Brain Research, 153 (1978) 55-77 (~ Elsevier/North-Holland Biomedical Press

55

E N Z Y M A T I C A N D M O R P H O L O G I C A L PROPERTIES OF PRIMARY RAT BRAIN ASTROCYTE CULTURES, AND E N Z Y M E D E V E L O P M E N T IN V1VO

H. K. K1MELBERG, S. NARUMI* and R. S. BOURKE Division of Neurosurgery and Department o] Biochemistr.v, Albany Medical College, Albany, N.Y. 12208 (U.S.A.)

(Accepted January 4th, 1978)

SUMMARY The development of (Na ~-~-K +) ATPase, carbonic anhydrase and HCO3--stimulated ATPase activity was studied in developing rat brain in vivo, and in primary astrocyte cultures from 1-3-day-old rat brain as a function of increasing cell growth. The primary cultures showed an increase in all the above enzyme activities during cell growth, with time courses which were qualitatively similar to their development in vivo. Cell cultures grown separately from the cerebellum plus brain stem regions showed greater carbonic anhydrase activity than cerebral cultures over the entire 4week growth period, corresponding to development of this activity in these same regions in vivo. HCO3--stimulated ATPase activity was slightly greater in cerebellar cultures and (Na ~÷ K +) ATPase activity was greater in cerebral cultures up to the second week of growth, resembling development of the same enzyme activities in vivo. C6 glioma and neuroblastoma cells showed no and 10-fold lower carbonic anhydrase activities respectively, compared to the primary astrocyte cultures. Addition of l mMN6-2'-O-dibutyryladenosine-3',5'-monophosphate(DBcAM P) in the presence of serum caused marked formation of cellular processes and increased carbonic anhydrase and (Na ~ + K +) ATPase activity. Maximum effects were found 2 h after addition of 1 m M DBcAMP and thereafter declined. In the absence of serum such effects persisted for at least 24 h. Electron microscope studies showed large numbers of microtubule ( ~ 20 nm diameter) and filamentous structures (~< 10 nm diameter) in the cytoplasm, which showed changes in distribution in cells treated with DBcAMP. This study suggests that the increase in ATPase and carbonic anhydrase activities in rat brain with increasing age may be in part a reflection of proliferation and development of astroglia cells. Together with the morphological data, it also * Present address: Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd. Yodogawaku, Osaka 532, Japan.

56 provides additional evidence that primary cultures derived from neonatal rats may closely resemble developing astroglia in vivo.

INTRODUCTION There is currently considerable interest in the functions of astroglial cells in the central nervous system. In the mature brain they may well be involved in the modulation of neuronal activity, and changes in such activity could be sensed by astrogha cells through alterations in the extracellular environment. One important constituent of the extracellular fluid that astroglia are responsive to is K ~. and levels of this ion can increase up to l0 mM during intense neuronal activity 20, and up to 70 m M during hypoxia and ischemia 57,5a. Our interest has focused on the responsiveness o f astroglial cells to extracellular K ~, and it has been found that K ~ levels greater than tO mM cause swelling both in vivo and in vitro. This swelling involves intracellular uptake of K ~', Na ~ and C l - , and has been localized to astroglia 7-1°,22,48. Previous studies a-t° suggested that carbonic anhydrase and (Na+q-K ~) ATPase, both of which have been reported to be present in higher levels in glial cells as compared to neurons17,21.a6,a% ~o. and possibly a HCO3--stimulated ATPase found in brain 2a as well as othel tissues (see refs. 14, 24 and references therein), may be involved. One problem with studying astroglial cells in the intact mammalian central nervous system or brain slices has been in clearly distinguishing the responses of the glial from the neuronal compartment. Use of bulk-isolated neuronal perikarya and glial cells has proved of some use. These preparations do have certain drawbacks, however, such as low yield, possibilities of contamination with other fractions and damage during tissue disruption and isolation, which may involve both alterations of membrane permeability and loss of cell processes a6. Cells grown in tissue culture answer many of these problems, but it must still be established how closely they resemble the related differentiated cell in vivo. It therefore seems likely that at present most information will result from studies using and comparing all the available preparations. A further approach to distinguishing glial and neuronal properties is a developmental one, which can be conveniently examined in the rat, where both astrogila proliferation16, e2 and K~-induced swelling 1°,48 develop in brain cortex within a 10-30-day period after birth. Previous studies have also reported that, in the rat, carbonic anhydrase 4 and (Na ~ - K +) ATPase activity 1,47 increases with increasing age. We have therefore, in this study, compared the appearance of carbonic anhydrase, (Na t q-K +) ATPase and HCO3--stimulated ATPase activity in developing rat brain with the appearance of the same enzymic activities in primary astrogtial cultures. Under normal conaitions, however, these cultures consist of cells which have a predominantly fiat, polygonal shape, unlike that of mature astrocytes in vivo. They are therefore often referred to as astroglial precursors or astroblasts a4,u8,52.56. Upon addition of adenosine 3',5'-monophosphate (cAMP) analogues a°'al'3a'~a'a~'Sa'Sa or transforming factors aa,a4,52 extracted from rat brain, however, these cultures show

57 marked and sometimes rapid alterations in morphology to cells possessing numerous processes more closely resembling astrocytes in vivo. It was thus of interest to see if such morphological transformation was accompanied by significant alterations in enzyme activities. METHODS AND MATERIALS

Preparation and homogenization of bra#t tissue Late stage Sprague-Dawley pregnant rats were obtained from commercial suppliers. After birth the offspring were decapitated at various ages. The entire cerebrum, cerebellum and brain stem was removed, rinsed once in 0.32 M sucrose, blotted dry and then homogenized as a 10 ~, w/v suspension in 0.32 M sucrose using a Potter Eveljehn homogenizer. Protein was determined by the method of Lowry et al. 35. Aliquots of this suspension were assayed directly. In some experiments the cerebrum and cerebellum plus brain stem were homogenized separately. Cell culture Primary cell cultures were set up from the brains of 1-3-day-old Sprague-Dawley rats essentially according to the method of Booher and Sensenbrenner 6 and Schousboe et al. 49. This procedure has been reported to result in predominantly pure cultures of undifferentiated astroglia after 2-3 weeks in culture 6,38,49,5e. Our procedure involved removal of the whole brain under sterile conditions from 4-6 rats. The brains were then placed in a total volume of 10 ml of 0 . 2 5 ~ trypsin at 37 °C. The whole procedure was performed in a Baker Biogard laminar flow hood. The tissue was cut up with scissors and then left for 10 rain at room temperature. For some experiments the cerebrum and cerebellum plus brain stem were cultured separately. Mesh size 73 ,um nylon bolting cloth (Tobler, Ernst and Traber, Elmsford, N.Y.) was secured to the cut, open end of a plastic, sterile 20 ml syringe and the trypsinized brain mixture was slowly pushed through the syringe. The filtered mixture was then brought to a final volume of 200 ml for 6 brains (for T flasks) or 250 ml for 4 brains (for Petri dishes) with Eagle's Basal Medium (BME) containing glutamine, Earle's salts, and 26 mM NaHCO3 (G1BCO cat. no. 101 G). The medium was supplemented with the following additions; 20 ~ fetal calf serum (FCS), 4 ~ (100 x ) BME vitamin solution, 2 ~o (100 ×) BME amino acids, l ~ 0.7 M glucose and 1 ~ penicillin (10,000 units/ml)streptomycin (10,000 #g/ml). All per cent values are in v/v. The 200 ml of brain suspension was then plated in twenty 75 sq.cm T-flasks (Corning) and the 250 ml suspension was plated in fifty 20 sq.cm Petri dishes (Corning). Media was initially changed 24 h after first plating the flasks, and thereafter twice weekly. The cultures were grown at 37 °C in a 95 ~ air/5 ~ CO2 humidified incubator. Cells were seeded at an initial density of l05 cells/sq.cm, but the initial media change removed a large proportion of the cells, since on the fifth day of growth only 3-4 × 103 cells/sq.cm were present. After 28 days in culture the density had almost levelled off to around 4 × 105 cells/sq.cm (0.02 mg protein). In such primary cultures considerable variability in cell density of around 20~/o was found, even though the

58 number of brains seeded per flask or dish was kept constant. Cells were removed from culture flasks by treating them for 10-20 min with 0.25 9o trypsin (GIBCO), or by scraping with a rubber policeman. The cells were then centrifuged in complete media in a bench top centrifuge. For enzyme assays the cells were washed twice in 0.32 M sucrose and then sonicated for 1.5 min in 1-2 ml 0.32 M sucrose in a test tube immersed in a bath-type sonicator (Cole-Parmer, 80 W). When cells were treated with DBcAMP growth media was replaced with complete media containing DBcAMP without FCS 24 h before examination. For short-term treatment in media containing 20~,,, FCS, medium was changed 24 h before I m M DBcAMP was added. Cell cultures were examined using a Nikon Model MS phase contrast inverted microscope and photographed with the EFM 35 m m camera attachment.

Enzyme assays (Na + ~-K ~) ATPase activity was the activity inhibited by 1 m M ouabain at pH 7.5 and Mg z~ ATPase activity was the ouabain-msensitive component. Carbonic anhydrase activity was determined by measuring decreasing p H due to COz hydration in a veronal buffer at p H 8.3. HCO--z ATPase activity was the Mg 2 ~-dependent activity stimulated by 20 m M NaHCO3 at p H 8.5. The methods for measuring these activities have all been described in detail elsewhere 8,2~-z9 Blood content of brain Blood content of brain was measured by intracardiac injection of 0.2/~Ci of l~5llabelled albumin in a total volume of 0.05 ml of Hank's balanced salt solution (GIBCO). After 20-30 min the animal was decapitated and blood immediately collected from the body by holding the cut neck over heparinized tubes. The brain and brain stem were then removed and rinsed according to the same procedure used for preparing tissue for enzymatic assay. Portions of tissue of around 100 mg were digested with protosol. Aliquots of blood of 0.1 ml were digested with protosol and decolorized with HzO2. The [125I]albumin content of brain tissue and blood was then counted in a Packard 3330 liquid scintillation counter, correcting for quenching using the external standard. Electron microscopy Media was poured off cells growing in Petri dishes and they were then fixed in 5 ~, glutaraldehyde for l h. washed twice in sucrose buffer, postfixed in Millonig's osmium tetroxide 4a. washed 3 times in buffer, and stained in 2?/0 aqueous uranyl acetate for 20 min. The cells were washed 3 times in water and then run through graded alcohols (35~o, 50.%, 75~0, 9 0 ~ and 100~) for 5 min each. They were then washed 3 times for 10 min each with an Epon mixture and left in a 37 ~C oven overnight in Epon, and then placed in a 60 °C oven for 24 h. After this the Epon was removed from each dish. A cork borer was used to remove small areas of the cultured cells, which were then mounted on Epon blocks for sectioning using a Dupont-Sorvall Type MT2 'Porter-Blum' Ultra microtome. Silver-gray sections were picked up on 200 mesh copper grids (E. F. Fullam, Schenectady, N.Y.). Sections were stained with 2 o:~,

59 uranyl acetate in 100 % methanol for 15 rain, rinsed in distilled water and stained with lead citrate 44 for 25 min. They were then rinsed and dried. Grids were viewed on a JOEL Electron Microscope 100B (Japan Electron Optics Laboratory, Tokyo).

Materials Crystalline ATP (disodium or Tris salt, Sigma grade, from equine muscle) and ouabain were obtained from Sigma Chemical (St. Louis, Mo.). Sucrose and Tris base were ultrapure reagents flora Schwarz/Mann (Orangeburg, N.Y.). DBcAMP monosodium salt and sodium butyrate (anhydrous n-butyric acid) neutralized by NaHCO3 were from Sigma Chemical. All other compounds were at least of analyzed, reagent grade quality. Tissue culture media was from Grand Island Biologicals (GIBCO), Grand Island, N.Y. and Corning, disposable plastic tissue ware was obtained from Fisher Chemical. RESULTS

Development of A TPase and carbonic anhydrase activity in vivo Fig. IA shows the increase in the (Na+ + K +) ATPase specific activity and the

,4,2I

B

[

A

MgP-ATPase

,21

k.

~.

8

~

4

u~

~-~ 'K[

(No+-K*)-ATPose

; ,6

2

io A6E (DAYS)

WEEKS AFTER BIRTH Fig. 1. D e v e l o p m e n t of ( N a + 4 K +) a n d Mg 2 ~ A T P a s e activity ill rat brain. A : whole rat brains including cerebellum a n d brain stem were r e m o v e d a n d h o m o g e n i z e d as described in Materials a n d Methods. ( N a + + K +) A T P a s e activity was the activity inhibited by 1 m M o u a b a i n a n d the M g 2T activity was the ouabain-insensitive c o m p o n e n t . A s s a y o f the h o m o g e n a t e after t r e a t m e n t with 0.06 % deoxycholate for 20 rain at r o o m temperature, followed by a 30-fold dilution in the reaction m e d i u m , caused a I. 5-fold increase in activity, but did n o t change the pattern of the developing activity. Activities are s h o w n as the m e a n ~_ S.E.M. where n - 3-4. O , (Na + + K +) A T P a s e ; /,, Mg2+-ATPase. B: (Na + ÷ K ~) A T P a s e activities were determined on h o m o g e n a t e s o f the separated cerebrum and cerebellum Flus brain stem regions. T h e age o f the animals t r o m which brains were removed, given in weeks, refers to the last d a y of the period, i.e. 7, 14, 2l a n d 28 days after birth. Values s h o w n are m e a n s ± S.E.M. (n 3-4). O , cerebellum ~ brain s t e m ; A , cerebrum.

60 ouabain-insensitive M g 2+ A T P a s e activity in whole brain with increasing age. There was a r e p r o d u c i b l y higher ( N a + - ~ K +) A T P a s e activity on the first d a y a f t e r b i r t h , b u t the activity then d r o p p e d to a level t h a t was three-fold lower t h a n this o n d a y 2, A sharp increase in activity was first seen between d a y 6 a n d 8, which then c o n t i n u e d up to 18 days a n d r e m a i n e d essentially c o n s t a n t for at least 80 d a y s after birth. T h e M g z~ A T P a s e activity was higher b e t w e e n d a y s 1 a n d 6 a n d did n o t show the earl) increase between d a y s 6 a n d 8, b u t did show a rise in activity b e t w e e n days 8 a n d 20. The ( N a t. -~ K +) A T P a s e activity was increased a b o u t 50 ~o when the h o m o g e n a t e s were p r e t r e a t e d with 0.06 ~o (v/v) d e o x y c h o l a t e for 20 min at r o o m t e m p e r a t u r e a n d then d i l u t e d 30fold in the reaction m e d i u m 25, but the p a t t e r n o f the d e v e l o p i n g activity was unchanged. A t later times a slight increased activity was seen in the d e v e l o p m e n t o f ( N a + 4 - K +) A T P a s e in the cerebellum plus b r a i n stem c o m p a r e d to the cerebral region (Fig. IB), b u t was otherwise t h e same. M e a s u r e m e n t o f c a r b o n i c a n h y d r a s e activity in whole brain is c o m p l i c a t e d by the high c o n t e n t o f c a r b o n i c a n h y d r a s e in r e d b l o o d cells. The specific activity o f red b l o o d cells, as shown in Table 1. is a b o u t 10-fold greater t h a n the activity in nonperfused b r a i n tissue after 14 days ( c o m p a r e Fig. 6 a n d Table I), a n d the differences were even greater at earlier ages. Since it is very difficult to perfuse very y o u n g rats to remove b l o o d from the cerebral vasculature, we decided to d e t e r m i n e w h e t h e r there was any variation o f b o t h b l o o d c a r b o n i c a n h y d r a s e activity a n d b l o o d c o n t e n t o f b r a i n with increasing age. T a b l e I shows t h a t the specific activity o f c a r b o n i c a n h y d r a s e in b l o o d is essentially c o n s t a n t for up to at least 60 days, and t h a t the a p p a r e n t b l o o d c o n t e n t o f b o t h c e r e b r u m a n d cerebellum plus b r a i n stem based on [125I]-albumin c o n t e n t was a b o u t t w o - f o l d greater on a w w basis at early times (up to

TABLE 1 Carbonic anhydrase activity in blood and blood content o f developing rat brain

As described in Materials and Methods, ml of whole blood/g wet wt of brain tissue was determined 20-30 min after intracardial injection of [125Ilalbumin; mg blood protein/rag brain protein was calculated from this based on 8 and 20 ~ protein per g wet wt of both cerebrum and cerebellum and per ml of blood respectively at both days 7 and 14. Values means -z S.E.M. where indicated. Days after birth

5 8 10 15 29 60

Whole blood carbonic anhydrase activity (tool CO~' 1/see/mg protein) 10 z

ml Whole blood/g wet wt o f brain tissue

259 205 208 247 241 323

0.020 0.019 0.019 0.011 0.011

mg BIood protein/ ¢ng brain protein

Per cent carbonic anhydrase activity due to bloodcontent

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cerebrum

cerebellum • brain stem

± 0.007 0.038 ~_ 0.007 z: 0.008 0.021 ~ 0,007 ± 0.004 0.022 ~z 0.011 0.012 d- 0.005 0.019 ~ 0.003

eerebruin

cerebellum cere- brain bruin stem

cerebellure -brain ~tem

0.050 0.048 0.048 0.027 0.027

0.095 0.053 0.056 0.029 0.047

100 75 45 32 22

100 91 71 62 29

61 10 days) as compared to later times, presumably related to development of the blood-brain barrieH 5. Taken together, these data suggest that the increasing carbonic anhydrase activity o f developing rat brain was not due to increasing carbonic anhydrase activity of blood or increasing blood content of brain. In order to subtract the carbonic anhydrase activity expressed per mg blood protein to obtain corrected values for brain activity, the relative protein concentrations per g wet wt of brain and per ml of whole blood had to be determined, and were found to be 8 ~),/, and 20 °,~i respectively. The per cent of blood content in mg blood protein/mg brain protein is thus 2.5-fold greater than the v/w figures as shown in Table I. The actual percentage contributions o f blood carbonic anhydrase activity to total brain activity can then be calculated from the specific activities, and are shown in Table I for increasing ages. The carbonic anhydrase activities of separated cerebrum and cerebellum plus brain stem regions, corrected for activity due to blood content using the preceding data, are shown in Fig. 2A. Since the blood content was only determined for 5 time periods, as shown in Table I, each value has been assumed to represent the entire time period between it and the previous value. The figure at day 29 is also used to calculate the blood content at 60 days. It can be seen that activity attributable to brain tissue only appears at around day 7. A gradual increase in activity up to day 29 is seen for both regions, but the activity in the cerebellar and brain stem region is 3 4 times

,7-.--

~.

4

B

CEREBELLUM~ 4°IAEBE'UM2 CE

30j BR:NT

2O

0

/5 2'o

3'o"

AGE (DAYS) WEEKS AFTER BERTH Fig. 2. Increasing carbonic anhydrase and HCOa -stimulated ATPase activity in cerebrum and cerebellum plus brain stem regions. A : the carbonic anhydrase activity shown is the activity in total homogenates of cerebrum and cerebellum plus brain stem with the activity calculated as due to blood content (Table I) subtracted. Values shown are means ± S.E.M. (n 3 4). B: HCOa -stimulated ATPase activity was determined, as described in Materials and Methods, for total homogenates of the separated cerebrum and cerebellum plus brain stem regions.

62 greater than that from the cerebrum. Between days 29 and 60 the cerebellar and brain stem activity declines, while the cerebrum activity still increases. Fig. 2B shows increasing HCO3--stimulated ATPase activity in the cerebrum and cerebellum plus brain stem region with age. The cerebellum plus br~tin stem also shows greater activity up to 3 weeks after birth. Thereafter, like carbonic anhydrase, H C O z - ATPase activity declines :in the cerebellum and brain stem area, becoming equivalent to the cerebral activity.

Development of A TPase and carbonic anhydrase activities in culture The growth of primary cultures from t-3-day-old rat brain in T flasks is shown in Fig. 3, where growth in terms of total protein is compared for both a single rat brain in vivo and for cultured cells in vitro derived f r o m one rat brain, Duration of time in both cases is referred to the same zero time point of birth of the animal, which for the astrocytes is also equivalent to time in culture. The cultured cells have around 10-fold less protein content than whole brain in vivo but the rate of growth is quite similar. Fig. 4 shows the (Na ~ + K ') ATPase (lower panel) and ouabain-insensitive Mg 2 ATPase (upper panel) activity of cultured astrocytes after increasing times in culture. The development of the same activities in rat brain in vivo is also shown for comparison. In the case of the (Na ~+ K ~) ATPase, it can be seen that there is a similar increase both in vitro and in vivo between the first and second weeks (14 days), with the specific activity of the cells being about one-half that of the brain in vivo (note difference in scales). However, in contrast to the behavior in vivo, after the second week the activity of cultured astrocytes declined, falling to about one-tenth of the in

~

100

s.r ,oEz

2~ C.3

CULTURED

RAT BRAIN

~ 2o

-.q

".M

o i

4 WEEKS AFTER BIRTH

Fig. 3. Comparison of growth of one rat brain in vivo and of cultured astroeytesderived from one brain~ The whole brain was removed from animals at the various times indicated after birth, homogenized and its total protein content determined. The growth in the total protein content of primarycuttures derived from a single one-day-old rat brain was also determined. All values are means i S.E.M. (n = 3-4).

63

(Mg2+ATPase), ~

10

12

.g::

IO

~2

~

4 I

I

I

I

k ~5 k

g..

2~ .-...

2

4o

o I WEEKS AFTER BIRTH

Fig. 4. Comparison of the development of Mg 2+ ATPase (upper panel) and (Na + ÷ K +) ATPase (lower panel) activity in whole rat brain in vivo and primary astrocytes cultured from whole brain. Activity in rat brain was determined on homogenates of whole brain and derived from the same data as in Fig. 1A. Primary cultures were started from whole brain and activities determined on sonicates of whole cells as described in Materials and Methods. The times shown are for the same time in culture as the age of the rat for the in vivo experiments. The (Na + + K +) ATPase activities shown were increased proportionally by about 50 % by pretreatment with 0.06 % deoxycholate for both brain tissue and cultured cells as described in Fig. 1.

vivo activity at 4 weeks. The Mg 2+ ATPase activity showed a similar development both in vivo and in vitro, but with no decline in the activity in vitro at later times. The percentage of the total astrocyte ATPase activity that was ouabain-sensitive dropped from 30 % in the second week of growth to 13 % in the fourth week. In contrast, the percentage of the total ATPase activity of rat brain in vivo that was ouabain-sensitive reached a maximum of 54 % in the fourth week. After trypsinization and replating, the astrocyte cultures showed a constant low specific activity for the (Na+-FK +) ATPase of 0.5-1.0 #moles Pi/mg/h. The ( N a + + K +) ATPase activity was also found to vary

64 (rag protein/crn2)x10-2 0.5

1.0

,

1.5

i

2.0

i

2.5 ,

~b

I

1

I

2

I

I

I

I

I

3 4 5 6 7 mg protein/4 T FLASKS

8

Fig. 5 Variation of (Na ÷ ÷ K ÷) ATPase activity with varying cell densities in two-week astrocyte cultures. Activities of whole homogenates of different two-week-old astrocyte cultures, at varying celI densities as indicated by protein content, were determined as described in Materials a n d Methods. The lower scale refers to the total amount of protein meaSured from four 75 sq.cm T-flasks a n d t h e upl~er scale shows the same values expressed per sq.cm of growing area.

with the density of cells at a fixed growth time. This is shown in Fig. 5, where activity is plotted as a function of the varying density of cells as determined by protein content for a number of different two-week cultures. The activity was measured in all cases at the optimum pH of 7.5 using homogenized, fresh cells. The range of activities seen are similar to those reported by others 49. Carbonic anhydrase activity also showed a rise in activity in cultured astrocytes with increasing growth, the maximum activity occuring at 3 weeks after seeding (Fig.

B

A 4o

so

CEREBELLUM*~ /

TU R EY DTE~/ AC SU TL R O C

BRAIN STEM/

K

:I

\

/

\

I

QI

~,

o

I WEEKS AFTER BIRTH

2

3

4

W E E K S IN CULTURE

Fig. 6. Comparison of the development of carbonic anhydrase activity in vivo and in vitro, A: carbonic anhydrase activity was determined at the times shown on homogenates of whole rat brain or cultured astrocytes, from non-perfused whole rat brain. Values shown are means ::: S.E.M. (n - 3 ~ ) . B: carbonic anhydrase activity was determined on homogenates o f ceils cultured from theeerebrum or cerebellum plus brain stem regions o f one-day-old rats. Values shown are means ~: S.E.M. (n =: 3).

65 6A). The specific carbonic anhydrase activity is about one-fifth of that found in whole brain tissue in vivo between 2 and 4 weeks after seeding, when the contribution due to blood (see Table I) is subtracted from the in vivo brain values. At one week the difference between the activity in cells and net brain activity is only two-fold. The carbonic anhydrase activity was also quite labile, and after trypsinization and replating the secondary cultures showed no detectable activity. Fig. 6B shows the increase in carbonic anhydrase specific activity in separate astrocyte cultures from the cerebrum, and cerebellum plus brain stem regions. It can be seen that the increased activity oftBsue obtained from the cerebellum plus brain stem region for up to 28 days after birth (see Fig. 2A) is duplicated in the astrocyte cultures also derived from these two regions. Furthermore, the decline in cerebellar plus brain stem activity seen at later times in vivo also occurs in the in vitro cell culture system. The increased HCO3- ATPase activity in the cerebellum plus brain stem at earlier times, and a similar activity at later times seen in vivo (Fig. 2B), was also seen in cultured ceils (Fig. 7A), although the differences were not as marked in the latter case. In contrast, the ( N a + + K +) ATPase activity was higher in astrocyte cell cultures derived from the cerebrum compared to cerebellum plus brain stem during the second week of culture, but were similar at later times (Fig. 7B). The enzymatic activities of cultured astrocytes from cerebrum and cerebellum plus brain stem were compared to the corresponding activities in two cloned, permanent cell lines of neural origin, as shown in Table II. It can be seen that,

3..

2.0 B

4 ICEREBELLUM. %

p__.~ ',q:~

A

_.Q

BRAIN S T E M

RUM

6.5

,q:u

b.

--~

/CEREBELLUM /*BRAIN STEM

m-. %

0

I

3 4 WEEKS IN CULTURE

~

~.

0.5

2

WEEKS IN CULTURE

Fig. 7. D e v e l o p m e n t of HCO3 - a n d ( N a ~- -F K+)-stimulated A T P a s e activity in primary cultures from c e r e b r u m a n d cerebellum plus brain stem tissue. A : H C O ~ -stimulated A T P a s e activity was determined as described in Materials a n d M e t h o d s on cells grown f r o m the two separate brain regions. B : ( N a + + K+)-stimulated A T P a s e activity. Otherwise s a m e conditions as for A.

66 TABLE II Carbonic anhydrase activities of cultured cells under different growth conditions

Where indicated, serum-free media containing 1 mM DBcAM P was added to the cells which were then grown for 24 h before the activity determinations. Astrocytes were grown from Separate cerebral and cerebellar regions as indicated. Data is the mean of 2 determinations. HCO3- ATPase was the Mg'-'~dependent activity stimulated by 20 mMNaHCO3 at pH 8.5 and (Na + + K ÷) ATPase was the activity at pH 7.5.in the presence of Mg2+ + Na ~ + K + inhibited by 1 mMouabain, All determinations were done on fresh, homogenized cells. The total protein refers to the amount of cell protein per four 75 sq.cm T-flasks. Cell type

Rat astrocytes from cerebrum -~-DBcA M P) --Serum) Rat astrocytes from cerebellum -{ brain stem +DBcAMP) --Serum)

Total Cell protein/ density/ 4 T-flasks sq.cm

Carbonic anhydrase (mole C02/1/sec/mg protein) ,~ 10 -5

ATPase (itmoles/Pi/mg protein~h) ................ Mg 2+ (Na ~ ! K ~) HC03

7.0

92,667

1.5

9.0

1.8

():8

3.5

46,000

2.2

4.1

1.8

1.2

8.9

118,000

5.4

8.9

1.4

0.8

3.7

48,667

8.2

12.9

1.2

0.9

0

4.9

2.3

0.9

0

4.8

3.6

1.3

0.3

4.0

0.2

0.2

Rat glioma (C6) 5.7 +DBcAMP) --Serum) 2.8 Mouse neuroblastoma (NB4IA3) 13.8

although b o t h the primary and transformed glial lines show comparable ( N a ' ~-K ~) and HCO-3-stimulated ATPase activities, only the primary astrocyte cultures show carbonic anhydrase activity. Also, no carbonic anhydrase activity in the rat glioma line was detected after pretreatment with dibutyryl c A M P plus serum withdrawal for 24 h, which was f o u n d to increase activity in the primary cultures (see Table II). Such treatment was also found to result in marked process formation, such t h a t t h e cells ~ m o r p h o l o g y more closely resembled astrocytes in vivo. These effects will be described more fully later. The astrocytes samples were from two week cultures with carbonic anhydrase activities comparable to those shown in Fig. 6. The neuroblastoma line showed low (Na ÷ + K ÷) ATPase activities 26, and carbonic anhydrase activity which was 10-fold less than the primary astrocytes. Effects of DBcAMP

on e n z y m e activities in cultured cells

As briefly mentioned above, the addition o f 1 m M D B c A M P to cultures in serum-free media resulted in increased carbonic anhydrase activity, as well as morphological changes involving process f o r m a t i o n in a b o u t 90 ~ o f the cells (see Introduction). These changes persisted for up to at least 24 h, when the experiments were terminated. Addition o f 1 m M sodium butyrate inhibited c a r b o n i c anhydrase activity as well as cell growth, but had no effect on cell moiph01ogy. Serum removal

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Fig. 8. Enzyme activities and process formation at increasing times after treatment with I mMDBcA M P in media containing 20% FCS. A: DBcAMP was added to two-week-old growing cells 24 h after a media change, as described in Materials and Methods. At the indicated times cells were removed from the T-flasks by scraping and activities measured after sonication as described in Materials and Methods. The cultures contained an average of 2 3 mg protein/T-flask; 0.1-0.2 mg protein/l.5 ml and 3-4 mg protein/5:0 ml reaction medium were used for the ATPase and carbonic anhydrase activity measurements respectively. B: DBcAMP was added as described in A. At the various times indicated 50 100 cells in 5 randomly chosen fields were counted. Each cell was positively scored for process formation if it had at least two processes which were ~> twice the diameter of the cell body. The percentage of the total cells counted that this represented is plotted. Before addition of DBcAMP 5-10 % of the cells showed process formation by the same criteria, as indicated at 0 time. The one-, two- and 3-week-old cell cultures contained 1.5, 2.0 and 3.0 mg protein/75 sq.cm T-flask respectively.

alone resulted in a m a x i m u m of 20 % of the ceils showing morphological changes. After addition of I m M D B c A M P in media c o n t a i n i n g the n o r m a l a m o u n t of 20~o FCS, there was a transient increase in both ( N a + + K +) A T P a s e a n d Mg 2+ A T P a s e and also carbonic anhydrase activity. This reached a m a x i m u m increase of 4-fold for ( N a + + K ~) and Mg 2+ A T P a s e activities a n d a 2.6-fold increase for carbonic anhydrase activity 2 h after addition of D B c A M P , as shown in Fig. 8A for 2-week-old cultures from whole brain.

Morphological effects of DBcA MP on cultured cells Fig. 8B shows that morphological changes occur in parallel with the transient increases in enzyme activities caused by D B c A M P addition in media c o n t a i n i n g 20 ~,~ FCS. A total of 50-100 cells in at least 5 different, r a n d o m l y chosen fields was counted. The percentage of cells showing two or more processes, each of which was at least twice as long as the diameter of the cell body, are plotted at increasing times after D B c A M P a d d i t i o n in Fig. 8B. It can be seen that process f o r m a t i o n is quite marked at l h after D B c A M P addition, reaches a m a x i m u m at 2 h a n d then declines, reaching

68

] Fig. 9 Cell m o r p h o l o g y at increasing times after t r e a t m e n t with D B c A M P in m e d i u m c o n t a i n i n g 2 0 % FCS. A : 2-week culture u n t r e a t e d ; B : 2-week culture 1 m M D B c A M P for 2 h. : C: 2-week culture ~ 1 m M D B c A M P for 4 h. ; D : 2-week culture ~ 1 m M D B c A M P for 6 h. Final magnification o n original total 10.7 × 8.4 c m print was 190-fold for each individual print. P h o t o g r a p h s are f r o m different areas o f the s a m e culture.

69

Fig. 10. Electron micrographs of untreated cells. Two-week-old cultures growing in 60 mm Petri dishes were fixed and stained for electron microscopy as described in Materials and Methods. They were photographed at final magnifications of 7. 5300 (A), < 11,288 (B) and × 36,863 (C) respectively for 5 . 7 prints but reduced for publication. Size scales in/~m are shown on the prints. C is a part of B at higher magnification. A and B are different cells.

control levels again between 6 and 24 h. It also appears that process formation is greater in cell cultures of increasing age. Phase micrographs illustrating the typical morphology o f cells at increasing times after D B c A M P addition in complete media are shown in Fig. 9. In order to further analyze the m o r p h o l o g y of these cells, control cells and cells that had been exposed to 1 m M D B c A M P in FCS-free media were examined by electron microscopy. Typical control cells are shown in Fig. 10A-C. Fig. 10A is a relatively low magnification view o f a cell b o d y showing both nucleus and cytoplasm. Numerous mitochondria can be seen as dark elongate bodies, and also bundles o f filaments can be seen scattered within the cytoplasm and arranged as a discrete layer beneath the plasma membrane in the lower left hand corner. These filaments can be seen more clea~ly in the cytoplasm of another cell in Fig. 10B, where they form a dense criss-cross pattern. Fibrils of larger diameter are also seen in the cytoplasm. These structures are shown more clearly in the enlargement of a portion of the cell shown in Fig. 10B in Fig. 10C. The larger fibrils have the dimension o f microtubules, 20 nm, in diameter, while the diameter of the smaller filaments are 10 nm to 5 rim, which is close to the dimensions of glial filaments s4 and microfilaments respectively. Electron micrographs of a cell from a culture pretreated with 1 m M D B c A M P in FCS-free medium for 24 h are shown in Fig. I I A - C . They show the smaller cell body and some of the processes already seen in the phase photomicrographs in Fig. 9. In the areas B and C, in Fig. l l, shown at higher magnification, it can be seen that the microtubule-like structures with diameters of ~ 20 nm run along the length of the processes. Filaments which seem to vary from about 5 to 10 nm in diameter are aligned in a similar way. These structures are labelled MT and F respectively in Fig. l lB.

70

Fig. 11. Electron micrographs of cells treated with 1 m M DBcAMP in FCS-free medium for 2 4 h . Two-week-old cultures were grown in FCS-free medium containing I m M DBcAMP for 24 h and then fixed and treated for electron microscopy as described in Materials and Methods. A is at a magnification of 5300 for an 8 >~. 10 inch print. B and C are higher power ( × 48,000) photographs of portions of th e same cell as indicated by the marked areas in A. Size scales in t~m are also shown. MT, microtubules : F, filaments.

71 DISCUSSION Development of carbonic anhydrase and A TPase activity in vivo and in vitro An early study found no carbonic anhydrase activity in rat brain up to 3 days after birth and then increasing activity with age, with higher activities found in homogenates of the cerebellum plus brain stem region compared to the cerebrum 4. Our studies are in agreement with this finding, as well as the fact that differences in the activities of these two regions become smaller in the adult rat. The fact that we also find a qualitatively similar developmental pattern, including the higher activity in cerebellum plus brain stem region compared to the cerebr urn, in primary cultures from 1-3-day-old rat brain consisting predominantly of astrocytes is consistent with the viewpoint that carbonic anhydrase is mainly in glial cells 17,27,40. The specific carbonic anhydrase activities in cultured cells is however, less than that in vivo. Also, a glial localization need not be restricted to astroglia but could include oligodendroglia, since recent studies have shown that myelin fractions from adult rat brains contain relatively high specific activities of carbonic anhydrase 1~. A similar regional development of HCO3--stimulated ATPase and carbonic anhydrase activity in vivo, and to a lesser extent in vitro, may be due to some functional relationship between these two enzymes, or could simply be fortuitous. If there is a relationship it would imply some involvement of carbonic anhydrase in mitochondrial function, since mitochondrial ATPase is markedly stimulated by HCOs- ions 1~ and, in many tissues 24, including brain ')s, there seems to be little significant extramitochondrial HCO3 -stimulated ATPase activity. Several studies have shown that the ( N a ~ + K ~) ATPase activity of rat brain increases with age, with little or no detectable activity being present in neonates1, 47. Our results are clearly in agreement with these studies, and it is of interest that Medzihradsky et al. 37 found a 12-fold rise between 6 and 8 days after birth in bulkisolated glia but not neuronal fractions. We appear to have seen the same abrupt rise between 6 and 8 days in total homogenates of brain (Fig. IA), with specific activities about half those found in the bulk-isolated glial fraction aT. This correspondence, the oft-repeated finding of higher ( N a + + K +) ATPase activity in bulk-isolated glia cells compared to neuronal perikaryae2,z~,37, 40, and the parallel increase in (Na t + K ~) ATPase activity in vivo and in cultured astrocytes found in this study up to 14 days after birth, suggests that a considerable part of the early increase in ( N a + + K +) ATPase activity in rats is due to astrocyte proliferation and growth. Up to 14 days after birth the (Na ~ + K +) ATPase specific activity in cultured cells is about two-fold less than the average specific activity found in vivo, compared to the 5-fold lower levels of carbonic anhydrase in vitro as compared to in vivo. The growth and density-dependent decline in ( N a + + K +) ATPase activity found in this study has also been described by Schousboe et al. 49 in primary astrocyte cultures. However it has also been observed in 3T3 fibroblasts 29, suggesting that it may be a consequence of monolayer growth in culture. Schousboe et al. 49 also found that the decline in activity in the fourth week of culture was prevented if the cells were grown without serum during this period, which might be due to decreased cell growth and density.

72 The finding of carbonic anhydrase activity in primary astrocyte cultures in the present study is among the first reports of the presence of this enzyme in cultured glia cells (see also refs. 30 and 52), and further supports their glial origin. It is of interest that the C6 glioma cell line (see Table 11) has no detectable carbonic anhydrase activity under a variety of culture conditions, The carbonic anhydrase activity is very labile in primary astrocyte cultures, being lost after trypsinization and subculturing. Such correlative studies cannot be used alone, however, to exclusively ascribe increased ATPase and carbonic anhydrase activity to astroglia cells, since many developmental changes take place in the rat during the first 20 days. Thus, proliferation and development of oligodendroglia associated with myelination of brain axons begins during this time, and because of the high specific activity of carbonic anhydrase in brain myelin 12 some of the increased carbonic anhydrase activity may be due to this. Also, although the neuronal cell population of rat brain cortex, and to a lesser extent other areas, is established before birth 2,11,22,46, continued growth and proliferation of neuronal processes 4~ and synapses 1 could also contribute to the gradual continued increase in ( N a + + K ~) ATPase activity after day 10. As previously mentioned the decline in the (NaE-+ K ÷) ATPase activity of the astrocyte cultures after two weeks may be a specific response to in vitro conditions. The contribution due to synapse development is unlikely to predominate, however, since we have found that the specific ( N a + + K +) ATPase activity of synaptosomes is less than the glial fraction zT. The quantitative contribution of axons and dendrites to total brain ( N a ~ + K ~) ATPase activity, in spite of recent histochemical studies ~5, remains unresolved. Morphological and enzyme changes due to DBcA M P addition This study supports a number of other reports, mentioned in the introduction, in which treatment with DBcAM P or brain extract changes the cells in prlmary cultures of neonatal rat brains from flat polygonal cells with few processes to cells resembling astrocytes with numerous processes, and supports their identification as glial precursors or astroblasts 34,38,52,56. Such cells have been found to form a background for the development of neuronal cells and processes m embryonic rat brain cultures, and become the dominant cell type after 4 weeks 6°. In cultures from l-3-day-old rat brains the neurons seem to degenerate more rapidly than in cultures from embryonic rat brains and may be largely removed in the initial 24-h media change, and a predominantly astroblast culture seems to develop after 10 days 5~. Thus, gtial fibrillary acidic protein (GFA) is found at a level 47-fold greater than in adult brain after newborn rat brains were cultured for 21 days '5. Our results show two different types of morphological differentiation as indicated by process formation, depending on whether the medium contained FCS or was FCS-free. In amino acid and vitamin-supplemented BME medium containing no FCS, addition of 1 m M DBcAMP caused morphological differentiation which persisted for at least 24 h, beyond which time the cells were no longer examined. In the same BME medium but containing 20 ~ FCS. the effect of DBcAMP addition was transient, with morphological changes reaching a maximum 2 h after DBcAMP addition, and almost entirely reversing after 6 h. Moonen et al. as have reported that 1

73 m M DBcAMP caused marked morphological differentiation in FCS-free BME but had no effect in BME medium containing 20% FCS. In contrast, 1 m M DBcAMP caused 50-70 % morphological differentiation in MEM medium containing 20 % FCS and these changes were irreversible. A possible reason for the fact that DBcAMP caused transient effects in BME medium containing 20% FCS is that our medium contains an additional 4-fold increased concentration of vitamins and two-fold increased amino acids, as previously used for cells cultured in both Eagle's BME 6 and Eagle's MEM 4~. Indeed, Moonen et al. as suggested that one of the differences between MEM and BME that might be responsible for differences in behavior was the higher concentrations of amino acids in MEM. The long-term morphological differentiation in FCS-free BME after addition of 1 m M DBcAMP was associated with small or no changes in enzyme activity. However in BME medium containing FCS, addition of 1 m M DBcAMP resulted in parallel changes in morphology and enzyme activity. Furthermore, the increases in enzyme activity were relatively large. The reversibility of the DBcAMP-induced morphological and enzymatic changes is similar to the fall in cAMP levels seen in cultured cells within 6 h of addition of norepinephrine la, and it was suggested that this was due to rapid synthesis of a protein which inhibited adenyl cyclase. If the effects of DBcAMP partly involve activation of this enzyme, and since serum promotes cell growth and protein synthesis in cultured cells 2a, synthesis of an inhibitory protein could well be inhibited in serum-free medium.

Mechanisms of the DBcAMP-induced changes Effects of DBcAMP on cell morphology and enzyme activity are usually interpreted as being due to increased intracellular cAMP, in part due to uptake of DBcAMP. The many effects of cAMP in the nervous system have been recently reviewed 42. In particular the rapid changes in shape observed by us and others may well be mediated by cAMP-dependent structural changes in microtubules and filaments which, as seen by electron microscopy in Figs. l0 and 1 l, are to be found in these cultured cells in considerable amounts. Like Lira et al. 34 we can find filaments both about 5 nm as well as 10 nm in diameter, and we also find that these structures align themselves along the processes formed after DBcAMP treatment with the filaments arranged to some extent in bundlesa4, 39. Morphological changes, which were attributed to such structures, have also been reported in cultured neurons 45 and nonneuronal 59 cells. The increase in carbonic anhydrase activity is similar to the effect of cAMP on the soluble, type I enzyme in erythrocytes41, where it was found that up to 4.6-fold increased activity could be obtained. The characteristics of this activation were consistent with it being due to cAMP-dependent, protein kinase-mediated, phosphorylation of the enzyme. In cultured astrocytes we found 36% of the carbonic anhydrase activity to be associated with the total particulate fractions and 65 ~ in the post 100,000 g × 60 rain fraction, and the soluble fraction was activated directly by addition of cAMP al. It is unclear whether a similar mechanism can activate (Na++ K +) and Mg z+ ATPase activity. The increased ( N a + + K +) ATPase activity is,

74 however, unlikely to be simply a reflection of increased membrane area, since increased activity was not found in cells maintaining a differentiated state for 24 h after treatment with DBcAMP in FCS-free medium (Table 11).

Astrocytes in vitro and in vivo

While acceptable electrophysiological criteria have been established for astroglia cells, and this has enabled certain aspects of their behavior to be studied in the intact nervous system (e.g. ref. 19), biochemical and membrane transport studies require sufficient quantities of pure cells. Problems concerning the purity, yield, and especially viability and intactness of bulk-isolated glia have prompted many workers to use cultured glial cells as alternatives, especially the rapidly growing, cloned lines of transformed glial cells. Thus. studies on membrane potentials 32, (Na ~+ K ~) ATPase activities 26, neurotransmitter responsiveness is and uptake ~1 have all used the C6 rat glioma (astrocytoma) line as representative of normal astrocytes. A problem with such cultures, however, is to what degree their properties have been altered by viral transformation and prolonged maintenance in culture. Use of primary cultures should overcome these problems, especially if further passaging is avoided, but such cultures are more likely to show variability in growth rate as well as the inevitable variation in culture conditions. Thus. it is important to not only compare cells after the same time in culture but also as the same protein concentration and/or cell density. The studies described here indicate a fairly close correspondence between development of certain enzyme activities m vivo and m primary cultured astrocytes, and suggest that astrocytic proliferation and growth may be partly responsible for their occurrence in vivo. These primary cultured cells also contain large amounts of cytoplasmic filaments and microtubules and show carbonic anhydrase activity, which are all characteristic of astrocytes in vivo 17,~4. When DBcAMP is added the cells rapidly assume a more astrocyte-like morphology and show increased carbonic anhydrase and ATPase activities. These properties can then be added to a growing list of criteria including the presence of glial fibrillary acidic protein in primary cultures of both rat 5-~2 and human astrocytes 3 and mediated uptake of putative transmitters 5°. which suggests that these cultures are good models for normal astrocytes. Discrepancies, however, also exist and may well be a reflection of in vitro growth conditions. which cannot be expected to entirely mimic the in vivo state. Direct neuronal interaction with and, or chemical and hormonal effects on cultured astrocytes are no doubt required for further development. The marked changes induced in primary astrocyte cultures by addition of brain extracts or cyclic AMP analogues and/or serum removal does suggest that some of these developmental processes can be produced in vitro by these means.

ACKNOWLEDGEMENTS This work was supported by Grant 13042 from NINCDS.

75 W e s h o u l d like to t h a n k M r s . E l v i r a G r a h a m for t y p i n g the m a n u s c r i p t a n d Mrs. Sue E a s t o n for p e r f o r m i n g t h e e l e c t r o n m i c r o s c o p e studies. W e s h o u l d also like to t h a n k Dr. G. A. B a n k e r o f the D e p a r t m e n t o f A n a t o m y , A l b a n y M e d i c a l College, f o r his helpful c o m m e n t s on the m a n u s c r i p t .

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19 20

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