Soluble striatal extracts enhance development of mesencephalic dopaminergic neurons in vitro

Brain R~,search, 399 (1986) 111-124 Elsevier

111

BRE 12217

Soluble Striatal Extracts Enhance Development of Mesencephalic Dopaminergic Neurons in Vitro YASUKO TOMOZAWA and STANLEY H. APPEL

Department of Neurology and Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) (Accepted 13 May 1986)

Key words: Trophic factor - - Dopaminergic n e u r o n - Tissue culture - - Striatal extract

Recent studies have suggested that diffusible factors released by neural target tissues enhance survival, growth, and differentiation of neurons within the central, as well as the peripheral, nervous system. In this report, we use catecholamine cytofluorescence to demonstrate that a soluble factor from the striatum produces a 4-fold increase in number of catecholamine cytofluorescent-positive dopaminergic neurons in dissociated mesencephalon cultures prepared from embryonic 14-day-old rats. The same soluble extract enhances the number of neurites per cell and the length of neurites, and also produces a greater than 3.5-fold stimulation of high affinity dopamine uptake into neurons. Such stimulation is significantly reduced following trypsin treatment. The trophic effects on dopaminergic neurons are maximal in extracts of the striatum, but are also found in extracts of the hippocampus-entorhinalcortex-amygdaloid nucleus and the cerebral cortex, although they are less in extracts of the cerebellum, negligible in the olfactory bulb, and absent in the liver. With molecular sieving chromatography, the soluble factors stimulating high affinity dopamine uptake are partially separable from the factors stimulating neuronal high affinity GABA uptake. The approximate molecular weight of the factors influencing dopaminergic neurons is 1500-2200 Da.

INTRODUCTION Trophic factors released by neural target tissues exert an important influence on n e u r o n a l growth and development 4'5"s'31'32'61'64'65. The best example of such a trophic factor and the only well-characterized one is N G F . N G F is synthesized in the innervated target tissues of sympathetic and spinal sensory neurons 21'5z and in the innervated tissues of central cholinergic n e u r o n s 27'53'66, and acts in a retrograde fashion on such n e u r o n s 45'5°'59. In i m m a t u r e animals, N G F anti-serum produces destruction of sympathetic and spinal sensory n e u r o n s 14'24'3°. A d m i n i s t r a t i o n of exogenous N G F during the period of naturally occurring cell death reduces the loss of n e u r o n s in both sympathetic and sensory ganglia 16'2°'25'39. N G F can also prevent the effects of axotomy on developing sympathetic and sensory n e u r o n s 2°. In the developing central nervous system, exogenous N G F increases choline acetyltransferase activity35, while

N G F anti-serum inhibits d e v e l o p m e n t of choline acetyltransferase activity4°. In addition, N G F can prevent the effects of axotomy on central cholinergic neurons 17. Three other such target-tissue derived neuronotrophic factors have b e e n highly purified. Ciliary n e u r o n o t r o p h i c factor (CNTF), which supports survival of embryonic chick ciliary ganglionic neurons as well as sensory and sympathetic ganglionic n e u r o n s in vitro, has been purified from chick embryonic c h o r o i d - i r i s - c i l i a r y body 1'2. The glycoprotein which controls n e u r o t r a n s m i t t e r choice in sympathetic n e u r o n s has b e e n purified from heartconditioned m e d i u m t2. The other acts predominantly on sensory n e u r o n s 3. However, their in vivo significance is less well understood. Other putative trophic factors have been partially purified from different systems and have some degree of specificity for their innervating neurons in vitro. Factors affecting process extension as well as cholinergic activity of fetal rat ventral spinal cord neu-

Correspondence: Y. Tomozawa, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

112 rons have been isolated from conditioned media as well as from skeletal muscle extracts 55'56. Hippocampal extracts have been demonstrated to enhance acetylcholine synthesis in septal explant cultures 37. Dopaminergic neurons in the substantia nigra project primarily to neuronal targets in the striatum; and the striatum, in turn, may well serve as a source of retrograde trophic factors. This dopaminergm neuronal pathway has been extensively studied with respect to its anatomical relations and development 9,13' 29,36.38,46,51,57,58 and biochemistry 28'49. Prochiantz et al. 44 have previously demonstrated that striatal membrane fractions enhance dopaminergic differentiation in rat mesencephalic neurons in vitro. In the present study, we report that soluble factors from the striatum increase the number of cytofluorescent-positive neurons and stimulate the morphological differentiation of dopaminergic neurons and high affinity dopamine uptake in explant and dissociation cultures of fetal rat mesencephalon. MATERIALS AND METHODS Precisely timed pregnant Sprague-Dawley rats were obtained from Texas Animal Specialties (Humble, Texas 77338). Dulbecco's modified Eagle, H a m ' s F12 media and sera were purchased from G I B C O Labs. Other chemicals for tissue culture and bioassays were obtained from Sigma Chemical Corp. Biogel P4 was from Bio-Rad Labs. 7S-NGF was from Collaborative Research. Neurotensin was from Peninsula Lab. Somatostatin-14 was from Sigma Chemical Corp. [3H]Dopamine (3,4-dihydroxyphenyl[7-3H(N)]ethyl amine), specific activity: 20-30 Ci/mmol) and [3H]y-aminobutyric acid (specific activity 75 Ci/ mmol) were from New England Nuclear and ICN, respectively. Anti-GFAP serum was purchased from Accurate Chemical and Scientific Co. Tissue culture Explant and dissociation cultures of the mesencephalon were obtained from 14-day-old rat embryos. Tyro sine hydroxylase-immunoreactive cells appear for the first time on day E l 2 of rat brain. At day E l 4 all tyrosine hydroxylase(TH)-positive neurons in the mantle layer of the ventral mesencephalon were not labelled by [3H]thymidine 46. The first terminal field

of TH-immunoreactive dendrites of dopaminergic neurons was detected in the rudimentary striatum at day E14.5 (ref. 57). At embryonic day 14, therefore, the dopaminergic neurons in the mesencephalon are predominantly postmitotic 46, but have not yet innervated their striatal target 57. For explant cultures, the mesencephalon was dissected, and the ventral region fragment was dissected into 0.3-0.4 mm pieces in the culture medium. Approximately 30 pieces were cultured on poly-L-lysine (Sigma, 300,000 M.W. polymerization) coated 35 mm Falcon tissue culture plates in a standard culture medium consisting of 50% high glucose DMEM-50% F12 medium supplemented with insulin (5 ~g/ml), transferrin (100 ~g/ml), progesterone (20 nM), putrescine (100 ~M), selenium (30 nM), glutamine (4 raM), gentamicin (50 gg/ml) and H E P E S (5 raM) 6 without serum and maintained at 37 °C in a 95% a i r - 5 % CO 2 humidified incubator. Dissociation cultures were prepared by triturating mesencephalic cells in the presence of DNase (1 mg/ml, Sigma) and trypsin (0.2 mg/ml, Sigma) 33. After washing twice with culture medium containing 4% heat-inactivated horse serum, the cells were plated at 250,000 cells/well on poly-L-lysine coated Falcon 24 multiwell plates or on coverslips in the same culture medium unless stated otherwise. Twenty-four hours after plating, the soluble factors were added to the medium together with cytosine arabinoside (2 BM) to kill proliferating glia. Prior to application, the soluble factors were activated for 30 min with fl-mercaptoethanol (0.5 mM) and added to cultures at concentrations described in figure legends. The final concentration of fl-mercaptoethanol in all culture media was 20 ~M. Control cultures contained the same volume of PBS or column effluent and the same final concentration of/3-mercaptoethanol. Treatment of the soluble factor with fl-mercaptoethanol stimulated high affinity dopamine uptake. Under these culture conditions, dopaminergic neurons survived and differentiated for more than three weeks. Glial conditioned medium A glial cell line RT4-D6 derived from RT4-AC, a neuronal-glial stem cell of peripheral neurotumor RT4, was chosen for preparation of glial conditioned medium 23. The glial properties of RT4-D6 include glial marker proteins, glial fibrillary acidic protein

113 (GFAP) and S100P. RT4-D6 also lacks the neuronal properties of voltage-dependent Na + and K + channels 62'63. The cells were cultured in the same culture medium for 5 days. The glial conditioned medium was harvested and filtered with a 0.2/~m Acrodisc filter.

of neurites per cytofluorescent-positive neurons was counted from 12 x 9 cm 2 photographs. Counts of number of neurites per cell were based only on neurites that originated from the cell body. The neurite length of cytofluorescent-positive cells was measured from the photographs by tracing the neurite with thread.

Preparation of PBS extract The striatal tissue from 2-week-old rat brains was homogenized in 2 ml of PBS (140 mM NaC1/2.6 mM KC1/1 mM KHzPO4/1 mM Na2HPO4, pH 7.0) per g of wet weight with 20 strokes of a Teflon-glass homogenizer. This homogenate was spun at 100,000 g at 4 °C for 90 min and filtered through a 0.2/~m Acrodisc filter. Supernatant fractions from this centrifugation were assayed for trophic activity. Protein concentration was assessed by the modified method of Lowry 47.

Molecular sieving column chromatography The PBS extract of the striatum was applied to preequilibrated Biogel P4 columns (200-400 mesh, 2.5 cm x 70 cm column) with 25 mM ammonium bicarbonate buffer, pH 6.5, and eluted with the same buffer.

Cell number and morphological analyses Morphological analysis of explants. For the morphological analysis of explants, dying explants corresponding to 5% of total were eliminated. A quarter of the remaining explants were randomly selected for photography. In order to facilitate counting of the processes, a circle was drawn around the explant at a radius of 5 cm from the edge of the explant in an 18 x 24 cm 2 print. Then perpendicular lines were drawn, dividing the circle into 4 sectors. Processes in a representative sector for that explant were counted, and multiplied by 4 to get the number of processes for that explant. Two persons scored all explants independently.

Cell number and morphometric analyses of catecholamine cytofluorescent-positive neurons in dissociation cultures. The total number of cytofluorescentpositive neurons was counted in the whole area of the multi-chamber under a fuorescence microscope. For morphometric analysis, 8-12 randomly distributed fields corresponding to 4 - 6 % of the total area for each condition were photographed. The number

Dopaminergic activity High affinity [3H] dopamine uptake. Assays of high affinity [3H]dopamine uptake were performed by the method reported by Prochiantz 44. After culture for 5 days in the presence and absence of factors, the cells were incubated at 37 °C for 15 rain with 50 nM [3H]dopamine (New England Nuclear, 20-30 Ci/mmol) in 0.7 ml of PBS (140 mM NaC1/2.6 mM KC1/0.75 mM MgC1J0.75 mM CaC12/1 mM KHzPOJ1 mM Na2HPO 4 containing 6 mg/ml glucose, I mg/ml BSA, p H 7.0) in the presence of pargyline (100/~M), an inhibitor of monoamine oxidase. Assays were terminated by washing cells four times with ice-cold PBS without CaC12 or MgC12. Cells were lysed by the addition of 0.3 ml of 0.5 N N a O H for two hours at room temperature. Incorporated [3H]dopamine was measured by a liquid scintillation counter. In studies of sodium dependence and energy requirements, H E P E S buffer was substituted for phosphate buffer and the pH was adjusted with ammonium hydroxide to 7.0. As an assay for the sodium dependence of high affinity dopamine uptake, Li was substituted for Na. To assess the requirement for energy, two conditions were chosen: assay at 0 °C, and pretreatment of cells for 5 rain with the metabolic inhibitor iodoacetic acid (100 ~M) followed by assay with iodoacetic acid at 37 °C. High affinity [3H]dopamine uptake by dopaminergic neurons was verified by specific inhibition by benztropine and desmethylimipramine at 1 tiM 44. In the presence of both inhibitors high affinity dopamine uptake was inhibited by 87%. The sodium dependence and energy requirements of high affinity dopamine uptake are similar to that described for norepinephrine ~s. High affinity dopamine uptake was linear for 30 min under the standard condition. To determine the non-specific dopamine uptake by glia, glial cultures were prepared from 1-day-old rat cerebral cortex according to the method of McCarthy and de Vellis 33. The cultured population was 98% astroglia by GFAP stain-

114 ing. Non-specific d o p a m i n e u p t a k e in these glial-enriched dissociation cultures was less than 3% of the PBS control of m e s e n c e p h a l o n cultures. T h e r e f o r e , m o r e than 90% of d o p a m i n e u p t a k e u n d e r these conditions was c o n s i d e r e d to be due to n e u r o n a l high affinity d o p a m i n e u p t a k e . Catecholamine cytofluorescence. Dissociated cultured m e s e n c e p h a l o n cells were analyzed by catecholamine fluorescence using a glyoxylic acid technique 6°. Cells grown on glass m u l t i c h a m b e r slides were rinsed in ice-cold PBS (NaC1, 0.1 M p h o s p h a t e , p H 7.4) and i m m e d i a t e l y placed in a l % - b u f f e r e d glyoxylic acid solution (1% glyoxylic acid, 0.1 M p h o s p h a t e , p H was a d j u s t e d to p H 7.4 with N a O H at 4 °C) for 5 min. Excess glyoxylic acid solution was removed. The slides were dried for 5 min u n d e r warm air and then h e a t e d for 10 min at 95 °C. Fluorescence

was viewed u n d e r a Nikon fluorescence microscope with V filter block (IF 399-425 interference excitation filter and 470 b a r r i e r filter). Control cells were t r e a t e d with cold PBS for the same period. To intensify cytofluorescence, the cells were p r e t r e a t e d five hours with 100 ktM L - D O P A in 570 ktM ascorbic acid and 100 ktM pargyline. W i t h o u t this intensification, catecholamine cytofluorescence was too low to detect most of catecholaminergic neurons t r e a t e d with PBS. Glial cells did not show any specific glyoxylic acid-induced catecholamine fluorescence u n d e r these conditions. High affinity [3H]GABA uptake. Since G A B A e r gic neurons are also present in the m e s e n c e p b a l o n , the striatal extract was tested for its ability to enhance neuronal high affinity G A B A uptake. M e s e n cephalic cells were incubated at 37 °C for 15 min with

Fig. 1. Morphological effect of striatal extract on mesencephalon explant cultures. Explants of mesencephalon tissues from embryonic 14-day-old rat brains were cultured on poly-L-lysine-coated Falcon tissue culture plates in serum-free defined medium. Forty-eight hours after plating, the cultures were supplemented with striatal extract (20 pg protein/ml, 40 pg protein/ml) from 2-week-old rat brains, with ovalbumin (40/~g/ml), or with the same volume of PBS. All cultures contained the same amount of fl-mercaptoethanol. Photographs were taken 72 h after application of (a) PBS, (b) ovalbumin (40/2g protein/ml), (c) striatal extract (20 pg protein/ml) and (d) striatal extract (40/~g protein/ml). The scale bar equals 100 pm.

115 10 nM [3H]7-aminobutyric acid ( G A B A ) (ICN, 75 Ci/mmol) in PBS, p H 7.4, in the presence of fl-alanine (2 m M ) , an inhibitor of glial high affinity G A B A u p t a k e 11. High affinity [ 3 H ] G A B A u p t a k e specific for neurons were verified with inhibition by L-2,4diaminobutyric acid (1 m M ) 42. N e u r o n a l high affintiy G A B A u p t a k e was expressed as the difference of high affinity G A B A u p t a k e in the presence of both L-2,4-diaminobutyric acid and fl-alanine from total high affinity G A B A u p t a k e in the presence of/3-alanine alone. RESULTS

Morphological effects o f soluble factors f r o m the striatum: The mesencephalic explants grown in s t a n d a r d m e d i u m without serum attached to the culture substratum and e x t e n d e d processes within 24 h after plating. The addition of striatal extract (20 and 40/~g protein/ml) a p p e a r e d to increase b o t h the n u m b e r and length of cellular processes (Fig. lc, d) c o m p a r e d to cultures t r e a t e d with PBS (Fig. l a ) or an equivalent a m o u n t of ovalbumin (40 pg/ml) (Fig. l b ) . The average n u m b e r of processes longer than 280 p m in the presence of striatal extract was 362 _+ 55 at 20 p g protein/ml and 357 _+ 37 at 40 p g protein/ml, comp a r e d with controls of 53 + 22 with PBS and 102 _+ 25 in ovalbumin 40/~g/ml at 72 h after application. T h e

addition of striatal extract increased average process length from 246-288 ,um in PBS and ovalbumin to 459 p m at striatal extract 20/zg protein/ml and 588/~m at striatal extract 40/~g protein/ml (Table I).

Effects o f striatal extract on differentiation and number o f dopaminergic neurons To investigate w h e t h e r soluble striatal extract stimulated differentiation of dopaminergic neurons and increased the n u m b e r of d o p a m i n e r g i c neurons, both high affinity [3H]dopamine u p t a k e 44 and glyoxylic acid-induced catecholamine cytofluorescence 6° were assayed in mesencephalic dissociation cultures. Striatal extract stimulated specific high affinity dopamine u p t a k e 3 . 0 - 3 . 5 - f o l d in dissociation cultures in a d o s e - d e p e n d e n t m a n n e r (Fig. 2). A similar stimulation was o b s e r v e d with explant cultures. Stimulation reached a m a x i m u m at 100/~g protein/ml in dissociation cultures. High affinity [3H]dopamine u p t a k e was a s o d i u m - d e p e n d e n t and energy-requiring process (Fig. 3). High affinity [3H]dopamine u p t a k e in these experiments was inhibited by 87% in the presence of both 1/zM b e n z t r o p i n e and i p M desmethylimipramine, specific inhibitors for high affinity dopamine u p t a k e 44 (Fig. 3). Stimulation of high affinity d o p a m i n e u p t a k e continued to increase with longer exposure to striatal extract. Striatal extract did not exhibit any short-term effects on stimulation of high affinity d o p a m i n e u p t a k e when cells were cultured

TABLE I

Morphological effects of striatal extracts on mesencephalon explant cultures Twenty-five to 30 explants of mesencephalon tissues from embryonic 14-day-old rat brains were cultured on poly-L-lysine coated tissue culture plates in serum-free defined medium. Forty-eight hours after plating, the cultures were supplemented with striatal extracts, ovalbumin or the same volume of PBS. Seventy-two hours after supplementing, morphological effects were observed. For the morphological analysis of explants, dying explants, especially in PBS control cultures, were eliminated. A quarter of the remaining explants were randomly selected for photography. As described in the Methods section, the number of processes per explant and average process length were analysed. The number of processes per explant in cultures with PBS and ovalbumin represented the actual number of processes present. However, the number of processes per explant in cultures with striatal extracts gave a minimum value due to the fact that the processes became too dense to count each one individually. No statistical difference in both number of processes and average process length was found between PBS and ovalbumin cultures (P > 0.2). The difference between cultures containing PBS (or ovalbumin) and cultures containing striatal extract was highly significant for both criteria (P < 0.002).

Condition

Number of processes per explant (process > 280 pm)

Average process length (ktm)

PBS Ovalbumin (40pg protein/ml) Striatal extract (20/~g protein/ml) Striatal extract (40/~g protein/ml)

53 + 102 + 362 + 357 +

246 + 288 + 459 + 588 +

22 (n = 25 (n = 55 (n = 37 (n =

8) 6) 8) 8)

28 22 28 39

116 cells c u l t u r e d with striatal extract.

o o ¢.) ..~ 0') ot,n

In o r d e r to assess effects of striatal extract on dif-

4.0

f e r e n t i a t i o n , we c o u n t e d the n u m b e r of n e u r i t e s p e r cell as well as t h e n e u r i t e l e n g t h in the p r e s e n c e and

3.0

a b s e n c e of e x t r a c t . T h e c r u d e striatal extract in°t-_ o

E®

g~ °c~

o v

c r e a s e d the n u m b e r of n e u r i t e s p e r cell as well as n e u -

2.0

rite length up to 2.0-fold and 2.5-fold, r e s p e c t i v e l y .

1.0 0 0

Protein nature of the soluble factors stimulating high affinity dopamine uptake by dopaminergic neurons I 20

I 40

I 60

I 80

I I00

T h e striatal e x t r a c t was t r e a t e d with trypsin (1/~g

protein ( ) u g / m l medium)

Fig. 2. Effect of striatal extract on high affinity dopamine uptake of mesencephalon cultures. Dissociated mesencephalic cells from embryonic 14-day-old rats were plated at 250,000 cells/well on poly-L-lysine coated 24 multiwell plate (Falcon). A given amount of the striatal extract was applied to the medium 24 h after plating in the absence (O C)) and presence (0---------0) of cytosine arabinoside (2 /~M), and cultured for 5 days. The stimulatory effect was expressed as the relative increase of high affinity dopamine uptake over PBS control (fold increase). The PBS control value assigned to 1.0 in the absence of cytosine arabinoside (C) C)) was 161.1 fmol/well. The PBS control value assigned to 1.0 in the presence of cytosine arabinoside (0---------0) was 72.2 fmol/ well. The data represent mean values + S.E.M. obtained from two sets of triplicate experiments. The stimulatory effect of the striatal extract was statistically highly significant (P < 0.001).

3.0

G3

.--

o

g~ o

d

for six days in the a b s e n c e o f e x t r a c t and t h e n w e r e t r e a t e d w i t h striatal e x t r a c t for 20 m i n p r i o r to assay (Fig. 4). Striatal e x t r a c t , t h e r e f o r e , has t r o p h i c effects on m e s e n c e p h a l i c d o p a m i n e r g i c n e u r o n s in culture. H i g h affinity [ 3 H ] d o p a m i n e u p t a k e i n c r e a s e d with i n c r e a s i n g cell density in dissociation cultures. C o n v e r s e l y , e n h a n c e m e n t o f high affinity d o p a m i n e upt a k e by soluble striatal e x t r a c t d e c r e a s e d w i t h h i g h e r plating density. T h e effects of striatal extract was assessed o n the n u m b e r of m e s e n c e p h a l i c d o p a m i n e r g i c n e u r o n s . In dissociation cultures of m e s e n c e p h a l o n , t h e n u m b e r of c a t e c h o l a m i n e

cytofluorescent-positive

neurons

i n c r e a s e d up to 4-fold in the p r e s e n c e of striatal extract ( T a b l e II; Fig. 5). Similarly, an i n c r e a s e d n u m b e r of c a t e c h o l a m i n e c y t o f l u o r e s c e n t - p o s i t i v e n e u rons was n o t e d with striatal extract e v e n in t h e abs e n c e of c y t o s i n e a r a b i n o s i d e . S o m e c y t o f l u o r e s c e n t n e u r o n s in P B S c o n t r o l cultures w e r e well differe n t i a t e d and s h o w e d a similar f l u o r e s c e n t i n t e n s i t y to

v - -~,'=-=-- -'F-'-=~-~ - 0

20

40 Striatal (jug

protein

60

80

extract / ml)

Fig. 3. Sodium and energy requirements of high affinity dopamine uptake and its inhibition by benztropine and desmethylimipramine. Dissociated mesencephalic cells were plated at 250,000 cells/well on poly-L-lysine coated 24-well plates. A given amount of striatal extracts was applied to the medium 24 h after plating in the presence of cytosine arabinoside (2 ~M) and cultured for 5 days. Control cultures contained the same volume of PBS. On the sixth day, high affinity dopamine uptakes were assayed under the following conditions: (a) the standard condition as described in the method section, except phosphate buffer was substituted with HEPES buffer at 37 °C (~C)--); (b) NaCI was replaced with LiCI and other conditions were the same with (a) (--C]--); (c) the same condition with (a) except at 0 °C ( ~ ) ; (d) the same condition with (a) except addition of 100/~M iodoacetic acid ( - - A - - ) ; (e) the same condition with (a) except addition of i/~M benztropine and 1 /xM desmethylimipramine ( - - × - - ) . Stimulatory effects were expressed as the relative increase of high affinity dopamine uptake over PBS control (fold increase). The PBS control value under the standard condition (a) was 123.7 fmol/ well. The data represents the mean +_ S.E.M. obtained from triplicate experiments. The differences between standard conditions and other conditions were statistically highly significant (P < 0.005).

117 T A B L E II Effect o f crude striatal extract on number and differentiation o f cytofluorescent-positive neurons in dissociated mesencephalon cultures Dissociated mesencephalon cells were plated on poly-L-lysine coated glass multi-chamber slides at 15,000 cells/chamber in standard medium supplemented with 4% heat-inactivated horse serum. Twenty-four hours after plating, crude striatal extract was added to the culture medium at concentrations of 0, 20, 40 and 80 ~g protein/ml in the presence of cytosine arabinoside (2/~M). All cultures contained the same volume of PBS and fl-mercaptoethanol in final. On the sixth day, half of medium was changed with fresh medium containing the same amount of crude extract. O n the twelfth day, glyoxylic acid-induced cytofluorescence assay was performed. A m o u n t o f extract (ktg protein/ml)

Total cytofluorescentpositive neuron number per chamber ~

Number o f neurites per cellb

Neurite length c (~m)

0 20 40 80

43.0 + 118.0+ 196.5+ 162.3 +

2.7 + 0.8 (n = 8) 4 . 6 + 0 . 4 ( n = 15) 5.2+0.4(n=16) 5.4 + 0.5 (n = 11)

142 +_+_18 (n = 22) 267__20(n=43) 316+25(n=37) 348 + 23 (n = 35)

4.2 (n = 4) 7.1(n=4) 2.8(n=4) 16.3 (n = 2)

Total cytofluorescent-positive neurons per chamber were counted under fluorescence microscope, n represented total number of chambers scored. b Eight to twelve randomly distributed fields corresponding to 4 to 6% of the total area were photographed. Each photograph contained one to six cytofluorescent-positive neurons. In the presence of striatal extract, more elaborately branched neurites, and two or three strongly fluorescent neurites and additional weakly fluorescent neurites were observed. Number of neurites per cell were based on neurites that originated from the cell body. Neurites that branched off from a primary process were not counted, n represented cytofluorescent-positive neurons scored. c Neurite length of cytofluorescent-positive cells was measured from the photographs by tracing the neurite with thread, n represented number of neurites scored. a

The statistical significance of the results was as follows. The difference in numbers of total cytofluorescent-positive neurons per chamber between PBS and experimental cultures with striatal extracts was highly significant (P < 0.002). The difference in number of neurites per cell between PBS and experimental cultures with striatal extract 20, 40, 80 ~g protein/ml was significant (P < 0.05, P < 0.01, and P < 0.01, respectively). The difference in average neurite length between PBS and experimental was highly significant (P < 0.001).

per 100 gg protein) at 37 °C. Trypsin digestion was terminated by the addition of soybean trypsin inhibitor (2/~g trypsin inhibitor per 1 ktg trypsin). Fortytwo, 58, and 78% of the stimulatory effect of the extract were lost by trypsin treatment for 1, 2 and 3 h, respectively. These results suggest that the major stimulatory components from the striatal extract are peptides (Table III).

0 C:

o

~.0_

~g

3.0 2.0

E~ ~P

0

1.0 0

L 2o go 8; ,;o

$triatal extract (.,~.g proteinlml medium)

Two peptides having similar molecular weight and affecting dopaminergic systems, namely neurotensin 41 and somatostatin-14 (ref. 7), were tested for activity. Neither peptide was active when applied to culture medium at concentrations up to 5/~g/ml for neurotensin (Fig. 6c) and 0.5 ktg/ml for somatostatin-

Fig. 4. Trophic effect of striatal extract on mesencephalic dopaminergic cells. Dissociated mesencephalic cells were plated at 250,000 cells/well on poly-L-lysine-coated 24-multiwell plates. A given amount of the striatal extract was applied to the medium 4 h after plating in the presence of cytosine arabinoside (2/~M), and cells were cultured for 5 days ( ~ ) . The equivalent amount of PBS with extract was added to medium in the presence of cytosine arabinoside, and cells were cultured for 5 days and then treated with a given amount of the striatal extract for 20 rain in cultures prior to assaying for high affinity dopamine uptake (O O). Stimulatory effects were expressed as the relative increase of high affinity dopamine uptake over PBS control (fold increase). The PBS control value assigned to 1.0 was 123.4 fmol/well. The data represent the mean value _+ S.E.M. obtained from two sets of triplicate experiments.

118

d

Fig. 5. Effect of striatal extract on number and differentiation of dopamincrgic neurons in dissociated mcsencephalon cultures. Dissociated mesencephalic cells were plated on poly-J-lysine-coated glass multichamhcr slides (Lab-Tec) at 15,000 cells/chamber in standard medium supplemcnted with 4% heat-inactivated horse serum. Twenty-four hours after plating, striatal extract was added to the culture medium at concentrations of 20, 40. and 80 !~g protein/ml in the presence ol cytosine arabinoside (2 !~M), and the same vohunc of PBS and/J-mercaptoethanol was added to control cultures. On the sixth day in culture, half of the medium was changed with fresh medium which contained extract. Olyoxylic acid induced catecholamine fluorescence assay was perfonncd on the twelfth day in culture. Fluorescence was viewed under a Nikon fluorescence microscope with V filter block (IF 399-425 interference excitation filter and 470 barrier filter), a: PBS. b: 20!zg protein/ml, c: 4()/~gprotein/ml, d: 80ug protein/ml. All photographs werc exposed, developed, and printed under the same conditions. Thc scale bar equals 100 am.

14 (Fig. 6d). At 2,ug somatostatin-14 had minimal effects (15% increase in high affinity d o p a m i n e uptake). N G F (0.5-50 nM) had no effect on high affinity dopamine uptake (Fig. 6b). Thus, the dopaminergic factors appear to be different from either neurotensin, somatostatin-14, or N G F .

Specificity of trophic factor source To assess the specificity of the source of trophic factor, extracts of different tissues of 2-week-old rats were tested for their abilities to alter high affinity dopamine uptake. As shown in Fig. 7, the highest specific activity was observed in extracts of the striatum, followed by extracts of the hippocampus and the cerebral cortex. Extracts of the cerebellum and the ol-

factory bulb had lesser effects o11 high affinity dopamine uptake. Liver extract somewhat suppressed cell growth and demonstrated lower uptake than the PBS controls.

Neuronal specificity of soluble factors from the striaturn To test the n e u r o n a l target specificity of soluble factors from the striatum, effects on cultured mesencephalic dopaminergic neurons and G A B A e r g i c n e u r o n s were compared. Each activity was assessed by high affinity d o p a m i n e uptake and neuronal high affinity G A B A uptake. Crude striatal extract stimulated both high affinity dopamine uptake and neuronal high affinity G A B A uptake. However, factors

119 TABLE III

I I

The effect of trypsin digestion of striatal extract on high affinity dopamine uptake

2 I

3

PBS striatum

Trypsin treatment of striatal extract (1 #g trypsin per 100/~g protein) was carried out in DMEM medium at 37 °C for 0, 1, 2 and 3 h, and the reaction was stopped by the addition of soybean trypsin inhibitor (2pg trypsin inhibitor perpg trypsin) and incubated at 37 °C for 60 min. PBS control contained the equivalent amount of PBS and fl-mercaptoethanol with the extract and also incubated at 37 °C for 60 rain in the presence of soybean trypsin inhibitor. Sixty #g striatal extract treated with trypsin was added to 1 ml of culture medium at 24 h after plating of dissociated El4 mesencephalon cells (250,000 cells/ well). High affinity dopamine uptake was assayed on the sixth day. The mean of the absolute values of high affinity dopamine uptake at times 0 for trypsin treatment of extract was 212.0 fmol/well. The value of PBS control cultures was 96.1 final/ well. All cultures were treated with trypsin inhibitor. Stimulation of high affinity dopamine uptake with extract over PBS control was expressed as 100%. The data were mean values + S.E.M. obtained from two sets of triplicate experiments. The differences of stimulatory activities between untreated striatal extract and extract treated with trypsin were statistically significant (P < 0.005, P < 0.005 and P < 0.001 for 1, 2 and 3 h, respectively).

Trypsin treatment time (hour)

Activity of high affinity dopamine uptake (%)

0 1 2 3

100.0 58.1 42.1 21.4

+ + + +

2.4 2.8 1.1 0.7

H

hippocampus cerebral

H

cortex

cerebellum olfactory

H

bulb

H

liver I I

I 2

I 3

Dopamine uptake (fold increase over PBS control)

Fig. 7. The effect of different tissue extracts on high affinity dopamine uptake of dissociated mesencephalon cultures. Dissociated mesencephalon cells were plated at 250,000 cells per well on poly-L-lysine coated 24 multiwell plates in standard culture medium containing 4% heat-inactivated horse serum. Fifty/xg of each tissue extract per ml of medium was added 24 h after plating in the presence of cytosine arabinoside (2/~M). High affinity dopamine uptake assays were performed on the sixth day. PBS control value, 1.0, represents 127.0 fmol per well. The data were expressed as mean numbers of relative stimulation of high affinity dopamine uptake + S.E.M. obtained from two sets of triplicate experiments. The differences between PBS and the striatum, the hippocampus, or the cerebral cortex were statistically significant (P < 0.005). However, the difference between PBS and the cerebellum was less significant (P < 0.05). There was no significant difference between PBS and the olfactory bulb (P < 0.1).

stimulating high affinity d o p a m i n e u p t a k e w e r e par2 '~

(o)

(b)

(c)

(d)

tially s e p a r a b l e f r o m factors s t i m u l a t i n g high affinity GABA

u p t a k e on B i o g e l P4 m o l e c u l a r sieving col-

u m n c h r o m a t o g r a p h y (Fig. 8b, c). F r a c t i o n s 6 5 - 6 9 w e r e m o r e specific for d o p a m i n e r g i c n e u r o n s ; frac-

.1: O

0 ~

_ o

tions 9 3 - 1 0 2 and 1 0 3 - 1 0 8 w e r e m o r e specific for

1.0

0

I ~ 2 s 4

0 20,*060e0100 Striatal

extract

(~g/ml)

7S-NGF

(x IO'eM)

Neurotensin

(./,tg/ml)

GABAergic neurons. Fractions 72-85 were common 00.20-40.60.81.0 S o m o t o s t o t i n - 14 (./ag/rnl)

Fig. 6. Effects of striatal extract, 7S-NGF, neurotensin and somatostatin-14 on high affinity [SH]dopamine uptake by dopaminergic neurons in mesencephalic dissociation cultures. Dissociated mesencephalic cells were plated at 250,000 cells/well on poly-L-lysine coated 24-well plates. A given amount of either striatal extracts, 7S-NGF, neurotensin or somatostatin14 was applied to the medium 24 h after plating in the presence of cytosine arabinoside (2/~M) and cultured for 5 days. On the sixth day, high affinity dopamine uptake was assayed. The sets of cultures were (a) striatal extract, (b) 7S-NGF, (c) neurotensin, and (d) somatostatin-14. All cultures contained the same amount of fl-mercaptoethanol. The data represents the mean value + S.E.M. obtained from quadruplicate experiments. Unit 1.0 represents 80 fmol high affinity dopamine uptake/well.

for b o t h n e u r o n s . Sixty-flue to 7 5 % of high affinity d o p a m i n e u p t a k e activity of the c r u d e extracts was r e c o v e r e d in t h e s e t w o m a j o r fractions on m o l e c u l a r sieving c h r o m a t o g r a p h y (Fig. 8b). T h e m a j o r p e a k s stimulating

high

affinity d o p a m i n e

uptake

were

e l u t e d at a p p r o x i m a t e l y 1 5 0 0 - 2 2 0 0 D a , and stimulation of high affinity d o p a m i n e u p t a k e by these m a j o r p e a k s was d o s e - d e p e n d e n t . DISCUSSION T h e effects of the t a r g e t striatal tissue u p o n the dev e l o p m e n t of nigral n e u r o n s w e r e first d e m o n s t r a t e d

120 CO ~J 0

0 o

~I"

vo

°3

1.5

A

.E ~

~l~

0

~, (0

~ O~ "6o

~ rO ~

© -i-

~_.

.~

z

o

q O

1.0

N

W

0.5

0

20 -

40

60

80

I00

120

140

160

0

2O

40

60

80

I00

120

140

160

0

20

40

60

80

I00

120

i 140

2000

~.0

~

.E E Ea. o o

I000

0

0

i

..~

i

1500

0

m ~

I000

~E

Z

0 Fraction

number

Fig. 8. Neuronal specificity of effect of soluble factors from striatum. The PBS extract of the striatum (10 ml) was applied on the preequilibrated Biogel P4 column (200-400 mesh, 2.5 × 70 cm column) with 25 mM ammonium bicarbonate buffer, pH 6.5, and eluted with the same buffer. The eluate (3.0 ml) was collected and protein concentrations were assessed by E2s0 (A). Fractions were lyophilized and dissolved in the culture medium containing fl-mercaptoethanol (500 #M) and filtered with 0.2 #m Acrodisc filter. Twenty-four hours after plating, an amount corresponding to 60kd of each fraction was added to the mesencephalic dissociation cultures (1.5 ml medium per well). At the same time, cytosine arabinoside (2#M) was added to the medium. On the sixth day in culture, high affinity dopamine uptake (B) and neuronal high affinity G A B A uptake (C) were assayed. Neuronal G A B A uptake was expressed as the difference of high affinity G A B A uptake in the presence of fl-alanine, and high affinity G A B A uptake in the presence of both fl-alanine and 2,4-diaminobutyric acid (inhibitor of neuronal G A B A uptake). The data represents mean values + S.E.M. obtained from triplicate cultures.

121 in co-cultures of dissociated embryonic mesencephalic dopaminergic neurons with striatum or frontal cortex. In these studies, enhancement of both high affinity dopamine uptake and elongation of cellular processes were noted 1°'19'43. It was subsequently demonstrated that particulate membrane fractions, and not soluble factors, were responsible for such morphologic and neurotransmitter alterations 44. This effect was observed with membrane fractions from 2-3week-old animals, but not from 1-week-old animals. A non-diffusible factor also appeared responsible for the proliferation and/or maintenance of dopaminergic axons and increased survival of dopaminergic neurons in aggregated mesencephalon cell cultures, but was present in embryonic tissue is. In our own studies, soluble factors from the striatum increased the number and length of cytofluorescent-positive mesencephalic neurons, and enhanced high affinity dopamine uptake. This effect was observed with extract from 15-day embryos as well as later stages (unpublished data). Our extracts were prepared by centrifugation for 90 min at 100,000 g and filtration of the supernatant through a 0.2 ktm filter, thereby assuring their soluble character. In explant cultures, soluble components enhanced high affinity dopamine uptake up to 4.5-fold, while membrane fractions enhanced high affinity dopamine uptake to a lesser extent (up to 2-fold). Our PBS homogenates were prepared in the absence of protease inhibitors, thus we cannot rule out a membrane origin for our soluble factors. Whether such soluble factors are derived from the membrane fractions can only be definitely answered with complete purification of both components or the availability of specific antibodies against such factors. However, at present our factors differ from those reported in other studies by their solubility and their presence in embryonic as well as postnatal striatum ls'44. The soluble factors derived from the striatum appeared to enhance both the number and differentiation of the mesencephalic dopaminergic neurons as assayed by glyoxylic acid-induced catecholamine fluorescence and high affinity [3H]dopamine uptake. At this time, it is difficult to segregate effects on differentiation from effects on survival, especially since the enhancing effects on number of cytofluorescentpositive neurons are of the same order of magnitude as the enhancing effects on dopamine uptake. Cyto-

fluorescent cell number could increase either because the trophic factor increases the concentration of catecholamine to threshold levels of detectability or because cell numbers themselves increase. The enhanced dopamine uptake cannot be totally explained by survival since the number of neurites per cell, neurite length, and catecholamine fluorescence intensity are enhanced in individual cells cultured in the presence of striatal extract. Thus, factors which enhance the number of cytofluorescent-posirive neurons and factors which enhance dopaminergic differentiation are both present in striatal extract. Whether both of these effects reside in the same molecule or whether such effects reside in different molecules is presently unresolved. Stimulation of high affinity dopamine uptake by striatal extract is present both in the presence and absence of cytosine arabinoside. However, the absolute stimulation of dopamine uptake by soluble striatal factor is larger in the absence of cytosine arabinoside, thereby suggesting that glial cells can influence cell differentiation and/or survival of dopaminergic neurons. Direct evidence was provided by the stimulatory effect of glial-conditioned medium on high affinity dopamine uptake in the presence of cytosine arabinoside. Striatal extract stimulated high affinity dopamine uptake over and above the stimulation by glial conditioned medium. The effect of glia may differ from the effect of striatal extract since precoating substratum with glial-conditioned media synergistically enhanced high affinity dopamine uptake by striatal extract (data not shown) 34. Moreover, the major fractions stimulating [3H]thymidine incorporation into glia in dissociated culture in the absence of cytosine arabinoside were found in fractions of Biogel P4 chromatography other than those which stimulated high affinity dopamine uptake. Thus, enhancement of high affinity dopamine uptake by soluble striatal factors appears different from its enhancement by glia. Crude striatal extract stimulated both high affinity dopamine uptake and neuronal high affinity G A B A uptake. However, partially purified factors stimulating dopaminergic effects and factors stimulating GABAergic effects were partially separable using molecular sieving chromatography. These chromatographic patterns were observed in PBS extract from young adult rat striatum and in calf caudate-puta-

122 men. Whether such stimulating effects are specific for dopaminergic neurons in the mesencephalon or are common to all dopaminergic neurons is not known. Nor is it clear whether such factors only influence dopaminergic neurons, or can effect other types of catecholaminergic neurons. The chemical nature of the soluble factor is not known. Just as with crude striatal extracts, the major active peaks of high affinity dopamine uptake were trypsin sensitive. Fraction numbers 65-69 (Fig. 8) lost 89%, and fraction numbers 71-81 (Fig. 8) lost 48% of stimulatory activity of high affinity dopamine uptake following trypsin treatment for two hours. These results suggest a peptidic nature of small striatal soluble factors (molecular weight: 1500-2200 Da) stimulating dopaminergic activity. Whether such factors are new or are previously described peptides is not clear. Peptides having similar molecular sizes and influencing dopaminergic systems, such as neurotensin and somatostatin-14, were tested as candidates. Neurotensin receptors are located in high density in the limbic system and fibers originating from the locus coeruleus, the ventral tegmental tract, and the substantia nigra 26'41. Somatostatin is known to influence dopamine release 7 and is located in high density in the hippocampus, the cerebral cortex, the amygdala and the hypothalarnus 26'54. However, neither neurotensin nor somatostatin-14 had stimulatory effect on high affinity dopamine uptake in our system. Thus, the activity of our peptide(s) appears to reside in a constituent different from either neurotensin or somatostatin. The soluble factors stimulating the survival and differentiation of dopaminergic neurons do not appear to be NGF. NGF synthesis in the CNS has been demonstrated to be highest in the hippocampus followed by cerebral cortex, but is low in the striat u r n 27'53'66. Furthermore, the molecular weights of factors stimulating dopaminergic differentiation and for survival are primarily confined to a 1500-2200 Da range, which is far smaller than active units of NGF. Finally, NGF (0.5 x 10 -9 M to 0.5 x 10-7 M)

REFERENCES 1 Adler, R., Landa, K.B., Manthorpe, M. and Varon, S., Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons, Science, 204 (1979) 1434-1436.

had no effect on high affinity dopamine uptake in dissociated mesencephalon cultures. NGF and NGF antiserum do not change tyrosine hydroxylase activity in the substantia nigra or in the locus coeruleus a8. The trophic activity enhancing survival of dopaminergic neurons and their differentiation in mesencephalon cultures is highest in the striatum, next highest in the hippocampus-entorhinal cortex-amygdaloid nucleus area and in the cerebral cortex, but lowest in the cerebellum and the olfactory bulb. Substantia nigra and ventral tegmental dopaminergic neurons project their fibers primarily to the neostriatum, and also to the limbic cortex (medial prefrontal, entorhinal cortex, etc.) and other limbic structures (the septum, the nucleus accumbens, the amygdaloid cortex, etc) 36. Preliminary results suggest that mesencephalic dopaminergic neurons from early embryonic development are more sensitive to the striatal extracts and stimulated high affinity dopamine uptake. However, at early ontogeny, the striatum is observed as one of the most developed tissues in the telencephalon compared with the hippocampus and other cerebral cortical structures. Thus, considering the developmental sequences in rat brain, it would appear reasonable to suggest the potential importance of a factor(s) derived from the striatum which enhanced the survival and differentiation of mesencephalic dopaminergic neurons. Further characterization of the dopaminergic trophic factor must await purification and development of appropriate immumological and molecular probes.

ACKNOWLEDGEMENTS The authors thank Mr. E.S. Zetka and Miss A. Rader for technical assistance and Drs. D. Giulian, S. Sakato, and W. Strittmatter for their helpful advice. We are grateful to the R.J., Jr. and H.C. Kleberg Foundation and the Harkins Foundation for support of these studies.

2 Barbin, G., Manthorpe, M. and Varon, S., Purification of the chick eye ciliary neuronotrophic factor, J. Neurochem.., 43 (1984) 1468-1478. 3 Barde, Y.-A., Edgar, D. and Thonen, H., Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1 (1982) 549-553.

123 4 Barde, Y.-A., Edgar, D. and Thoenen, H., New neurotrophic factors, Annu. Rev. Physiol., 45 (1983) 601-612. 5 Berg, D.K., New neuronal growth factors, Annu. Rev. Neurosci., 7 (1984) 149-170. 6 Bottenstein, J.E. and Sato, G.H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 514-517. 7 Chesselet, M.-F. and Reisine, T.D., Somatostatin regulates dopamine release in rat striatal slices and cat caudate nuclei, J. Neurosci., 3 (1983) 232-236. 8 Cowan, W.M. and Wenger, E., Degeneration in the nucleus of origin of the preganglionic fibers of the chick ciliary ganglion following early removal of the optic vesicle, J. Exp. Zool., 168 (1968) 105-124. 9 Coyle, J.T. and Henry, D., Catecholamines in fetal and newborn rat brain, J. Neurochem., 21 (1973) 61-67. 10 Denis-Donini, S., Glowinski, J. and Prochiantz, A., Specific influence of striatal target neurons on the in vitro growth of mesencephalic dopaminergic neurites: a morphological quantitative study, J. Neurosci., 3 (1983)2292-2299. 11 Frame, M.C., Freshney, R.I., Vaughan, P.F.T., Graham, D.I. and Shaw, R., Interrelationship between differentiation and malignancy-associated properties in glioma, Br. J. Cancer, 49 (1984) 269-280. 12 Fukada, K., Purification and partial characterization of a cholinergic neuronal differentiation factor, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8795-8799. 13 Golden, G.S., Prenatal development of the biogenic amine systems of the mouse brain, Dev. Biol. 33 (1973) 300-311. 14 Gorin, P.D. and Johnson, E.M., Experimental autoimmune model of nerve growth factor deprivation: effects on developing peripheral sympathetic and sensory neurons, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 5382-5386. 15 Greene, L.A. and Rein, G., Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells, Brain Research, 129 (1977) 247-263. 16 Hamburger, V. and Yip, J.W., Reduction of experimentally induced neuronal death in the spinal ganglia of the chick embryo by nerve growth factor, J. Neurosci., 4 (1984) 767-774. 17 Hefti, F. Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after injury, Abstract, 15th Neuroscience meeting at Dallas, 15 (1985) 197.7. 18 Heller, A. and Won, L., Selective association of dopaminergic (DA) axons with their striatal targets in vitro, Abstract, 15th Neuroscience meeting at Dallas, 15 (1985) 22.18. 19 Hemmendinger, L.M., Garber, B.B., Hoffmann, P.C. and Heller, A., Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 1264-1268. 20 Hendry, I.A. and Campbell, J., Morphometric analysis of rat superior of cervical ganglion after axotomy and nerve growth factor treatment, J. Neurocytol., 5 (1976) 351-360. 21 Heumann, R., Korsching, S., Scott, J. and Thoenen, H., Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues, E M B O J., 3 (1984) 3183-3189. 22 Hollyday, M. and Hamburger, V., Reduction of the naturally occurring motor neuron loss by enlargement of the periphery, J. Comp. Neurol., 170 (1976) 311-320. 23 Imada, M. and Sueoka, N., Clonal sublines of rat neurotumor RT4 and cell differentiation. I. Isolation and characte-

rization of cell lines and cell type conversion, Dev. Biol., 66 (1978) 97-108. 24 Johnson, E.M., Jr., Gorin, P.D., Brandeis, L.D. and Pearson, J., Dorsal root ganglion neurons are destroyed by exposure in utero to maternal antibody to nerve growth factor, Science, 210 (1980) 916-918. 25 Kessler, J.A. and Black, I.B., The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo, Brain Research, 189 (1980) 157-168. 26 Kobayashi, R.M., Brown, M. and Vale, W., Regional distribution of neurotensin and somatostatin in rat brain, Brain Research, 126 (1977) 584-588. 27 Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H., Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation, E M B O J., 4 (1985) 1389-1393. 28 Kotake, C., Hoffmann, P.C. and Heller, A., The biochemical and morphological development of differentiating dopamine neurons co-aggregated with their target cells of the corpus striatum in vitro, J. Neurosci., 2 (1982) 1307-1315. 29 Lauder, J.M. and Bloom, F.E., Ontogeny of monoamine neurons in the locus ceruleus, raphe nuclei and substantia nigra of the rat, J. Comp. Neurol., 155 (1974) 469-482. 30 Levi-Montalcini, R. and Booker, B., Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein, Proc. Natl. Acad. Sci. U.S.A., 46 (1960) 384-391. 31 Levi-Montalcini, R. and Angeletti, P.U., Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro, Dev. Biol., 7 (1963) 653-659. 32 Levi-Montalcini, R. and Angeletti, P.U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 33 McCarthy, K.D. and de Vellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 34 Manthorpe, M., Varon, S. and Adler, R., Neurite-promoting factor in conditioned medium from RN22 Schwannoma cultures: bioassay, fractionation and properties, J. Neurochem., 37 (1981) 759-767. 35 Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Buchanan, K., and Johnston, M.V., Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor, Science, 229 (1985) 284-287. 36 Moore, R.Y. and Bloom, F.E., Central catecholamine neuron systems: anatomy and physiology of the dopamine systems, Annu. Rev. Neurosci., 1 (1978) 129-169. 37 Ojika, K. and Appel, S.H., Neurotrophic effects of hippocampal extracts on medial septal nucleus in vitro, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)2567-2571. 38 Olson, L. and Seiger, A., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 137 (1972) 301-316. 39 Oppenheim, R.W., Maderdrut, J.L. and Wells, D.J., Cell death of motoneurons in the chick embryo spinal cord. VI. Reduction of naturally occurring cell death in the thoracolumbar column of Terni by nerve growth factor, J. Comp. Neurol., 210 (1982) 174-189. 40 Otten, U., Weskamp, G., Schlumpf, M., Lichtensteiger, W. and Mobley, W.C., Effects of antibodies against nerve growth factor on developing cholinergic forebrain neurons in rat. Abstract, 15th Neuroscience meeting at Dallas, 15

124 (1985) 197.10. 41 Palacios, J.M. and Kuhar, M.J., Neutotensin receptors are located on dopamine-containing neurons in rat midbrain, Nature (London), 249 (1981) 587-589. 42 Pastuszko, A., Wilson, D.F. and Erecinska, M., Net uptake of 7-aminobutyric acid by a high affinity system of rat brain synaptosomes, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 1242-1244. 43 Prochiantz, A., di Porzio, U., Kato, A., Berger, B. and Glowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5387-5391. 44 Prochiantz, A., Daguet, M.-C., Herbet, A. and Glowinski, J., Specific stimulation of in vitro maturation of mesencephalic dopaminergic neurons by striatal membranes, Nature (London), 293 (1981) 570-572. 45 Richardson, P.M. and Riopelle, R.J., Uptake of nerve growth factor along peripheral and spinal axons of primary sensory neurons, J. Neurosci., 4 (1984) 1683-1689. 46 Rothman, T.P., Specht, L.A., Gershon, M.D., Job, T.H., Teitelman, G., Pickel, V.M. and Reis, D.J., Catecholamine biosynthetic enzymes are expressed in replicating cells of the peripheral but not the central nervous system, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 6221-6225. 47 Schacterle, G.R., and Pollack, R.L., A simplified method for the quantitative assay of small amount of protein in biologic material, Anal. Bioehem., 51 (1973) 654-655. 48 Schwab, M.E., Otten, U., Agid, Y. and Thoenen, H., Nerve growth factor (NGF) in the rat CNS: absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra, Brain Research, 168 (1979) 473-483. 49 Schlumpf, M., Shoemaker, W.J. and Bloom, F.E., Explant cultures of catecholamine-containing neurons from rat brain: biochemical, histofluorescence, and electron microscopic studies, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4471-4475. 50 Seiler, M. and Schwab, M.E., Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat, Brain Research, 300 (1984) 33-39. 51 Sieger, ,~. and Olson, L., Late prenatal ontogeny of central monamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch.,-140 (1973) 281-318. 52 Shelton, D.L. and Reichardt, L.F., Expression of the /3nerve growth factor gene correlates with the density of sympathetic innervation in effector organs, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7951-7955. 53 Shelton, D.L. and Reichardt, L.F., Studies on the expression of the/3 nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as atrophic factor for

54

55

56

57

58

59

60

61 62

63

64

65

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several distinct populations of neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2714-2718. Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Iida, H., Minagawa, H., Hara, Y., Matsuzaki, T. and Tohyama, M., Ontogeny of somatostatin-containing neuron system of the rat: immunohistochemical analysis II. Forebrain and diencephalon, J. Comp. Neurol., 204 (1982) 211-224. Smith, R.G. and Appel, S.H., Extracts of skeletal muscle increase neurite outgrowth and cholinergic activity of fetal rat spinal motor neurons, Science, 219 (1983) 1079-1081. Smith, R.G., Vaca, K., McManaman, J. and Appel, S.H., Selective effects of skeletal muscle extract fractions on motoneuron development in vitro, J. Neurosci., 6 (1986) 439447. Specht, L.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Light-microscopic immunocytochemical localization of tyrosine hydroxylase in prenatal rat brain. I. Early ontogeny, Y. Comp. Neurol., 199 (1981) 233-253. Specht, L.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Light-microscopic immunocytochemical localization of tyrosine hydroxylase in prenatal rat brain. II. Late ontogeny, J. Comp. Neurol., 199 (1981) 255-276. St6ckel, K., Paravicini, U. and Thoenen, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-421. Sumners, C., Phillips, M.I. and Raizada, M.K., Rat brain cells in primary culture: visualization and measurement of catecholamines, Brain Research, 264 (1983) 267-275. Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. Tomozawa, Y. and Sueoka, N., In vitro segregation of different cell lines with neuronal and glial properties from a stem cell line of rat neurotumor RT4, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 6305-6309. Tomozawa, Y., Sueoka, N. and Miyake, M., Clonal sublines of rat neurotumor RT4 and cell differentiation. V. Comparison of Na + influx, Rb + effiux, and action potential among stem-cell, neuronal and glial cell types, Dev. Biol., 108 (1985) 503-512. Varon, S.S. and Bunge, R.P., Trophic mechanisms in the peripheral nervous system, Annu. Rev. Neurosci., 1 (1978) 327-361. Varon, S., Manthorpe, M. and Adler, R., Cholinergic neuronotrophic factors. I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia, Brain Research, 173 (1979) 29-45. Whittemore, S.R., Ebendal, T., L~irkfors, L., Olson, L., Seiger, ~., Str6mberg, I. and Persson, H., Developmental and regional expression of/3 nerve growth factor messenger RNA and protein in the rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 817-821.