Factors regulating the expression of ... - Semantic Scholar
Brain Research, 532 (1990) 131-139 Elsevier
131
BRES 16000
Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization Julia Dymshitz 1, Rafael Malach 1, Shimon Amir I and Rabi Simantov 2 Departments of INeurobiology and 2Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot (lsrael)
(Accepted 15 May 1990) Key words: Striatal culture; Acetylcholinesterase; Chemical depolarization; Differentiation; Neuronal survival
The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 gM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20/~M) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCI levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2÷ channels - - verapamil (1/~M) and nifedipine (1/~M) - - did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 gM) had a partial effect. When cultures treated with 5 gM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed. INTRODUCTION The survival and differentiation of neurons in tissue culture is influenced by a number of environmental factors including contact with other neuronal and nonneuronal cells 1,2'32, presence of trophic substances in culture medium 3'34 and the level of neuronal activation 5' 10. Though the physiological significance of these factors during the histogenesis of the central nervous system is not established yet, the in vitro studies are indicative of the potentially important regulatory processes that might occur during normal development. The evidence for the role of the electrical activity in neuronal survival has been obtained using cultures of both peripheral 4"31 and central neurons 1°'18, where the depolarizing effects of neuronal activation were mimicked by elevation of the extraceUular potassium chloride (KCI) levels or by a long-term application of sodium channel agonists. Thus in cultures of cerebellar granule cells KCl-induced chemical depolarization promoted the neuronal survival without affecting the morphological differentiation 18. In another in vitro model the blockade of a spontaneous electrical activity of cultured spinal cord neurons with tetrodotoxin (q'TX) resulted in a reduction
in a number of cells containing choline acetyltransferase (CHAT) 1°. There is also extensive evidence that chemical depolarization may alter selectively the expression of neurotransmitter-related enzymes and peptides 6'14'15'17'23. The activity of the catecholamine-synthesizing enzyme - tyrosine hydroxylase - - was markedly increased by chronic treatment with the sodium channel agonist veratridine in explants of locus coeruleus 15 as well as in dissociated cultures of substantia nigra 17. Similarly, both the synthetic and the degradative enzymes for acetylcholine - - C h A T and acetylcholinesterase (ACHE) - - were found to be enhanced by depolarization in neuronal striatal cultures 25 and in cultures of muscle cells 14, respectively. On the other hand, the same treatment which induced an increase in C h A T activity in striatal cultures diminished the levels of substance P and of somatostatin in peptidergic neurons, indicating that the effects of chemical depolarization are differential with regard to different neuronal populations in the same brain structure 25. In a number of studies 7'13'35'39 A C h E histochemistry has been used to identify a distinct population of striatal neurons which could be classified into several types
Correspondence: J. Dymshitz. Present address: Department of Physiology, Indiana Univ. Med. Sch., 635 Barnhill Drive, Indianapolis, IN 46223, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
132 according
to their
morphology
and
the
intensity
of
staining 7. It has b e e n suggested that large heavily stained cells b e l o n g e d to a class of aspiny cholinergic striatal i n t e r n e u r o n s v. In line with this suggestion, a c o m p l e t e o v e r l a p b e t w e e n i m m u n o l a b e l l i n g for C h A T and staining for A C h E has b e e n f o u n d in the rat striatum in situ 16. In the p r e s e n t study we used an in vitro a p p r o a c h to follow the d e v e l o p m e n t of A C h E - p o s i t i v e n e u r o n s in p r i m a r y n e u r o n a l cultures of e m b r y o n i c s t r i a t u m and to investigate the influence of c h e m i c a l d e p o l a r i z a t i o n
on the
d i f f e r e n t i a t i o n and survival of these cells. MATERIALS AND METHODS
Tissue culture Sprague-Dawley rat fetuses (E15-E16) were dissected on ice under sterile conditions in Dulbecco's modified Eagle's medium (DMEM, Gibco) to which penicillin (10 units/ml), streptomycin (0.1 mg/ml) and antimycotic n-butyl-p-hydroxybenzoate (2 mg/ml) were added. Excised striata were incubated with 0.25% trypsin for 5 min at 37 °C and then washed 3 times with DMEM containing 50 #M of the protease inhibitor phenylmethylsulfonyl fluoride (Sigma). The tissue was dissociated into single cells by trituration with a fire-polished Pasteur pipette and then filtered through a 100 × 100 /~m stainless steel net. This procedure resulted in 90% yield of viable cells, as determined by Trypan blue exclusion test. Striatal cells were resuspended in DMEM supplemented with 10% horse serum (Gibco) and plated in Nunclon 35-mm dishes precoated with poly-ornithine (10 :~g/ml, Sigma). In some experiments a confluent layer of striatal astrocytes was used as a substrate for neuronal cells. The plating density was 2.5-3 × 106 cells per dish. When serum-free medium was used (DMEM supplemented with 5/~g/ml bovine insulin, 1 /~g/ml human transferrin, 20 nM hydrocortisone-21-phosphate, 30 nM triiodothyronine, 2/~g/ml carnithine, 3/~g/ml linoleic acid, 135/~g/ml choline chloride, 1.35/tg/ml vitamin B12, 1/~g/ml biotin and the trace elements as described24), it was added 24 h after plating, the corresponding cultures being washed with DMEM prior to medium change. Subsequently, the medium was changed every 4-5 days. Primary striatal astrocyte cultures were prepared from rat fetuses (E15-E16) and grown in DMEM supplemented with 10% horse serum in 60-mm Nunclon dishes precoated with poly-ornithine. The initial plating density was 1 × 106 cells per dish. When cultures reached confluence (usually on day 10-11) they were trypsinized and replated in 35-mm dishes at a density of 0.1-0.2 × 106 cells/dish. Culture medium was changed every 3-4 days. These secondary cultures became confluent after 6-8 days in vitro. AChE histochemistry Cultures were fixed for 20 min with 4% paraformaldehyde (in PBS), rinsed briefly with distilled water and stained for acetylcholinesterase according to a procedure described by Geneser-Jensen and Blackstad 19. Optimal staining was observed with relatively long incubation times (20 h) at room temperature, which allowed a good visual discrimination of fine neuronal processes in AChE-positive cells. The neurons stained for AChE were counted in two diameter bands (2.5 x 33 mm) of each culture dish using a binocular microscope (×50). In experiments with different treatments the cells were counted using a blind procedure. lmmunocytochemistry Immunocytochemistry was performed with primary antibodies (Incstar; 1:10) against glial fibrillary acidic protein (GFAP) and against neuron specific enolase (NSE) using Vectastain ABC method (Vector Laboratories). Briefly, the cultures were fixed as described above, washed with PBS 3 times and incubated with
primary antibody overnight in TBS (Tris-buffered saline, 50 mM Tris-HCl in 0.9% NaCI, pH = 7.4) containing 1% normal goat serum and 0.1% Triton X-100. After being washed twice with PBS the cultures were incubated for 30 min with the biotinylated anti-rabbit IgG and then washed again. The preformed avidinbiotinylated peroxidase complex was applied for 30 min, after which the peroxidase activity was visualized by a reaction with 0.05% 3,3"-diaminobenzidine tetrahydrochloride (Sigma) and 0.03% H202 in TBS.
Binding assay The cultured cells were washed twice with PBS, removed from the plates with a rubber policeman and collected by centrifugation in PBS at 1000 g for 5 min. The pellet was frozen at -20 °C and used for binding assay within two weeks from the time of cell collection. On the day of the assay the pellet was thawed, resuspended in 2-3 ml of 5 mM Tris-HCl buffer (pH 7.4) and after incubation for 10-15 min at 4 °C was homogenized with a Teflon-glass homogenizer (15 strokes). An equal volume of 0.64 M sucrose was then added and following centrifugation at 28,000 g for 15 min the binding assay was conducted as described 36. Briefly, the homogenates (0.2-0.6 mg protein/ml) were incubated with appropriate ligands for 40 min (opiate binding) or 60 min (muscarinic binding) at 25 °C in a final volume of 0.5 ml. For determination of total opiate specific binding [3H]diprenorphine (34 or 43 Ci/mmol, Amersham) was used. Five ktM of unlabeled etorphine were used to determine the amount of non-specific binding. To evaluate the relative proportions of the 3 opiate receptor subtypes - - p , b and ~c, respectively, point concentrations of unlabeled DAGO (Tyr-D-Ala-Gly-NMe-Phe-Glyol, 100 nM), DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen, 200 nM) and U-50,488 ([trans-(dl)-3,4-dichloro-N-methyl-N- {2-(1-pyrrolidinyl)cylcohexyl}-benzenacetamide methane sulfonate hydrate], 200 nM) were added to displace [3H]diprenorphine. The assessment of muscarinic receptors was carried out with [3H]quinuclidinyl benzilate ([3H]QNB, 42 and 44 Ci/mmol) and 10/~M of atropine as a displacer. After incubation, bound radioligand was separated by filtration through Whatman GF/B filters, washed 3 times with 5 ml Tris-HCI buffer and measured in a liquid scintillation counter with 40% efficiency. Binding was performed in duplicates which differed from one another by less than 15%. Protein was determined by the method of Bradford 9 with bovine serum albumin as a standard. Statistical analysis" One-, two- or three-way analysis of variance (GLM or ANOVA) was performed on cell counts; individual groups were compared using Newman-Keuls post-hoc comparisons or Student's t-test for independent samples. In binding studies paired t-test was applied in order to neutralize the variation between different cultures. RESULTS U n d e r t h e s t a n d a r d culturing c o n d i t i o n s u s e d in the p r e s e n t study - - s e r u m - f r e e m e d i u m and p o l y - o r n i t h i n e as a plating
substrate
--
striatal
cultures
consisted
p r i m a r i l y of n e u r o n a l cells. T h e i m m u n o s t a i n i n g of the cultures for G F A P after 10 days in v i t r o ( D I V ) i n d i c a t e d that astrocytes c o m p r i s e d less t h a n 5 % of the total cell population. A C h E - p o s i t i v e n e u r o n s c o u l d be first i d e n t i f i e d after 4 - 5 D I V , t h o u g h at this stage t h e staining of t h e cell bodies was pale and t h e n e u r o n a l p r o c e s s e s w e r e scanty. A f t e r an a d d i t i o n a l 5 days t h e intensity of A C h E labelling m a r k e d l y i n c r e a s e d and a l m o s t e v e r y stained cell possessed a n u m b e r of n e u r i t e s w h i c h c o u l d b e f o l l o w e d for a relatively l o n g d i s t a n c e (Fig. 1 A , B ) . In c u l t u r e s g r o w n
133
Fig. 1. Striatal neuronal cultures grown in serum-free medium on polyornithine and stained for AChE after 10 (A,B) and 30 (C,D,E) DIV. Stained cell bodies are indicated by arrows. See text for description. Bars: (A,B) = 30/zm; (C-E) = 200 ~m.
134 for 15 days, the n u m b e r of A C h E - p o s i t i v e cells was similar to that found on the 10th DIV, but the dendritic arborization was m o r e prominent. W h e n striatal cultures were kept for longer periods in serum-free medium their condition gradually d e t e r i o r a t e d , as judged by a disintegration of the neuronal network and cell necrosis. Nevertheless, the remaining A C h E - p o s i t i v e neurons in these cultures had even more d e v e l o p e d dendritic trees and their dendrites seemed to come into contact with numerous cell aggregates which could be distinguished from the surroundings by a d a r k e r brown staining (Fig. 1C-E). The morphological a p p e a r a n c e of A C h E - p o s i t i v e neurons suggested that the culturing conditions used here were a p p o p r i a t e for their differentiation; however, the n u m b e r of these neurons was lower than might be expected ( 0 . 0 5 - 0 . 1 % of seeded cells) given a high initial plating density of striatal cells (2.5-3 x 106 cells per plate). Consequently, we decided to investigate the influence of two additional variables which might promote survival/differentiation of neurons expressing ACHE. For this purpose 4 experimental conditions were used: striatal cells were seeded on astrocytes or polyornithine and kept in defined medium or in m e d i u m containing serum. As can be seen in Fig. 2, the n u m b e r of A C h E - p o s i t i v e cells was maximal when neurons were plated on astrocytes and grown in serum-free m e d i u m ,
I00
Fig. 3. Effect of veratridine on AChE-positive cells. Neuronal striatal cultures grown in serum-free medium on poly-ornithine were exposed to 5 #M of veratridine for 5 days (from 6 DIV to 10 DIV) and stained for AChE on day 10. The majority of AChE-containing cells in treated cultures apparently lost their neurites, so that many denuded cell bodies were observed (arrowheads); in certain cases degenerating processes could be seen (thin arrows). Bar = 50/zm.
while the lowest n u m b e r of these cells was seen in serum-free m e d i u m and in the absence of astrocytes. The values for cultures grown with serum were intermediate regardless of a substrate on which they were plated. A statistical analysis of these data by a two-way A N O V A revealed a significant effect of substrate (F1,32 = 33.5, P < 0.001 at 5 D I V ; F1,32 = 15.3, P < 0.001 at 10 D I V ) and a significant interaction b e t w e e n substrate and m e d i u m (F1,32 = 15.2, P < 0.001 at 5 D I V ; F1,32 = 23.1, P < 0.0001 at 10 D I V ) . A l t h o u g h the main effect
I0 DIV
80 SERUM-FREE 60
{3
40
20
w= u
i
Cells with
[ ~ 1 Cell b o d i e s without neudtes I
60 40 u
20
5 DtV
6O
oQ . i
LIJ
)l
SERUM
6C"
~" 4c z
neurites
N 4O
20'
-As
*As
i -As *Asj Z
Serum
-
-
Serum-free
Fig. 2. Effects of medium and astrocytes on the expression of AChE-positive cells after 5 and 10 days in culture. Cultures were grown either in serum-free or in serum-containing medium, on poly-ornithine or on a monolayer of astrocytes. Each bar represents mean + S.E.M. of 9 determinations from 3 independent experiments. * Significantly different from cultures grown in serum-free medium on poly-ornithine (Newman-Keuls comparisons, P < 0.05).
Verotridine
-
5 20
i
,i
-As
5 20 1
.
i
*As
Fig. 4. Effect of veratridine on the number of AChE-positive cells in striatal cultures grown under 4 possible combinations of medium and substrate (see Fig. 2 for details). Each bar represents mean +-_ S.E.M. of 8-9 determinations from 3 independent experiments. * Significantly different from control (Newman-Keuls comparisons,
P < 0.05).
135
Fig. 5. Immunostaining of neuronal striatal cultures with an antibody directed against neuronal specific enolase. Cultures were grown in serum-free medium on poly-ornithine for 10 days. A: control culture; B,C: cultures treated with 5/aM or 20/aM of veratridine, respectively (from 6 DIV to 10 DIV). Bar = 25/am.
of substrate reached statistical significance, it seemed to result primarily from an increase in AChE-positive cells number in a particular combination of serum-free medium and astrocytes. Post-hoc comparisons between the individual groups confirmed this impression, since no difference was found at any timepoint between cultures grown on different substrates and maintained in serum, while cell counts in cultures grown on astrocytes in serum-free medium were consistently higher than cell counts under any other experimental condition. The influence of neuronal activation on the expression of AChE-positive cells was evaluated by means of a prolonged chemical depolarization with a sodium channel agonist. Two concentrations of veratridine (5 and 20/aM) were applied for 5 consecutive days (from 5 D I V to 10 D I V ) , using the 4 combinations of medium and substrate as above. The inspection of the cultures under phasecontrast microscope during the period of treatment did not reveal any gross morphological changes or signs of neuronal degeneration. When chronically depolarized cultures were examined again after being stained for ACHE, it turned out that this treatment had a detrimental effect on AChE-positive cells, especially in serum-free medium. At 5 / a M of veratridine these cells seemed to lose their neurites so that many denuded cell bodies were observed (Fig. 3), which occasionally were surrounded by disintegrating neuronal processes. At a higher veratridine concentration of 20/aM, however, the number of stained cell bodies was also reduced. The effects of veratridine appeared to be selective, since, as has been mentioned
earlier, unstained neurons looked normal. To get a more quantitative estimate of the effect, AChE-positive cells were classified either as neurite-bearing cells or as denuded cell bodies, and neurons from both categories were counted (Fig. 4). It was found that 5 /aM of veratridine produced a drastic (6-fold) reduction in a number of cells with dendrites, without affecting cell body number. This effect was dependent on the medium, since the loss of neurites elicited by 5 /aM was less pronounced in cultures grown with serum. The statistical analysis by a three-way A N O V A (medium x substrate x t r e a t m e n t ) u s i n g two dependent
TABLE I Effects of chemical depolarization on opiate and muscarinic receptors
[3H]Diprenorphine and [3H]QNB were used at 2-3 nM and at 0.9-1.4 nM, respectively. Binding values are means ___S.E.M. based on 4-6 independent cultures. The asterisk indicates a value significantly different from control (paired t-test, P < 0.05). Parameter measured
Total [3H]diphrenorphine bound (fmol/mg protein) Apparent binding top 6 r [3H]QNB bound (fmol/mg protein) Protein Q.~g/lO6 seeded cells)
Control
Veratridine treatment O/aM)
(20~aM)
232 _+20
211 + 25
223 _+26
114_+5 52_+4 41+6
1 0 7 _ + 1 0 110+7 44_+7 41_+10 39_+8 37_+5
137 _+27 60 _+6
188 _+34* 64 _+6
106 _+19 54 -+ 5
136
=•
18)
IOC - -
~ 5 8c
*
4
(8}
~% 60 113) E._~ 4C o 13,..
2C
,
Ld .Iz
131
BRES 16000
Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization Julia Dymshitz 1, Rafael Malach 1, Shimon Amir I and Rabi Simantov 2 Departments of INeurobiology and 2Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot (lsrael)
(Accepted 15 May 1990) Key words: Striatal culture; Acetylcholinesterase; Chemical depolarization; Differentiation; Neuronal survival
The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 gM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20/~M) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCI levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2÷ channels - - verapamil (1/~M) and nifedipine (1/~M) - - did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 gM) had a partial effect. When cultures treated with 5 gM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed. INTRODUCTION The survival and differentiation of neurons in tissue culture is influenced by a number of environmental factors including contact with other neuronal and nonneuronal cells 1,2'32, presence of trophic substances in culture medium 3'34 and the level of neuronal activation 5' 10. Though the physiological significance of these factors during the histogenesis of the central nervous system is not established yet, the in vitro studies are indicative of the potentially important regulatory processes that might occur during normal development. The evidence for the role of the electrical activity in neuronal survival has been obtained using cultures of both peripheral 4"31 and central neurons 1°'18, where the depolarizing effects of neuronal activation were mimicked by elevation of the extraceUular potassium chloride (KCI) levels or by a long-term application of sodium channel agonists. Thus in cultures of cerebellar granule cells KCl-induced chemical depolarization promoted the neuronal survival without affecting the morphological differentiation 18. In another in vitro model the blockade of a spontaneous electrical activity of cultured spinal cord neurons with tetrodotoxin (q'TX) resulted in a reduction
in a number of cells containing choline acetyltransferase (CHAT) 1°. There is also extensive evidence that chemical depolarization may alter selectively the expression of neurotransmitter-related enzymes and peptides 6'14'15'17'23. The activity of the catecholamine-synthesizing enzyme - tyrosine hydroxylase - - was markedly increased by chronic treatment with the sodium channel agonist veratridine in explants of locus coeruleus 15 as well as in dissociated cultures of substantia nigra 17. Similarly, both the synthetic and the degradative enzymes for acetylcholine - - C h A T and acetylcholinesterase (ACHE) - - were found to be enhanced by depolarization in neuronal striatal cultures 25 and in cultures of muscle cells 14, respectively. On the other hand, the same treatment which induced an increase in C h A T activity in striatal cultures diminished the levels of substance P and of somatostatin in peptidergic neurons, indicating that the effects of chemical depolarization are differential with regard to different neuronal populations in the same brain structure 25. In a number of studies 7'13'35'39 A C h E histochemistry has been used to identify a distinct population of striatal neurons which could be classified into several types
Correspondence: J. Dymshitz. Present address: Department of Physiology, Indiana Univ. Med. Sch., 635 Barnhill Drive, Indianapolis, IN 46223, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
132 according
to their
morphology
and
the
intensity
of
staining 7. It has b e e n suggested that large heavily stained cells b e l o n g e d to a class of aspiny cholinergic striatal i n t e r n e u r o n s v. In line with this suggestion, a c o m p l e t e o v e r l a p b e t w e e n i m m u n o l a b e l l i n g for C h A T and staining for A C h E has b e e n f o u n d in the rat striatum in situ 16. In the p r e s e n t study we used an in vitro a p p r o a c h to follow the d e v e l o p m e n t of A C h E - p o s i t i v e n e u r o n s in p r i m a r y n e u r o n a l cultures of e m b r y o n i c s t r i a t u m and to investigate the influence of c h e m i c a l d e p o l a r i z a t i o n
on the
d i f f e r e n t i a t i o n and survival of these cells. MATERIALS AND METHODS
Tissue culture Sprague-Dawley rat fetuses (E15-E16) were dissected on ice under sterile conditions in Dulbecco's modified Eagle's medium (DMEM, Gibco) to which penicillin (10 units/ml), streptomycin (0.1 mg/ml) and antimycotic n-butyl-p-hydroxybenzoate (2 mg/ml) were added. Excised striata were incubated with 0.25% trypsin for 5 min at 37 °C and then washed 3 times with DMEM containing 50 #M of the protease inhibitor phenylmethylsulfonyl fluoride (Sigma). The tissue was dissociated into single cells by trituration with a fire-polished Pasteur pipette and then filtered through a 100 × 100 /~m stainless steel net. This procedure resulted in 90% yield of viable cells, as determined by Trypan blue exclusion test. Striatal cells were resuspended in DMEM supplemented with 10% horse serum (Gibco) and plated in Nunclon 35-mm dishes precoated with poly-ornithine (10 :~g/ml, Sigma). In some experiments a confluent layer of striatal astrocytes was used as a substrate for neuronal cells. The plating density was 2.5-3 × 106 cells per dish. When serum-free medium was used (DMEM supplemented with 5/~g/ml bovine insulin, 1 /~g/ml human transferrin, 20 nM hydrocortisone-21-phosphate, 30 nM triiodothyronine, 2/~g/ml carnithine, 3/~g/ml linoleic acid, 135/~g/ml choline chloride, 1.35/tg/ml vitamin B12, 1/~g/ml biotin and the trace elements as described24), it was added 24 h after plating, the corresponding cultures being washed with DMEM prior to medium change. Subsequently, the medium was changed every 4-5 days. Primary striatal astrocyte cultures were prepared from rat fetuses (E15-E16) and grown in DMEM supplemented with 10% horse serum in 60-mm Nunclon dishes precoated with poly-ornithine. The initial plating density was 1 × 106 cells per dish. When cultures reached confluence (usually on day 10-11) they were trypsinized and replated in 35-mm dishes at a density of 0.1-0.2 × 106 cells/dish. Culture medium was changed every 3-4 days. These secondary cultures became confluent after 6-8 days in vitro. AChE histochemistry Cultures were fixed for 20 min with 4% paraformaldehyde (in PBS), rinsed briefly with distilled water and stained for acetylcholinesterase according to a procedure described by Geneser-Jensen and Blackstad 19. Optimal staining was observed with relatively long incubation times (20 h) at room temperature, which allowed a good visual discrimination of fine neuronal processes in AChE-positive cells. The neurons stained for AChE were counted in two diameter bands (2.5 x 33 mm) of each culture dish using a binocular microscope (×50). In experiments with different treatments the cells were counted using a blind procedure. lmmunocytochemistry Immunocytochemistry was performed with primary antibodies (Incstar; 1:10) against glial fibrillary acidic protein (GFAP) and against neuron specific enolase (NSE) using Vectastain ABC method (Vector Laboratories). Briefly, the cultures were fixed as described above, washed with PBS 3 times and incubated with
primary antibody overnight in TBS (Tris-buffered saline, 50 mM Tris-HCl in 0.9% NaCI, pH = 7.4) containing 1% normal goat serum and 0.1% Triton X-100. After being washed twice with PBS the cultures were incubated for 30 min with the biotinylated anti-rabbit IgG and then washed again. The preformed avidinbiotinylated peroxidase complex was applied for 30 min, after which the peroxidase activity was visualized by a reaction with 0.05% 3,3"-diaminobenzidine tetrahydrochloride (Sigma) and 0.03% H202 in TBS.
Binding assay The cultured cells were washed twice with PBS, removed from the plates with a rubber policeman and collected by centrifugation in PBS at 1000 g for 5 min. The pellet was frozen at -20 °C and used for binding assay within two weeks from the time of cell collection. On the day of the assay the pellet was thawed, resuspended in 2-3 ml of 5 mM Tris-HCl buffer (pH 7.4) and after incubation for 10-15 min at 4 °C was homogenized with a Teflon-glass homogenizer (15 strokes). An equal volume of 0.64 M sucrose was then added and following centrifugation at 28,000 g for 15 min the binding assay was conducted as described 36. Briefly, the homogenates (0.2-0.6 mg protein/ml) were incubated with appropriate ligands for 40 min (opiate binding) or 60 min (muscarinic binding) at 25 °C in a final volume of 0.5 ml. For determination of total opiate specific binding [3H]diprenorphine (34 or 43 Ci/mmol, Amersham) was used. Five ktM of unlabeled etorphine were used to determine the amount of non-specific binding. To evaluate the relative proportions of the 3 opiate receptor subtypes - - p , b and ~c, respectively, point concentrations of unlabeled DAGO (Tyr-D-Ala-Gly-NMe-Phe-Glyol, 100 nM), DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen, 200 nM) and U-50,488 ([trans-(dl)-3,4-dichloro-N-methyl-N- {2-(1-pyrrolidinyl)cylcohexyl}-benzenacetamide methane sulfonate hydrate], 200 nM) were added to displace [3H]diprenorphine. The assessment of muscarinic receptors was carried out with [3H]quinuclidinyl benzilate ([3H]QNB, 42 and 44 Ci/mmol) and 10/~M of atropine as a displacer. After incubation, bound radioligand was separated by filtration through Whatman GF/B filters, washed 3 times with 5 ml Tris-HCI buffer and measured in a liquid scintillation counter with 40% efficiency. Binding was performed in duplicates which differed from one another by less than 15%. Protein was determined by the method of Bradford 9 with bovine serum albumin as a standard. Statistical analysis" One-, two- or three-way analysis of variance (GLM or ANOVA) was performed on cell counts; individual groups were compared using Newman-Keuls post-hoc comparisons or Student's t-test for independent samples. In binding studies paired t-test was applied in order to neutralize the variation between different cultures. RESULTS U n d e r t h e s t a n d a r d culturing c o n d i t i o n s u s e d in the p r e s e n t study - - s e r u m - f r e e m e d i u m and p o l y - o r n i t h i n e as a plating
substrate
--
striatal
cultures
consisted
p r i m a r i l y of n e u r o n a l cells. T h e i m m u n o s t a i n i n g of the cultures for G F A P after 10 days in v i t r o ( D I V ) i n d i c a t e d that astrocytes c o m p r i s e d less t h a n 5 % of the total cell population. A C h E - p o s i t i v e n e u r o n s c o u l d be first i d e n t i f i e d after 4 - 5 D I V , t h o u g h at this stage t h e staining of t h e cell bodies was pale and t h e n e u r o n a l p r o c e s s e s w e r e scanty. A f t e r an a d d i t i o n a l 5 days t h e intensity of A C h E labelling m a r k e d l y i n c r e a s e d and a l m o s t e v e r y stained cell possessed a n u m b e r of n e u r i t e s w h i c h c o u l d b e f o l l o w e d for a relatively l o n g d i s t a n c e (Fig. 1 A , B ) . In c u l t u r e s g r o w n
133
Fig. 1. Striatal neuronal cultures grown in serum-free medium on polyornithine and stained for AChE after 10 (A,B) and 30 (C,D,E) DIV. Stained cell bodies are indicated by arrows. See text for description. Bars: (A,B) = 30/zm; (C-E) = 200 ~m.
134 for 15 days, the n u m b e r of A C h E - p o s i t i v e cells was similar to that found on the 10th DIV, but the dendritic arborization was m o r e prominent. W h e n striatal cultures were kept for longer periods in serum-free medium their condition gradually d e t e r i o r a t e d , as judged by a disintegration of the neuronal network and cell necrosis. Nevertheless, the remaining A C h E - p o s i t i v e neurons in these cultures had even more d e v e l o p e d dendritic trees and their dendrites seemed to come into contact with numerous cell aggregates which could be distinguished from the surroundings by a d a r k e r brown staining (Fig. 1C-E). The morphological a p p e a r a n c e of A C h E - p o s i t i v e neurons suggested that the culturing conditions used here were a p p o p r i a t e for their differentiation; however, the n u m b e r of these neurons was lower than might be expected ( 0 . 0 5 - 0 . 1 % of seeded cells) given a high initial plating density of striatal cells (2.5-3 x 106 cells per plate). Consequently, we decided to investigate the influence of two additional variables which might promote survival/differentiation of neurons expressing ACHE. For this purpose 4 experimental conditions were used: striatal cells were seeded on astrocytes or polyornithine and kept in defined medium or in m e d i u m containing serum. As can be seen in Fig. 2, the n u m b e r of A C h E - p o s i t i v e cells was maximal when neurons were plated on astrocytes and grown in serum-free m e d i u m ,
I00
Fig. 3. Effect of veratridine on AChE-positive cells. Neuronal striatal cultures grown in serum-free medium on poly-ornithine were exposed to 5 #M of veratridine for 5 days (from 6 DIV to 10 DIV) and stained for AChE on day 10. The majority of AChE-containing cells in treated cultures apparently lost their neurites, so that many denuded cell bodies were observed (arrowheads); in certain cases degenerating processes could be seen (thin arrows). Bar = 50/zm.
while the lowest n u m b e r of these cells was seen in serum-free m e d i u m and in the absence of astrocytes. The values for cultures grown with serum were intermediate regardless of a substrate on which they were plated. A statistical analysis of these data by a two-way A N O V A revealed a significant effect of substrate (F1,32 = 33.5, P < 0.001 at 5 D I V ; F1,32 = 15.3, P < 0.001 at 10 D I V ) and a significant interaction b e t w e e n substrate and m e d i u m (F1,32 = 15.2, P < 0.001 at 5 D I V ; F1,32 = 23.1, P < 0.0001 at 10 D I V ) . A l t h o u g h the main effect
I0 DIV
80 SERUM-FREE 60
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20
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Cells with
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20
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SERUM
6C"
~" 4c z
neurites
N 4O
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-As
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Serum
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-
Serum-free
Fig. 2. Effects of medium and astrocytes on the expression of AChE-positive cells after 5 and 10 days in culture. Cultures were grown either in serum-free or in serum-containing medium, on poly-ornithine or on a monolayer of astrocytes. Each bar represents mean + S.E.M. of 9 determinations from 3 independent experiments. * Significantly different from cultures grown in serum-free medium on poly-ornithine (Newman-Keuls comparisons, P < 0.05).
Verotridine
-
5 20
i
,i
-As
5 20 1
.
i
*As
Fig. 4. Effect of veratridine on the number of AChE-positive cells in striatal cultures grown under 4 possible combinations of medium and substrate (see Fig. 2 for details). Each bar represents mean +-_ S.E.M. of 8-9 determinations from 3 independent experiments. * Significantly different from control (Newman-Keuls comparisons,
P < 0.05).
135
Fig. 5. Immunostaining of neuronal striatal cultures with an antibody directed against neuronal specific enolase. Cultures were grown in serum-free medium on poly-ornithine for 10 days. A: control culture; B,C: cultures treated with 5/aM or 20/aM of veratridine, respectively (from 6 DIV to 10 DIV). Bar = 25/am.
of substrate reached statistical significance, it seemed to result primarily from an increase in AChE-positive cells number in a particular combination of serum-free medium and astrocytes. Post-hoc comparisons between the individual groups confirmed this impression, since no difference was found at any timepoint between cultures grown on different substrates and maintained in serum, while cell counts in cultures grown on astrocytes in serum-free medium were consistently higher than cell counts under any other experimental condition. The influence of neuronal activation on the expression of AChE-positive cells was evaluated by means of a prolonged chemical depolarization with a sodium channel agonist. Two concentrations of veratridine (5 and 20/aM) were applied for 5 consecutive days (from 5 D I V to 10 D I V ) , using the 4 combinations of medium and substrate as above. The inspection of the cultures under phasecontrast microscope during the period of treatment did not reveal any gross morphological changes or signs of neuronal degeneration. When chronically depolarized cultures were examined again after being stained for ACHE, it turned out that this treatment had a detrimental effect on AChE-positive cells, especially in serum-free medium. At 5 / a M of veratridine these cells seemed to lose their neurites so that many denuded cell bodies were observed (Fig. 3), which occasionally were surrounded by disintegrating neuronal processes. At a higher veratridine concentration of 20/aM, however, the number of stained cell bodies was also reduced. The effects of veratridine appeared to be selective, since, as has been mentioned
earlier, unstained neurons looked normal. To get a more quantitative estimate of the effect, AChE-positive cells were classified either as neurite-bearing cells or as denuded cell bodies, and neurons from both categories were counted (Fig. 4). It was found that 5 /aM of veratridine produced a drastic (6-fold) reduction in a number of cells with dendrites, without affecting cell body number. This effect was dependent on the medium, since the loss of neurites elicited by 5 /aM was less pronounced in cultures grown with serum. The statistical analysis by a three-way A N O V A (medium x substrate x t r e a t m e n t ) u s i n g two dependent
TABLE I Effects of chemical depolarization on opiate and muscarinic receptors
[3H]Diprenorphine and [3H]QNB were used at 2-3 nM and at 0.9-1.4 nM, respectively. Binding values are means ___S.E.M. based on 4-6 independent cultures. The asterisk indicates a value significantly different from control (paired t-test, P < 0.05). Parameter measured
Total [3H]diphrenorphine bound (fmol/mg protein) Apparent binding top 6 r [3H]QNB bound (fmol/mg protein) Protein Q.~g/lO6 seeded cells)
Control
Veratridine treatment O/aM)
(20~aM)
232 _+20
211 + 25
223 _+26
114_+5 52_+4 41+6
1 0 7 _ + 1 0 110+7 44_+7 41_+10 39_+8 37_+5
137 _+27 60 _+6
188 _+34* 64 _+6
106 _+19 54 -+ 5
136
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