246 (1975) 64--79. 20 Vitry, F. ce, Attempts to induce ADH release from a clone of mouse hypothalamic neurosecv¢ ~ory cells, to be published.