Neuroscience Letters, 140 (1992) 75 77 ~'~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940925115.00
75
NS1, 08667
Leukemia inhibitory factor (LIE) mediated increase of choline acetyltransferase activity in mouse spinal cord neurons in culture M a k o t o M i c h i k a w a , Seiji K i k u c h i and S e u n g U. Kim Division o[" Neuroh~J4v, D~7~artmenl ~[' Medicine, Univer~'it v ~! Brili.~/I ('olumhia. I ~mcouv~'r. B. ( "l ( "~l[J(l(h[]
(Received 18 Februar5 1992:Accepted 9 March 1992) Key words:
Cholineaeetyltransferase: Cholinergic neuronal differentiation factor: l.eukemia inhibitory factor: Spinal cord" Tissue cullurc
The effects of leukemia inhibitory thctor (LIF) on choline acetyltranstkrase (CHAT)enzymeactivity m cultured mouse spinal cord neurons were examined. The administration of L1F to cultures at concentrations of 10 U/ml and higher enhanced ChAT acti'~ity approximatcl.~ 3- to 4-1kHdin cultured spinal cord neurons. Among neurotrophic factors tested, basic fibroblast growth tEctor (bFGF) and insulin-like growth factor I (l(if:-ll stimulated the development of ChAT activity but to a smaller extent than LIE, while interlcukin 3 (I L-3), interleukin 6 (1L-6)and nerve gro\~th Lictor (NGF) showed no apparent effect on ChAT development, Our results indicate that LIF. which has nol been known to have an~ trophic effect on mammalian central nervous system neurons to date, acts as a potent differentiation lector for ('hAT in cholinergicneurons of mouse spinal cord in culture.
It is known that leukemia inhibitory factor (LIF), which is identical to the cholinergic neuronal differentiation factor (CDF) [12], induces acetylcholine synthesis and the development of ChAT activity in sympathetic neurons in culture [2, 7, 9-11]. It is reasonable to hypothesize that LIF might have similar activity on central nervous system cholinergic neurons (e.g. spinal motoneuron and forebrain septal neurons) in culture. However, studies of this nature have not been done in the past. In this study, we investigated the effect of LIF on expression of choline acetyltransferase (CHAT) activity in mouse spinal cord neurons grown in culture. Cultures enriched with neurons were prepared from spinal cords of 13 14-day-old CD-1 mouse fetuses, Spinal cords were dissected, freed of meninges and dorsal root ganglia, and diced into small pieces in Dulbecco's phosphate buffered saline (PBS). Spinal cord fragments were incubated then in PBS containing 0,25% trypsin and 20 #g/ml DNAse I at 36°C for 20 min. Following addition of heat-inactivated fetal bovine serum (FBS), cells were dissociated into single cells by gentle pipetting, washed three times with Eagle's minimum essential medium (MEM), suspended in the feeding medium and plated onto polylysine-coated Aclar plastic coverslips ( 12
Correspondence." S.U. Kim, Division of Neurology, University Hospital, University of British Columbia, Vancouver, B.C. V6T 2B5. Canada. Fax: (1) (604) 822-7897.
mm diameter) in Petri dishes at a cell density of 3 x 105 cells/coverslip. The feeding medium consisted of M EM containing 10% FBS, 5 mg/ml D-glucose. 100 U/ml penicillin and 100 #g/ml streptomycin. Eighteen h after plating, cultures were ted with a serum-free chemically defined medium (DM4) containing the trophic factors to be examined [4]. The serum-fl'ee medium consisted of M EM augmented with bovine insulin (10 #g/ml, Sigma), human transferrin (10 //g/ml, Sigma), sodium selenite (3 × 10 s M, Collaborative Research), hydrocortisone (3 x 10 s M, Sigma), triiodothyronine (3 x 10 s M, Sigma) [4[. The trophic factors examined were LIE (recombinant murine, Amrad, Vict., Australia), basic fibroblast growth factor (bFGF) (recombinant human, Zymogenetics, Seattle), insuline-like growth factor 1 (1GF-I) (recolnbinant human, Calbiochem), interleukin 3 (1L-3) (synthetic human, Dr. J. Schrader, Vancouver), interleukin 6 (IL-6) (synthetic human, Dr. J. Schrader, Vancouver) and nerve growth factor (NGE) (Sigma). The trophic factors and the medium were replaced every 3 days. ChAT activity was measured by the method of Fonnum [3] with slight modifications. In brief, the cultured cells were washed in PBS three times and sonicated in 100 #1 of 50 mM potassium phosphate buffer (pH 7.4) containing 0.5% Triton X-100. Twenty microliters of cell homogenate and 20 #l of substrate solution were mixed and incubated at 36°C for 60 rain. The substrate solution consisted of 124//M (29.8 MBq/mol) [l-~aC]acetyl-coenzyme A (CoA) (Amersham. 58 Ci/mol, specific radioac-
76
6oo~ 1500 500'
400'
300"
.>_ 200.
{,
1000
o
to
to
LI F (unit/ml) Fig. 2. Dose-response relationship of ChAT activity to LIF concentration in cultured spinal cord neurons. Dissociated spinal cord neurons from fetal mouse were cultured as described in the text. After 18 h in culture, the medium was substituted by a serum-free medium containing a different amount of LIF. After 8 days in culture, ChAT activity was measured in these samples. LIF was replaced every 3 days when medium was changed. The results shown represent the mean + I S.D. n = 3 each.
-g "~
500 -
O
I
0
I
1
4
I
I
8
Culture Time i Days ] Fig. 1. Time-dependent increase of ChAT activity by LIF in cultures of spinal cord neurons. Dissociated spinal cord neurons from fetal mouse were cultured as described in the text. After 18 h in culture, the medium was substituted by a serum-free medium containing LIF (1,000 U/ml). Data are expressed as mean + 1 S.D. Control (open circle), LIF (filled circle), n=3 each.
tivity was adjusted by the addition of non-labelled acetylCoA), 300 mM NaC1, 8 mM choline chloride and 0.2 mM physostigmine in 50 mM potassium phosphate buffer (pH 7.4). The magnitude of radioactivity of formed []4C]acetylcholine, which was extracted with 1 ml toluene scintillation cocktail, was counted by liquid scintillation counter (LS9000, Beckman). The culture time dependency of LIF-mediated increase of ChAT activity in spinal cord neurons in culture is shown in Fig. 1. ChAT activity of control neurons in a serum-free medium without LIF decreased on day 2 and increased gradually to 357 pmol/h/coverslip on day 8 (open circle). ChAT activity of neurons with LIF at a final concentration of 1,000 U/ml increased markedly and reached 1384 pmol/h/coverslip on day 8 (filled circle). This stimulating effect of LIF on the development of ChAT activity was significant as compared with the control group. The dose-response relationship of LIF to spinal cord neurons is shown in Fig. 2. The dose-response curve is saturated with LIF at a concentration of 100
U/ml. At a higher concentration than 10 U/mt of L[F, there is a 3-4-fold increase in the level of ChAT activity over that found in control cultures. Among the putative trophic factors tested, ChAT activity was elevated significantly by LIF, bFGF and IGFI in spinal cord neurons cultured for 8days (Fig; 3). IL-6 at a concentration of 100 ng/ml failed to increase ChAT activity. Similarly, no stimulative effect was observed by NGF and IL-3, which were tested at a concentration of 100 ng/ml and 200 ng/ml respectively (data not shown).
5c ~
o
40
3c .> "~ u
2O
,J¢
u
1(]
vv=.,
~|1
Lrr~
•
-i
. . . . .
FBS
Fig. 3. Comparison of various trophic factors and FBS on ChAT development in cultured spinal cord neurons. Dissociated fetal mouse spinal cord neurons were grown as described in the text. Eighteen hours after plating, cultures were exposed to the following reagents for 8 days: None (CONT), 1,000 U/ml LIF, 20/.tl/ml bFGF, 100 ng/ml IGF-I, 100 ng/ml IL-6 and 10% FBS. The values shown are the means _+ 1 S.D. from two separate experiments (n=3 each). *P < 0.001 :(t-test).
77
In this study, cholinergic expression, as mediated by LIF, was studied in dissociated neuron cultures obtained from fetal mouse spinal cord. These results indicate that LIF induced an increase of ChAT activity in cultured spinal cord neurons at concentrations of higher than 10 U/ml. The optimal concentration of LIF for the stimulation of ChAT activity in this study was 10-100 U/ml and is similar to that which was effective in rat sensory neuron survival in vitro [6]. LIF/CDF is known as a molecule that acts on postmitotic rat sympathetic neurons to induce acetylcholine synthesis and cholinergic function [2. 7, 9 11]. The results of the present study show that cholinergic spinal cord neurons are able to respond to LIF as demonstrated by the increased ChAT activity. There are three possibilities to explain the neurotrophic effect of LIF: (a) LIF specifically promotes the survival in culture of cholinergic neurons, (b) LIF elevates ChAT activity in neurons which already displayed a cholinergic phenotype, and (c) LIF induces ChAT in non-cholinergic neurons by switching phenotypic expression. Our experiments do not allow favoring any one of these possibilities. Spinal cord neurons were initially cultured in the absence of LIF for 8 days and then exposed to LIF at a concentration of 1,000 U/ml for 3, 5, 15 and 60 min. When ChAT activity was measured in these cultures, there was no increase in ChAT activity (data not shown). This suggests that LIF is unlikely to regulate the ChAT activity in a short-term manner. In the present study, spinal cord neurons were cultured in a serum-free medium to exclude the possible interference by unknown substance(s) in serum which are known to increase ChAT activity [8, 12] and may induce the elevation of the ChAT m R N A [7]. In agreement with previous studies, FBS at 10% concentration in the medium showed as strong a potency as LIF in the increase of ChAT activity in cultured spinal cord neurons (Fig. 3). The present study showed bFGF and IGF-I, as described previously [1, 5], stimulated the development of ChAT activity in spinal cord neurons in culture, bFGF and IGF-I had consistent but smaller effects in developing ChAT activity than those of LIF. We thank Grace Kim for her technical assistance. This
work was supported by the Medical Research Council of Canada and Amyotrophic Lateral Sclerosis Society of Canada. 1 Arakawa, Y., Sendtner~ M. and Thoenen, H.. Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic lectors and cytokines, J. Neurosci., 10 (1990) 35(17 3515. 2 Fukuda, K., Purification and partial characterization o f a cholinergic neuronal differentiation factor, Proc. Natl. Acad. Sci. U.S,A., 82 (1985)8795 8799. 3 Fonnum, F.. A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407 409. 4 Kim, S.U.. Stern, J.. Kim, M.W. and Pleasure. D., Culture of purified rat astrocytes in serum-free medium supplemented with mitogen, Brain Res., 274 (1983) 79 86. 5 MacManaman, J., Crawfold, F., Clark, R., Richker, J, and Fuller, F., Multiple neurotrophic factors from skeletal muscle; demonstration of effects of basic fibroblast growth factor and comparisons with 22-kilodalton choline acetyltransferase development factor, J. Neurochem.,53(1989) 1763 1711. 6 Murphy, M., Reid. K.. Hilton. D.J. and Bartlette, PH., Generation of sensory neurons is stimulated by leukemia inhibitory factor, Proc. Natl. Acad. Sci. U.S.A.. 88 (1991) 3498 3501. 7 Nawa, H., Nakanishi, S. and Patterson. P.H.. Recombinant cholinergic differentiation factor (leukemia inhibitory factor) regulates s~mpathetic neuron phenotype by aherations m the size and amounts of neuropeptide mRNAs. J. Neurochem.. 56 ( 1991 ) 2147 2150. 8 Nawa, H. and Sah, D.W,, Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons, Neuron, 4 (199(1) 279 287. 9 Patterson, P,H. and Chum L.L.Y., The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons, Proc. Natl. Acad. Sci. U.S.A,,71 {1974) 3607 3610. 10 Patterson, P.H. and Chun. k.L.Y.. The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons, Dev. Biol., 56 (1977) 263 280. 11 Potter, D.D,, Landis, S.C.. Matsumoto, S.G. and |:urshpan. E.J., Synaptic functions in rat sympathetic neurons ii'J inicrocultures. 11. Adrenergic/cholinergic dual status and plasticity. ,I. Neurosci., 5 (1986) 1080- 1098. 12 Wolinsky, E.J. and Patterson, PH., Rat serum contains a dexelopmentally regulated cholinergic inducing activity. J. Neurosci., 5 (1985) 1509 1512. 13 Yamamori, T., Fukada, K., Aebersold, R.. Korsching, S., Fann, M.J. and Patterson. P.H., The cholinergic neuronal differentiation ft~ctor from heart cells is identical to leukemia inhibitory factor, Science, 246(1989) 1412 1416.
75
NS1, 08667
Leukemia inhibitory factor (LIE) mediated increase of choline acetyltransferase activity in mouse spinal cord neurons in culture M a k o t o M i c h i k a w a , Seiji K i k u c h i and S e u n g U. Kim Division o[" Neuroh~J4v, D~7~artmenl ~[' Medicine, Univer~'it v ~! Brili.~/I ('olumhia. I ~mcouv~'r. B. ( "l ( "~l[J(l(h[]
(Received 18 Februar5 1992:Accepted 9 March 1992) Key words:
Cholineaeetyltransferase: Cholinergic neuronal differentiation factor: l.eukemia inhibitory factor: Spinal cord" Tissue cullurc
The effects of leukemia inhibitory thctor (LIF) on choline acetyltranstkrase (CHAT)enzymeactivity m cultured mouse spinal cord neurons were examined. The administration of L1F to cultures at concentrations of 10 U/ml and higher enhanced ChAT acti'~ity approximatcl.~ 3- to 4-1kHdin cultured spinal cord neurons. Among neurotrophic factors tested, basic fibroblast growth tEctor (bFGF) and insulin-like growth factor I (l(if:-ll stimulated the development of ChAT activity but to a smaller extent than LIE, while interlcukin 3 (I L-3), interleukin 6 (1L-6)and nerve gro\~th Lictor (NGF) showed no apparent effect on ChAT development, Our results indicate that LIF. which has nol been known to have an~ trophic effect on mammalian central nervous system neurons to date, acts as a potent differentiation lector for ('hAT in cholinergicneurons of mouse spinal cord in culture.
It is known that leukemia inhibitory factor (LIF), which is identical to the cholinergic neuronal differentiation factor (CDF) [12], induces acetylcholine synthesis and the development of ChAT activity in sympathetic neurons in culture [2, 7, 9-11]. It is reasonable to hypothesize that LIF might have similar activity on central nervous system cholinergic neurons (e.g. spinal motoneuron and forebrain septal neurons) in culture. However, studies of this nature have not been done in the past. In this study, we investigated the effect of LIF on expression of choline acetyltransferase (CHAT) activity in mouse spinal cord neurons grown in culture. Cultures enriched with neurons were prepared from spinal cords of 13 14-day-old CD-1 mouse fetuses, Spinal cords were dissected, freed of meninges and dorsal root ganglia, and diced into small pieces in Dulbecco's phosphate buffered saline (PBS). Spinal cord fragments were incubated then in PBS containing 0,25% trypsin and 20 #g/ml DNAse I at 36°C for 20 min. Following addition of heat-inactivated fetal bovine serum (FBS), cells were dissociated into single cells by gentle pipetting, washed three times with Eagle's minimum essential medium (MEM), suspended in the feeding medium and plated onto polylysine-coated Aclar plastic coverslips ( 12
Correspondence." S.U. Kim, Division of Neurology, University Hospital, University of British Columbia, Vancouver, B.C. V6T 2B5. Canada. Fax: (1) (604) 822-7897.
mm diameter) in Petri dishes at a cell density of 3 x 105 cells/coverslip. The feeding medium consisted of M EM containing 10% FBS, 5 mg/ml D-glucose. 100 U/ml penicillin and 100 #g/ml streptomycin. Eighteen h after plating, cultures were ted with a serum-free chemically defined medium (DM4) containing the trophic factors to be examined [4]. The serum-fl'ee medium consisted of M EM augmented with bovine insulin (10 #g/ml, Sigma), human transferrin (10 //g/ml, Sigma), sodium selenite (3 × 10 s M, Collaborative Research), hydrocortisone (3 x 10 s M, Sigma), triiodothyronine (3 x 10 s M, Sigma) [4[. The trophic factors examined were LIE (recombinant murine, Amrad, Vict., Australia), basic fibroblast growth factor (bFGF) (recombinant human, Zymogenetics, Seattle), insuline-like growth factor 1 (1GF-I) (recolnbinant human, Calbiochem), interleukin 3 (1L-3) (synthetic human, Dr. J. Schrader, Vancouver), interleukin 6 (IL-6) (synthetic human, Dr. J. Schrader, Vancouver) and nerve growth factor (NGE) (Sigma). The trophic factors and the medium were replaced every 3 days. ChAT activity was measured by the method of Fonnum [3] with slight modifications. In brief, the cultured cells were washed in PBS three times and sonicated in 100 #1 of 50 mM potassium phosphate buffer (pH 7.4) containing 0.5% Triton X-100. Twenty microliters of cell homogenate and 20 #l of substrate solution were mixed and incubated at 36°C for 60 rain. The substrate solution consisted of 124//M (29.8 MBq/mol) [l-~aC]acetyl-coenzyme A (CoA) (Amersham. 58 Ci/mol, specific radioac-
76
6oo~ 1500 500'
400'
300"
.>_ 200.
{,
1000
o
to
to
LI F (unit/ml) Fig. 2. Dose-response relationship of ChAT activity to LIF concentration in cultured spinal cord neurons. Dissociated spinal cord neurons from fetal mouse were cultured as described in the text. After 18 h in culture, the medium was substituted by a serum-free medium containing a different amount of LIF. After 8 days in culture, ChAT activity was measured in these samples. LIF was replaced every 3 days when medium was changed. The results shown represent the mean + I S.D. n = 3 each.
-g "~
500 -
O
I
0
I
1
4
I
I
8
Culture Time i Days ] Fig. 1. Time-dependent increase of ChAT activity by LIF in cultures of spinal cord neurons. Dissociated spinal cord neurons from fetal mouse were cultured as described in the text. After 18 h in culture, the medium was substituted by a serum-free medium containing LIF (1,000 U/ml). Data are expressed as mean + 1 S.D. Control (open circle), LIF (filled circle), n=3 each.
tivity was adjusted by the addition of non-labelled acetylCoA), 300 mM NaC1, 8 mM choline chloride and 0.2 mM physostigmine in 50 mM potassium phosphate buffer (pH 7.4). The magnitude of radioactivity of formed []4C]acetylcholine, which was extracted with 1 ml toluene scintillation cocktail, was counted by liquid scintillation counter (LS9000, Beckman). The culture time dependency of LIF-mediated increase of ChAT activity in spinal cord neurons in culture is shown in Fig. 1. ChAT activity of control neurons in a serum-free medium without LIF decreased on day 2 and increased gradually to 357 pmol/h/coverslip on day 8 (open circle). ChAT activity of neurons with LIF at a final concentration of 1,000 U/ml increased markedly and reached 1384 pmol/h/coverslip on day 8 (filled circle). This stimulating effect of LIF on the development of ChAT activity was significant as compared with the control group. The dose-response relationship of LIF to spinal cord neurons is shown in Fig. 2. The dose-response curve is saturated with LIF at a concentration of 100
U/ml. At a higher concentration than 10 U/mt of L[F, there is a 3-4-fold increase in the level of ChAT activity over that found in control cultures. Among the putative trophic factors tested, ChAT activity was elevated significantly by LIF, bFGF and IGFI in spinal cord neurons cultured for 8days (Fig; 3). IL-6 at a concentration of 100 ng/ml failed to increase ChAT activity. Similarly, no stimulative effect was observed by NGF and IL-3, which were tested at a concentration of 100 ng/ml and 200 ng/ml respectively (data not shown).
5c ~
o
40
3c .> "~ u
2O
,J¢
u
1(]
vv=.,
~|1
Lrr~
•
-i
. . . . .
FBS
Fig. 3. Comparison of various trophic factors and FBS on ChAT development in cultured spinal cord neurons. Dissociated fetal mouse spinal cord neurons were grown as described in the text. Eighteen hours after plating, cultures were exposed to the following reagents for 8 days: None (CONT), 1,000 U/ml LIF, 20/.tl/ml bFGF, 100 ng/ml IGF-I, 100 ng/ml IL-6 and 10% FBS. The values shown are the means _+ 1 S.D. from two separate experiments (n=3 each). *P < 0.001 :(t-test).
77
In this study, cholinergic expression, as mediated by LIF, was studied in dissociated neuron cultures obtained from fetal mouse spinal cord. These results indicate that LIF induced an increase of ChAT activity in cultured spinal cord neurons at concentrations of higher than 10 U/ml. The optimal concentration of LIF for the stimulation of ChAT activity in this study was 10-100 U/ml and is similar to that which was effective in rat sensory neuron survival in vitro [6]. LIF/CDF is known as a molecule that acts on postmitotic rat sympathetic neurons to induce acetylcholine synthesis and cholinergic function [2. 7, 9 11]. The results of the present study show that cholinergic spinal cord neurons are able to respond to LIF as demonstrated by the increased ChAT activity. There are three possibilities to explain the neurotrophic effect of LIF: (a) LIF specifically promotes the survival in culture of cholinergic neurons, (b) LIF elevates ChAT activity in neurons which already displayed a cholinergic phenotype, and (c) LIF induces ChAT in non-cholinergic neurons by switching phenotypic expression. Our experiments do not allow favoring any one of these possibilities. Spinal cord neurons were initially cultured in the absence of LIF for 8 days and then exposed to LIF at a concentration of 1,000 U/ml for 3, 5, 15 and 60 min. When ChAT activity was measured in these cultures, there was no increase in ChAT activity (data not shown). This suggests that LIF is unlikely to regulate the ChAT activity in a short-term manner. In the present study, spinal cord neurons were cultured in a serum-free medium to exclude the possible interference by unknown substance(s) in serum which are known to increase ChAT activity [8, 12] and may induce the elevation of the ChAT m R N A [7]. In agreement with previous studies, FBS at 10% concentration in the medium showed as strong a potency as LIF in the increase of ChAT activity in cultured spinal cord neurons (Fig. 3). The present study showed bFGF and IGF-I, as described previously [1, 5], stimulated the development of ChAT activity in spinal cord neurons in culture, bFGF and IGF-I had consistent but smaller effects in developing ChAT activity than those of LIF. We thank Grace Kim for her technical assistance. This
work was supported by the Medical Research Council of Canada and Amyotrophic Lateral Sclerosis Society of Canada. 1 Arakawa, Y., Sendtner~ M. and Thoenen, H.. Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic lectors and cytokines, J. Neurosci., 10 (1990) 35(17 3515. 2 Fukuda, K., Purification and partial characterization o f a cholinergic neuronal differentiation factor, Proc. Natl. Acad. Sci. U.S,A., 82 (1985)8795 8799. 3 Fonnum, F.. A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407 409. 4 Kim, S.U.. Stern, J.. Kim, M.W. and Pleasure. D., Culture of purified rat astrocytes in serum-free medium supplemented with mitogen, Brain Res., 274 (1983) 79 86. 5 MacManaman, J., Crawfold, F., Clark, R., Richker, J, and Fuller, F., Multiple neurotrophic factors from skeletal muscle; demonstration of effects of basic fibroblast growth factor and comparisons with 22-kilodalton choline acetyltransferase development factor, J. Neurochem.,53(1989) 1763 1711. 6 Murphy, M., Reid. K.. Hilton. D.J. and Bartlette, PH., Generation of sensory neurons is stimulated by leukemia inhibitory factor, Proc. Natl. Acad. Sci. U.S.A.. 88 (1991) 3498 3501. 7 Nawa, H., Nakanishi, S. and Patterson. P.H.. Recombinant cholinergic differentiation factor (leukemia inhibitory factor) regulates s~mpathetic neuron phenotype by aherations m the size and amounts of neuropeptide mRNAs. J. Neurochem.. 56 ( 1991 ) 2147 2150. 8 Nawa, H. and Sah, D.W,, Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons, Neuron, 4 (199(1) 279 287. 9 Patterson, P,H. and Chum L.L.Y., The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons, Proc. Natl. Acad. Sci. U.S.A,,71 {1974) 3607 3610. 10 Patterson, P.H. and Chun. k.L.Y.. The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons, Dev. Biol., 56 (1977) 263 280. 11 Potter, D.D,, Landis, S.C.. Matsumoto, S.G. and |:urshpan. E.J., Synaptic functions in rat sympathetic neurons ii'J inicrocultures. 11. Adrenergic/cholinergic dual status and plasticity. ,I. Neurosci., 5 (1986) 1080- 1098. 12 Wolinsky, E.J. and Patterson, PH., Rat serum contains a dexelopmentally regulated cholinergic inducing activity. J. Neurosci., 5 (1985) 1509 1512. 13 Yamamori, T., Fukada, K., Aebersold, R.. Korsching, S., Fann, M.J. and Patterson. P.H., The cholinergic neuronal differentiation ft~ctor from heart cells is identical to leukemia inhibitory factor, Science, 246(1989) 1412 1416.
