JOURNAL OF NEUROBIOLOGY, VOL. 6, NO. 6, PP. 597-608
Uptake of GABA by Neuronal and Nonneuronal Cells in Dispersed Cell Cultures of Postnatal Rat Cerebellum ROBERT S . LASHER Department
of
A n a t o m y , University of Colorado Medical School, Denver, Colorado 80220
SUMMARY
A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA,or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K , values for GABA neurons for high and low affinity uptake were 0.33 X M, respectively. For nonneuronal cells, the apparent K , M and 41.8 X for high affinity uptake was 0.29 X low6M. The apparent V,,, values for mol/g DNA/ GABA neurons for high and low affinity uptake were 28.7 X min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V,,, for high affinity uptake was 0.06 X mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X lop9 M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75-85%) of the ["HIGABA (4.4 X M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent. INTRODUCTION
Considerable evidence has accumulated which implicates y-aminobutyric acid (GABA) as an inhibitory transmitter in many regions of the mammalian CNS (Baxter, 1970; Iversen, 1972; Curtis and Johnston, 1973; Roberts, 1974). It has been suggested that rapid uptake into the presynaptic neuronal element, and possibly into the surrounding glial cells, is the main mechanism for the inactivation of GABA at mammalian CNS synapses (Iversen, 1971). Ra597 0 1975 by John Wiley & Sons, Inc.
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dioautographic evidence indicates that only certain types of neurons and glial cells are able to accumulate exogenous GABA rapidly (Lasher, 1973; Lasher, 1974; see Schon and Iversen, 1974). On the other hand, biochemical studies suggest that most if not all glial cells in the CNS have a high affinity uptake mechanism for GABA similar to that seen in brain slices (Henn and Hamberger, 1971; Hutchison, Werrbach, Vance, and Haber, 1974; Schrier and Thompson, 1974). This apparent inconsistency between the radioautographic and biochemical studies may be due t o the fact that the kinetic data obtained from the biochemical studies provides only a poor representation of the membrane properties of the specific neurons and glial cells involved. It may also be possible that GABA in a majority of the cells is selectively last during preparation for radioautography. These considerations indicated that for clarification of this problem more precise data were required on the properties of GABA neuron membranes. T o this end, a study has been made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neurons. METHODS
Culture methods Dispersed cell cultures of 2 day postnatal rat cerebellums were prepared as described previously (Lasher and Zagon, 1972; Lasher, 1974). Approximately 5 X lo5 cells were plated per dish. To obtain cultures without neurons, cells were fed with a medium containing 3 mM K ion (Kj cultures). Under these conditions, most neurons died between 7 and 14 days in uitro (DIV). To obtain cultures which included neurons, cells were fed with a medium containing 24 mM K ion (K24 cultures; Lasher and Zagon, 1972; also, see Results).
Radioautographic procedures and counting of labeled neurons For labeling with [3H]GABA ([2,3-"H]GABA, sp act = 10 Ci/mmol, New England Nuclear), cultures on 25 mm round glass cover slips (Arthur Thomas, Red Label) were rinsed once with 2 ml warm saline G (Puck, Cieciura, and Robinson, 1958) and then preincubated in 1 ml saline G M and the culfor 10-15 min. ["HIGABA was added to give a final concentration of 1.94 X tures were incubated for 30 min a t 23°C on a gyrotary shaker a t 80 rpm. They were then rinsed twice with 1.5 ml ice cold saline G for about 3 min and fixed in 0.16 M glutaraldehyde in 0.05 M Sorenson's phosphate buffer for 60 min (Zagon and Lasher, 1972), rinsed three times in 0.1 M Sorenson's phosphate buffer (made iso-osmotic to the fixative), dehydrated in 50, 70, 95, and 100% ethyl or isopropyl alcohol, and air dried. The cover slips were dipped in liquid Kotlak NTB-2 emulsion, exposed, developed, and stained as described previously (Lasher, 1974). T o determine the number of GABA neurons in a culture, a total of 4-5 cover slips for each time period from two separate experiments were completely scanned a t a magnification of 125 X using a micrometer grid eyepiece and all labeled neurons were counted.
T i m e course of GARA u p t a k e
To determine the time course of [?HH]GABAuptake, K3 and K24 cultures from a t least two separate experiments grown in 35 mm plastic dishes (Falcon) were rinsed once with 1.5 ml saline G (37°C) and then preincubated in 1 ml saline G a t 37OC for 10-15 min. [3H]GABA was added to replicate cultures of each type to give a final concentration of 4.8 X M (0.05 WCi). This very low concentration of GABA was chosen to accentuate any possible differences in high affinity uptake between GABA neurons and nonneuronal cells and avoid any overlap with the low affini-
NEURONAL AND NONNEURONAL GABA UPTAKE
599
t y uptake. The cultures were incubated for 1,5, 10, 20, or 40 rnin on a gyrotary shaker a t 80 rpm. Triplicate cultures of each type were also incubated as above on ice as a control for nonspecific binding and diffusion. At appropriate times, cultures were placed on a tray of ice, rinsed twice with 1.5 ml of ice cold saline G to remove nonincorporated ["HIGABA,and then 1 ml of distilled water was added to each dish. The dishes were placed in a refrigerator a t 4°C for several hours to osmotically shock the cells. The cells were then scraped off the dishes using a rubber or plastic policeman and the suspensions from the replicate cultures were pooled in a 10 X 75 mm tube. Approximately 350-400 pg BSA (Fraction V, Miles) were added to each tube in 0.1 ml distilled water to facilitate coprecipitation of DNA and then 1 ml of 30% trichloroacetic acid was added (final concentration, 10%). The suspensions were kept at 4°C for about 48 hr to permit complete precipitation. Tubes containing known amounts of DNA were prepared as above for each experiment for determination of a standard curve. The precipitates were centrifuged a t 1400 X g for 10 min a t 0°C in a Sorvall HB-4 rotor. Supernatants were collected in plastic tubes, and a 1 ml aliquot of each sample was placed in 10 ml Aquasol (New England Nuclear) and counted in a Beckman LS-133 scintillation counter. For DNA determinations, the fluorometric procedure of Hinegardner (1971) was used. Tubes containing precipitates were placed in an oven and dried under vacuum a t 50°C. Diaminohenzoic acid dihydrochloride (Eastman Kodak or Sigma, repurified, 0.1 ml, 0.4 mg/ml distilled water) was added to each tube; the tubes were incubated for 45 min at 60°C in a Temp-Blok heater and then 1.5 ml 1 N HCl was added to each tube. Readings were taken 1 hr after the addition of the HCl in an Aniinco-Bowman fluoro-colorimeter equipped with a mercury lamp, a Corning 7-51 filter in the primary position, and Wratten #4 and 1%neutral density filters in the secondary position. DNA values rather than protein values or cell counts were used to normalize data for a number of reasons. After 21 DIV, it is extremely difficult to completely dissociate all the cells in a culture, even after prolonged enzymatic treatment. In addition, the enzymatic treatment is quite traumatic and might invalidate any data on GABA uptake. Protein values could he influenced by the absorption of serum proteins from the culture medium and the increase in cell protein due to maturation processes. However, DNA values are also subject to possible error resulting from the presence of residual DNA derived from dead cells (Shashoua and Wolf, 1971). The potential for this problem has been examined in the cultures utilized in this study and has been found t o he neglegible (see Results). Kinetic analysis of G A R A uptake All cultures were treated as above, except that 4.8 X lo-' M ['HIGABA (0.5 pCi) and nonraM to dioactive GARA (Sigma) were added to give final concentrations varying from 9.8 X 0.4 mM. All cultures were incubated for 10 min. Controls ( O O C ) for binding and diffusion were run in triplicate for all concentrations.
Effect of sodium depletion and ouabain trpatment In some experiments, the NaCl in saline G was completely replaced with an equimolar amount of choline chloride (Sigma) and the phosphate buffer was replaced with 10 m M T E S (N-Tris[hydroxymethyl] methyl-2-aminoethane sulfonic acid, Sigma). Cultures were treated as above, except that the final concentration of ["HIGABA was 4.4 X M and the incubation time was 15 min. T o assess the importance of Na-K ATPase in GABA uptake, some cultures in the kinetics experiments were pretreated with 0.2 mM ouahain (Sigma) for 40 min and then incubated in saline M or 0.4 m M [3H]GABAfor 10 min. G plus ouahain and either 9.8 X Calculation of the GARA neuron component The following calculations were performed to determine the dpm/pg DNA (GABA uptake) for GABA neurons from the data obtained from K3 and K24 cultures.
600
LASHER K24 dpm
Kx dpm
fig total cell K24 DNA
fig total cell K:i DNA
GABA neuron dpm pg total cell K24 DNA
X
-
GABA neuron dpm
( a)
fig total cell K24 DNA
6.83 gg total cell DNA total GABA neuron dpm K24 culture Kz4 culture
total GABA neuron dpm/K24 culture 0.0247 fig GABA neuron DNA/K24 culture
-
(b)
GABA neuron dpm (C
p g GABA neuron DNA
1
T h e basic assumption involved in making these calculations is t h a t the difference in GABA uptake between cultures with (K24) and without neurons (K:j) is due only t o the presence of GABA neurons in K24 cultures (see Discussion). The values of 6.83 fig total cell DNA/Kz4 culture and 3674 GABA neurons K2,i culture have been experimentally determined in this study (see Itesults). The value of 6.73 pg DNA/cerebellar neuron has been reported elsewhere (McEwen, Flapinger, Wallach, and Magnus, 1972). T h e value of 0.0247 gg GABA neuron DNA/Kzd culture is derived from the following calculation: 3674 GABA neurons/K24 culture X 6.73 pg DNA/cerebellar cell
Statistical analysis
(x
All data are presented where possible as means f the standard error of the mean f SEM). For analyses of kinetic studies, a Hewlett-Packard HI’-65 calculator and linear regression program (STAT 1-05A and 1-22A) were used t o fit the best least squares estimates to the Lineweaver-Burk transformation of the Michaelis-Menten equation
All data were corrected for diffusion and binding by subtracting 0°C control values prior to performing the calculations. Where appropriate, relatedness of the data was determined using the Hewlett-Packard program for the statistic for two means (STAT 1-30A).
RESULTS
Estimation of GABA neurons and total DNA Table 1 gives the data for numbers of GABA neurons in K24 and K3 cultures after 7, 14, and 21 DIV. There was a significant loss of GABA neurons between 7 and 14 DIV in K3 cultures. By 2 1 DIV, GABA neurons, which were all in some stage of degeneration, represented only 1.7% of the total in K24 cultures. Conversely, there was only a small decrease in GABA neurons between 7 and 21 DIV in K24 cultures. However, a n estimated 25,000 total neurons are lost in K24 cultures after 2 1 DIV as compared with about 55,000 total neurons (essentially the whole neuronal population) in K3 cultures over the same time period. Mean values for total cell DNA were found t o be 6.83 f 0.29 pg/K2* culture and 6.72 f 0.29 pg/Ks culture (each based on 51 values). T o determine whether these values were in fact representative of the total cell population, estimates of total cells/K24 culture were made. T h e mean cell density for 49 areas of 4 X lo4 pm2 representing all regions from the edge t o the center of a culture was found t o be 50.6 f 3.1. T h e total surface area covered by cells a t 21 DIV was 8.55 X los pm2. This indicates there were about 1.082 x ]LO6 cells/K24 culture. If there are 6.73 pg DNA/cerebellar cell (McEwen e t al.,
NEURONAL AND NONNEURONAL GABA UPTAKE
601
TABLE 1 Numbers of Labeled Neurons in Cultures Fed with Medium Containing 24 mlM K Ion (KZ4)or 3 mM K Ion (K,) after Various Periods In Vitro" Days in uilro
7
Medium
K*4
4690 3794
K3
i i
498 532
14
21
4298 + 706 400 k 139
3674 i 561 62 + 22
"All values were obtained from a total of 4 cultures ( 5 cultures for K,, 21 DIV) from t w o different experiments, and are expressed as means f SEM. The difference between K,, and K, cultures was not significant a t 7 DIV, b u t was significant a t 1 4 DIV ( p < 0.01) and 21 DIV ( p < 0.001). Also, the decreases in GABA neurons in K, cultures from 7-14 DIV ( p < 0.001) and 14-21 DIV (p < 0.05) were significant. F o r details of labeling and radioautography see Methods.
1972) then 6.83 yg DNA/K24 culture is equivalent to 1.015 X lo6 cells, about 94% of the estimated value. Residual DNA from dead or dying cells should not make a significant contribution to the total DNA, since dead or dying cells detach from the substrate, and most are removed when the culture medium is changed completely during feeding each week. Determination of the total GABA neurons/K24 culture a t 21 DIV permitted calculation of the total GABA neuron DNA/K24 culture. Thus, 3674 GABA neurons/K24 culture X 6.73 pg DNA/cerebellar cell = 0.0247 yg GABA neuron DNA/K24 culture.
T i m e course of ["H]GABA
uptake
There was no significant difference between K24 and K3 cultures in uptake of 4.8 X M [3H]GABA over a 40 min period a t 7 DIV (Table 2). However, a t 14 DIV ( p < 0.05) and 21 DIV ( p I0.05) the differences were significant. A t 21 DIV, the amount of [3H]GABA taken up by GABA neurons was several orders of magnitude greater per cell than that taken up by nonneuronal cells for all time periods sampled (Fig. 1). Nonneuronal uptake was linTABLE 2 Cell: Medium Ratios for Cultures of Various Ages after Increasing Periods of Incubation with 4.8 x lO-'M [3H]GABAa Incubation time (min) Div Medium
7 14 21
K3
K*4 K3 K,, K, K,,
1
5
0 0
0.11 i 0.16i 0.13i 0.38 i 0.10 i 0.24 f
0.01 ? 0.02 0.11 + 0.03 0.03 * 0.01 0.11 + 0.05
10 0.04 0.02 0.06 0.06 0.06
0.03
0.23 i 0.04 0.30 + 0.09 0.17 i 0.04 0.57 i 0.13 0.20 i 0.09 0.53 i 0.17
20 0.32 i 0.23 i 0.36i 1.13 i 0.29 i 0.78 i
40 0.19 0.10 0.11 0.46 0.10 0.20
0.61 2 0.83 i 0.47 t 1.94 + 0.60 i 1.12 i
0.09 0.12 0.18 1.00 0.16 0.20
a Cultures were incubated a t 37°C o n a gyrotary shaker a t 80 rpm, o r a t 0°C with intermittent agitation. The data are presented as lo-* pmollyg DNA: pmol/ml medium ( X + SEM), have been corrected f o r binding and diffusion, and represent t w o experiments ( 4 cultures/point).
LASHER
14
12
v) I D
B
3 W 0 8
a
2
06
04
3
0 2
INCUBATION TIME (MIN.)
Fig. 1. Plots of the time course of 4.8 X lV9M [,'H]GABA uptake by nonneuronal cells in K i cultures (O--O) and CABA neurons in K24 cultures (.--O). For nonneuronal cells, cell: pmol GABA/pg DNA:pmol GABAlml, and for GABA medium ratios (C/M) are presented as neurons C/M values are given as pmol GABA/wg GABA neuron DNA:pmol GABA/ml. The apparent errors for GABA neuron values were not calculated.
ear through 40 min, but GABA neuron uptake was essentially complete by 20 min. At 21 DIV the 0°C control values were 13-3% of the 37°C values flor K24 cultures and 50-5% for K:l cultures as incubation time increased.
Kinetics of [3H]GABAu p t a k e The data indicate, that for any given concentration, GABA neurons in cudtures 21 DIV had an average velocity of uptake several orders of magnitude TABLE 3 Kinetic Constants for t h e Accumulation of [ 3HJ GABA in Cultures 21 DIV a n d in GABA Neurons (GN)a Apparent K , values (10 - 6 M)
Apparent V,,, values (pmollg D N A / m i n )
Culture o r cell type
High affinity uptake
L o w affinity uptake
High affinity uptake
K3 K*4 GN GN/K,
0.291 0.326 0.325 1.1
0 6329 4184 4184
0.058 0.169 28.74 496
L o w affinity uptake 0 833 151.5 x 151.5 x
lo3
lo3
a F o r high affinity uptake, best fits for regression lines were obtained for GABA concentrations of 0.005-1.048 X M (K,,, r 2 = 0.9997), 0.005-1.048 X M (K.,, r2 = 0.9995), a n d 0.005-1.048 X M (GN, r 2 = 0.9998). F o r low affinity uptake, best fits for regression lines were obtained for GABA concentrations of 10-400 x M (K,,, r 2 = 0.9993) a n d 10-400 X lo-' M (GN, r2 = 0.9994). Regression lines were c o m p u t e d using values of l / V vs. l [ S ] (see Fig. 2).
603
NEURONAL AND NONNEURONAL GABA UPTAKE
greater than that of nonneuronal cells. Examination of the kinetic constants for K:3 and K24 cultures and GABA neurons (Table 3), obtained from Lineweaver-Burk plots of 1/P versus l/[S] (Fig. 2 ) , indicated that the apparent K , for GABA neurons was about the same as that for nonneuronal cells for HIGH AFFINITY UPTAKE 8o
0
1
/K3
v 40
30 20
1
v, 0
-50
50
50
lo5x
1O'X
100
1
1 v
200
-50
0
1
50
105x
LOW AFFINITY
1
UPTAKE GN
lo
1 -5
10 X
-5
0
5
10
1
T
-5
0
5
10
20
1
103x
H
Fig. 2. Lineweaver-Burk plots ["HIGABA uptake during a 10 min incubation for K3 and KZ4 cultures 21 DIV. The GABA concentrations used and the "goodness of fit" of the regression lines ( r 2 )are given in the legend of Table 3. At least 4 data points were used in computing each regression line. Only the portion of the regression line near the intersection of the ordinate and abscissa is shown. Actual data points are indicated by 0 or 0. The GABA neuron (GN) values were calculated using the formula given in Methods.
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604
TABLE 4 Effect of Sodium Deficiency and Ouabain Treatment o n [ 'HIGABA Uptake" i,pm/pg DNA
____
~~
Treatment +Na, 37°C -Na, 37°C +Na, 0°C -0uabain +Ouabain
1457
i
368 * 412 i 2145 -t 1795 i
977 278 139 230 118
2176 481 309 4566 2120
k
i ii
I
905 270 14 606 202
% decrease
K,,
GN
K3
198.8 X l o 3 (227.3 X 1 O ' ) h 31.2 x 103 0 669.5 x 10' 0
-
-
-
75 75 72
78 78 86
84 84 100
K24
K3
-_
GN
-
-
-
16
54
100 ~~
a For
t h e sodium deficiency experiments, NaCl was replaced with choline chloride and the phosphate buffer with 1 0 mM TES. Cultures were preincubated in Na-deficient saline for 40-60 min. The [3H] GABA concentration was 4.4 x lo-' M. and incubation was for 1 5 min. Ouabain (0.2 mM) was added 40 min prior t o addition of t h e ['HIM), and incubation was f o r 1 0 min a t 37°C. +NA and -ouabain GABA (0.098 X values for K, and K,, cultures and GABA neurons are not comparable due t o the different concentrations of [ 3 H ]GABA and incubation times used. h GABA neuron uptake at 37°C after correcting the data for binding and diffusion at 0°C.
the high affinity uptake system. However, the apparent V,,, for GABA neurons was 496 times that for nonneuronal cells. After correcting the data for binding and diffusion, the low affinity uptake system appeared to be minimal or lacking in nonneuronal cells a t GABA concentrations above 4 x M, but had a large capacity and high velocity in GABA neurons.
Effect of sodium deficiency and ouabain o n [3H]GABAu p t a k e For cells in both K3 and K24 cultures, 72-86% of [3H]GABA (4.4 X lop6 M ) uptake appeared to be sodium and temperature dependent (Table 4). Treatment with 0.2 mM ouabain resulted in a significant decrease (54%) in uptake by K24 cultures ( p < 0.02), but had little or no effect on uptake by K3 cultures. Uptake of 9.8 X M ["H]GABA by GABA neurons was totally abolished by ouabain. However, a t a GABA concentration of 0.4 mM, ouabain had no effect on either K3 or K24 cultures. Radioautographic analysis of ["H]GABA accumulation (4.4 X lop6 M, 15 min, 37OC) in K24 cultures in the presence of 4.0 X lop5 M ouabain indicated an absence of label in most GABA neuron processes and a large decrease in label over most cell bodies. DISCUSSION
The ability to calculate data relating t o the properties of GABA neurons from the biochemical difference between K24 and K3 cultures depends on the validity of a number of assumptions. The first assumption is that the biochemical difference is due only to the absence or presence of neurons. The data indicate that culture of rat cerebellums in a medium with 3 m M K + ion results in the death of almost the entire neuronal population after 21 DIV, while neuron survival and differentiation is enhanced in a medium containing
NEURONAL AND NONNEURONAL GABA UPTAKE
605
24 mM K + ion. However, it is apparent from the DNA values that nonneuronal cells, which make up over 90% of the total, replicate at the same rate in both culture conditions. In addition, the biochemical and radioautographic data on the effect of ouabain, suggest that the difference in dpm/kg DNA for K:3 and K24 cultures is due almost entirely to a ouabain sensitive component of GABA neurons. However, the lack of a response to ouabain treatment in K3 cultures is somewhat puzzling, since data from other studies suggest that normal (Henn and Hamberger, 1971) and neoplastic glial cells (Snodgrass and Iversen, 1974) from the CNS have a ouabain sensitive [3H]GABA uptake system, as do glial cells from the peripheral nervous system (Davies and Johnston, 1974). However, in both of the CNS studies there was probably some contamination by neuronal endings or processes. On the other hand, in a study using cultured rat C-6 glioma, ouabain had only a minimal effect on [3H]GABA uptake (Schrier and Thompson, 1974). In addition, there are a number of studies using slices, minces, or explant cultures of vertebrate CNS tissue containing neurons and glial cells which indicate that ouabain treatment resulted in only 26-66% inhibition of ["H]GABA uptake, similar to the 54% inhibition seen in the K24 cultures (Tunnicliff, Cho, Blackwell, Martin, and Wood, 1973; Cho, Tunnicliff, and Martin, 1974; Davidoff and Adair, 1974; Kelly, Luttges, Johnson, and Grove, 1974). Also, fractions of CNS tissue enriched in neuronal membranes showed an increased sensitivity to ouabain inhibition of ["HIGABA uptake (Henn and Hamberger, 1971; Snodgrass, Hedley-White, and Lorenzo, 1973). These data suggest that in tissues containing both glial cells and neurons only part of [3H]GABA uptake is due to the presence of Na-K ATPase, or a t least one sensitive to ouabain. Furthermore, the bulk of ouabain-sensitive [3H]GABA uptake is probably neuronal. A second assumption required to calculate the GABA neuron component is that the number of GABA neurons is in fact equal t o the number of labeled cells (mean value/K24 culture). Light microscope (Lasher, 1974) and electron microscope (Burry and Lasher, 1975) radioautographic evaluation of cultures incubated with ["H]GABA indicates that exogenous GABA is taken up and accumulated rapidly almost entirely by stellate neurons and Purkinje cells. Granule cell neurons and most glial cells do not appear to accumulate GABA rapidly. While it is not yet possible to say either that there is an absolute correspondence between labeled cells and GABA neurons, or that all GABA neurons take up [3H]GABA,the approximation is probably quite close. The third assumption required is that the value of 6.73 pg DNA/cerebellar cell nucleus is a good approximation of total cerebellar cell DNA (McEwen, Plapinger, Wallach, and Magnus, 1972). This is probably true, since cytoplasmic DNA represents a very small part of total cell DNA (Granick and Gibor, 1967), and the average value of DNA/diploid rat cell is about 6.4 pg (e.g., see Sober, 1970) The validity of the calculated values also depends on the assumption that DNA is a good basis for normalizing the data. This assumption is supported by the close correspondence between the value for the mean total cells in a K24 culture estimated from actual cell counts and the value derived from the
606
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DNA determinations. In addition, the contribution of residual DNA from dead or dying cells to the total DNA values is probably no more than the standard error of the mean for these values. It would appear that the assumptions discussed above are valid, given the extent of present knowledge. It is, therefore, possible to calculate the values for the time course and kinetics of ["H]GABA uptake by GABA neurons. When these calculations are made a number of striking differences are found between GABA neurons and nonneuronal cells. In the study on the time course of uptake, GABA neurons accumulated 300-900 times more ["HJGABA (4.8 X lop9 M ) than nonneuronal cells for any time period up to 20 min. The kinetic studies indicated that GABA neurons also have a highly efficient low affinity uptake system which is not a result of simple diffusion. By substituting the data obtained from the kinetic studies into the Michaelis-Menten equation V = (V,,,) [S]/Km+ [S], it was possible to calculate the relative velocities for the high and low affinity transport of [3H]GABA into K3 cultures and GABA neurons. Thus, for 4.8 X lop9 M [3H]GABA the values were 0.42 pmol/g DNA/min (high) and 0.17 pmollg DNA/min (low) for GABA neurons; and 9.0 X pmol/g DNA/min (high) for nonneuronal cells. These values indicate that 99.8% of the label should be accumulated by GABA neurons; 71% is accumulated by the high affinity uptake system. In addition, it should take all the GABA neurons in one culture 20 min to accumulate 0.21 pmol of GABA. Data from the time course experiments indicate an uptake of 0.16 pmol of GABA/2O min, 76% of the predicted value. As uptake of 4.8 x M ["HIGABA leveled off by 20 min, neuronal metabolism or homoexchange of GABA may be important factors after this time. Also, evaluation of the apparent kinetic constants indicated that for virtually any concentration of exogenous GABA, over 99% of it should be accumulated by GABA neurons, given equal access of all cells to the label. Studies using brain slices, minces, or explant cultures do not indicate separate kinetics for neurons and nonneuronal cells, but it has often been assumed that the constants found are representative of neurons. The data from the present study suggest otherwise. The key to the problem may be equal access of all cells to the label. In slices, minces, or explant cultures, GABA neurons probably make up only a small proportion of the total neuronal population and they are generally within the tissue. Therefore, when label is presented exogenously, it has to diffuse between large numbers of nonneuronal cells before it is accessible to GABA neurons. Thus, the kinetics obtained are more a reflection of nonneuronal cell uptake than GABA neuron uptake. In a companion electron microscope radioautographic study (Burry and Lasher, 1975), where access of all cells to exogenous [3H]GABA was equal, the grain density over neuronal components (presynaptic terminals, perikerya, and processes) was 0.098 f 0.018/p2, while the over glial cells was 0.010 rt 0.004/p2 in K24 cultures 28 DIV incubated 5-15 min in 0.3 or 1 X low6M [3H]GABA. In these cultures, labeled GABA neurons and their synapses comprised about 10% of the total neuronal population. This indicates that
NEURONAI, AND NONNEURONAL GABA UPTAKE
607
probably 98% of the label was in fact in GABA neurons, given the specificity of uptake by these cells. This figure is in close agreement with that expected on the basis of kinetic constants. Patterns of label accumulation in numerous other radioautographic studies are also probably a reflection more of the relative uptake velocities of the cells involved than of metabolic turnover, especially in short incubations. This is supported by the observation that in the presence of 1.0 X lop5 M amino-oxyacetic acid, uptake of 0.25 X M [“H]GABA into the rat brain slices was not significantly affected, while GABA-transaminase activity was inhibited by 80% (Snodgrass and Iversen, 1973). Application of the kinetic values for low affinity uptake obtained in the present study on GABA neurons in culture indicate that the reuptake process is sufficiently powerful to reaccumulate all of the GABA estimated to be released per nerve impulse within the time duration of an inhibitory postsynaptic potential. T h e author thanks Althea Atherton, EM1 Nichols, and Sue Fraser for their excellent technical assistance during various phases of this study, Kristine McInvaille for drawing the graphs, and Dr. Floyd Bloom, Dr. Perry Molinoff, and Dr. Leslie Iversen for their critical evaluation of the manuscript and helpful suggestions. This research was supported by the National Institutes of Health Grant NS-09641 from the National Institute of Neurological Diseases and Stroke and by a grant from the Eli Lilly Company. REFERENCES
BAXTER,C. F. (1970). T h e nature of y-.sminobutyric acid. In: Handbook of Neurochemistry, vol. 3. A. Lajtha, Ed., Plenum Press, pp. 289-353. B~iiirtu,R. W. and LASHER,R. S. (1975). Uptake of GABA in dispersed cell cultures of postnatal rat cerebellum: An electron microscope autoradiographic study. Brain Res. 88: 502-507. CHO, Y. D., TUNNICLIFF, G. and MARTIN,R. 0. (1974). The uptake process for y-aminobutyric acid in cultures of developing chick cerebrum. E x p . Neurol. 44: 306-312. CURTIS,D. R. and JOHNSTON, G. A. R. (1973). Amino acid transmitters in the mammalian central nervous system. Ergebn. Physiol. 69: 97-188. DAVII)OFF,R. A. and ADAIR,R. (1974). High-affinity uptake of (3HH]gamma-aminobutyricacid into frog spinal cord slices. Brain Res. 76: 552-556. DAVIES, J . and JOHNSTON,G. A. R. (1974). T h e uptake of GABA into rat spinal roots. J . N e u rochem. 22: 931-935. GKANICK, S. and GIBOR,A. (1967). T h e DNA of chloroplasts, mitochondria, and centrioles. Progr. Nucl. Acid Res. Mol. Biol. 6: 143-186. HE”, F. A. and HAMBERGER,A. (1971). Glial cell function: uptake of transmitter substances. Proc. N a t . Acad. Sci. U.S. 68: 2686-2690. HINEGARDNER, R. T. (1971). An improved fluorometric assay for DNA. Anal. Biochem. 39: 197-201. HUTCHISON,H. T. WEKRRACH, K., VANCE:, C. and HARER,B. (1974). Uptake of neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. I. Transport of yaminobutyric acid. Brain Res. 66: 265-274. IVEIISEN,L. L. (1971). Role of transmitter uptake mechanisms in synaptic transmission. Br. J . Pharmacol. 41: 571-591. IVEIISEN,L. L. (1972). The uptake, storage, release and metabolism of GABA in inhibitory nerves. In: Perspectiues in Neuropharmacology. S. H. Snyder, Ed., Oxford University Press, pp. 75-111. KRILY,P., LLJTTGES,M., JOHNSON, T . and GROVE,W. (1974). Maturation-dependent alterations in [“HIGARAcompartmentalization in neural tissue i n uitro. Brain Res. 68: 267-280.
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LASHER,R. S. (1973). Uptake of "H-gamma-aminobutyric acid hy neurons in cultures of dissociated developing rat CNS. Expanded Abstracts, Society for Neuroscience, Third Annual Meeting, p. 25. LASHER,R. S. (1974). The uptake of ['HIGABA and differentiation of stellate neurons in cultures of dissociated postnatal rat cerebellum. Brain Res. 69: 235-254. LASHER,R. S. and ZAGON,I. S. (1972). The effect of potassium on neuronal differentiation in cultures of dissociated newborn rat cerebellum. Brain Res. 41: 482-488. S , (1972). Properties of cell nuMCEWEN,B. S., PLAPINGER,L., WALLACH,G. and M A G N ~ J C. clei isolated from various regions of rat brain: Divergent characteristics of cerebellar cell nuclei. J . Neurochem. 19: 1159-1170. P~JCK T., T., CIECIUHA, S. J. and ROBINSON,A. (1958). Genetics of somatic mammalian cells. 111. Long-term cultivation of euploid cells from humans and animal subjects. J . E x p . Med. 108: 945-956. ROBERTS,E. (1974). y-Aminobutyric acid and nervous system function-A perspective. Riochem. Pharmacol. 23: 2637-2649. SCHON,F. and IVERSEN, L. L. (1974). T h e use of autoradiographic techniques for the identification and mapping of transmitter-specific neurons in the brain. Life Sci. 15: 157-175. E. J. (1974). On the role of glial cells in the mammalian nerSCHKIEK,B. K. and THOMPSON, vous system. J . B i d . Chem. 249: 1769-1780. SHASHOUA, V. E. and WOLF, M. K. (1971). CNS tissue cultures: Changes in the ratio of incorporation of leucine and valine into proteins during myelinogenesis. J . Neurochpm. 18: 11491152. S. R., HEDLEY-WHITE,E. T. and LORENZO,A. V. (1973). GABA t,ransport by SNOLIGRASS, nerve ending fractions of cat brain. J . Neurochem. 20: 771-782. SNODGRASS, S. R. and IVERSEN,L. L. (1973). Effects of amino-oxyacetic acid on ["HIGABA uptake by rat brain slices. J . Neurochem. 20: 431-439. SNOIXRASS,S. R. and IVERSEN,L. L. (1974). Amino acid uptake into human brain tumors. Brain Res. 76: 95-107. SOBER, H. A. (1970). Handbook of Biochemistry, 2nd ed. The Chemical Rubber Co., Cleveland. N., MARTIN,R. 0. and WOOD, J. I). (1973). The TUNNICLIFF,G., CHO, Y. D., BLACKWELL, uptake of y-aminobutryate by organotypic cultures of chick spinal cord. Biochem. J . 134: 27-32. ZAGON,I. S. and LASHER,R. S. (1972). Fixation of developing rat cerebellum for electron microscopy: some experimental observations. Society for Neuroscience, Second Annual Meeting, p. 108. Accepted for publication May 8, 1975
Uptake of GABA by Neuronal and Nonneuronal Cells in Dispersed Cell Cultures of Postnatal Rat Cerebellum ROBERT S . LASHER Department
of
A n a t o m y , University of Colorado Medical School, Denver, Colorado 80220
SUMMARY
A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA,or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K , values for GABA neurons for high and low affinity uptake were 0.33 X M, respectively. For nonneuronal cells, the apparent K , M and 41.8 X for high affinity uptake was 0.29 X low6M. The apparent V,,, values for mol/g DNA/ GABA neurons for high and low affinity uptake were 28.7 X min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V,,, for high affinity uptake was 0.06 X mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X lop9 M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75-85%) of the ["HIGABA (4.4 X M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent. INTRODUCTION
Considerable evidence has accumulated which implicates y-aminobutyric acid (GABA) as an inhibitory transmitter in many regions of the mammalian CNS (Baxter, 1970; Iversen, 1972; Curtis and Johnston, 1973; Roberts, 1974). It has been suggested that rapid uptake into the presynaptic neuronal element, and possibly into the surrounding glial cells, is the main mechanism for the inactivation of GABA at mammalian CNS synapses (Iversen, 1971). Ra597 0 1975 by John Wiley & Sons, Inc.
598
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dioautographic evidence indicates that only certain types of neurons and glial cells are able to accumulate exogenous GABA rapidly (Lasher, 1973; Lasher, 1974; see Schon and Iversen, 1974). On the other hand, biochemical studies suggest that most if not all glial cells in the CNS have a high affinity uptake mechanism for GABA similar to that seen in brain slices (Henn and Hamberger, 1971; Hutchison, Werrbach, Vance, and Haber, 1974; Schrier and Thompson, 1974). This apparent inconsistency between the radioautographic and biochemical studies may be due t o the fact that the kinetic data obtained from the biochemical studies provides only a poor representation of the membrane properties of the specific neurons and glial cells involved. It may also be possible that GABA in a majority of the cells is selectively last during preparation for radioautography. These considerations indicated that for clarification of this problem more precise data were required on the properties of GABA neuron membranes. T o this end, a study has been made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neurons. METHODS
Culture methods Dispersed cell cultures of 2 day postnatal rat cerebellums were prepared as described previously (Lasher and Zagon, 1972; Lasher, 1974). Approximately 5 X lo5 cells were plated per dish. To obtain cultures without neurons, cells were fed with a medium containing 3 mM K ion (Kj cultures). Under these conditions, most neurons died between 7 and 14 days in uitro (DIV). To obtain cultures which included neurons, cells were fed with a medium containing 24 mM K ion (K24 cultures; Lasher and Zagon, 1972; also, see Results).
Radioautographic procedures and counting of labeled neurons For labeling with [3H]GABA ([2,3-"H]GABA, sp act = 10 Ci/mmol, New England Nuclear), cultures on 25 mm round glass cover slips (Arthur Thomas, Red Label) were rinsed once with 2 ml warm saline G (Puck, Cieciura, and Robinson, 1958) and then preincubated in 1 ml saline G M and the culfor 10-15 min. ["HIGABA was added to give a final concentration of 1.94 X tures were incubated for 30 min a t 23°C on a gyrotary shaker a t 80 rpm. They were then rinsed twice with 1.5 ml ice cold saline G for about 3 min and fixed in 0.16 M glutaraldehyde in 0.05 M Sorenson's phosphate buffer for 60 min (Zagon and Lasher, 1972), rinsed three times in 0.1 M Sorenson's phosphate buffer (made iso-osmotic to the fixative), dehydrated in 50, 70, 95, and 100% ethyl or isopropyl alcohol, and air dried. The cover slips were dipped in liquid Kotlak NTB-2 emulsion, exposed, developed, and stained as described previously (Lasher, 1974). T o determine the number of GABA neurons in a culture, a total of 4-5 cover slips for each time period from two separate experiments were completely scanned a t a magnification of 125 X using a micrometer grid eyepiece and all labeled neurons were counted.
T i m e course of GARA u p t a k e
To determine the time course of [?HH]GABAuptake, K3 and K24 cultures from a t least two separate experiments grown in 35 mm plastic dishes (Falcon) were rinsed once with 1.5 ml saline G (37°C) and then preincubated in 1 ml saline G a t 37OC for 10-15 min. [3H]GABA was added to replicate cultures of each type to give a final concentration of 4.8 X M (0.05 WCi). This very low concentration of GABA was chosen to accentuate any possible differences in high affinity uptake between GABA neurons and nonneuronal cells and avoid any overlap with the low affini-
NEURONAL AND NONNEURONAL GABA UPTAKE
599
t y uptake. The cultures were incubated for 1,5, 10, 20, or 40 rnin on a gyrotary shaker a t 80 rpm. Triplicate cultures of each type were also incubated as above on ice as a control for nonspecific binding and diffusion. At appropriate times, cultures were placed on a tray of ice, rinsed twice with 1.5 ml of ice cold saline G to remove nonincorporated ["HIGABA,and then 1 ml of distilled water was added to each dish. The dishes were placed in a refrigerator a t 4°C for several hours to osmotically shock the cells. The cells were then scraped off the dishes using a rubber or plastic policeman and the suspensions from the replicate cultures were pooled in a 10 X 75 mm tube. Approximately 350-400 pg BSA (Fraction V, Miles) were added to each tube in 0.1 ml distilled water to facilitate coprecipitation of DNA and then 1 ml of 30% trichloroacetic acid was added (final concentration, 10%). The suspensions were kept at 4°C for about 48 hr to permit complete precipitation. Tubes containing known amounts of DNA were prepared as above for each experiment for determination of a standard curve. The precipitates were centrifuged a t 1400 X g for 10 min a t 0°C in a Sorvall HB-4 rotor. Supernatants were collected in plastic tubes, and a 1 ml aliquot of each sample was placed in 10 ml Aquasol (New England Nuclear) and counted in a Beckman LS-133 scintillation counter. For DNA determinations, the fluorometric procedure of Hinegardner (1971) was used. Tubes containing precipitates were placed in an oven and dried under vacuum a t 50°C. Diaminohenzoic acid dihydrochloride (Eastman Kodak or Sigma, repurified, 0.1 ml, 0.4 mg/ml distilled water) was added to each tube; the tubes were incubated for 45 min at 60°C in a Temp-Blok heater and then 1.5 ml 1 N HCl was added to each tube. Readings were taken 1 hr after the addition of the HCl in an Aniinco-Bowman fluoro-colorimeter equipped with a mercury lamp, a Corning 7-51 filter in the primary position, and Wratten #4 and 1%neutral density filters in the secondary position. DNA values rather than protein values or cell counts were used to normalize data for a number of reasons. After 21 DIV, it is extremely difficult to completely dissociate all the cells in a culture, even after prolonged enzymatic treatment. In addition, the enzymatic treatment is quite traumatic and might invalidate any data on GABA uptake. Protein values could he influenced by the absorption of serum proteins from the culture medium and the increase in cell protein due to maturation processes. However, DNA values are also subject to possible error resulting from the presence of residual DNA derived from dead cells (Shashoua and Wolf, 1971). The potential for this problem has been examined in the cultures utilized in this study and has been found t o he neglegible (see Results). Kinetic analysis of G A R A uptake All cultures were treated as above, except that 4.8 X lo-' M ['HIGABA (0.5 pCi) and nonraM to dioactive GARA (Sigma) were added to give final concentrations varying from 9.8 X 0.4 mM. All cultures were incubated for 10 min. Controls ( O O C ) for binding and diffusion were run in triplicate for all concentrations.
Effect of sodium depletion and ouabain trpatment In some experiments, the NaCl in saline G was completely replaced with an equimolar amount of choline chloride (Sigma) and the phosphate buffer was replaced with 10 m M T E S (N-Tris[hydroxymethyl] methyl-2-aminoethane sulfonic acid, Sigma). Cultures were treated as above, except that the final concentration of ["HIGABA was 4.4 X M and the incubation time was 15 min. T o assess the importance of Na-K ATPase in GABA uptake, some cultures in the kinetics experiments were pretreated with 0.2 mM ouahain (Sigma) for 40 min and then incubated in saline M or 0.4 m M [3H]GABAfor 10 min. G plus ouahain and either 9.8 X Calculation of the GARA neuron component The following calculations were performed to determine the dpm/pg DNA (GABA uptake) for GABA neurons from the data obtained from K3 and K24 cultures.
600
LASHER K24 dpm
Kx dpm
fig total cell K24 DNA
fig total cell K:i DNA
GABA neuron dpm pg total cell K24 DNA
X
-
GABA neuron dpm
( a)
fig total cell K24 DNA
6.83 gg total cell DNA total GABA neuron dpm K24 culture Kz4 culture
total GABA neuron dpm/K24 culture 0.0247 fig GABA neuron DNA/K24 culture
-
(b)
GABA neuron dpm (C
p g GABA neuron DNA
1
T h e basic assumption involved in making these calculations is t h a t the difference in GABA uptake between cultures with (K24) and without neurons (K:j) is due only t o the presence of GABA neurons in K24 cultures (see Discussion). The values of 6.83 fig total cell DNA/Kz4 culture and 3674 GABA neurons K2,i culture have been experimentally determined in this study (see Itesults). The value of 6.73 pg DNA/cerebellar neuron has been reported elsewhere (McEwen, Flapinger, Wallach, and Magnus, 1972). T h e value of 0.0247 gg GABA neuron DNA/Kzd culture is derived from the following calculation: 3674 GABA neurons/K24 culture X 6.73 pg DNA/cerebellar cell
Statistical analysis
(x
All data are presented where possible as means f the standard error of the mean f SEM). For analyses of kinetic studies, a Hewlett-Packard HI’-65 calculator and linear regression program (STAT 1-05A and 1-22A) were used t o fit the best least squares estimates to the Lineweaver-Burk transformation of the Michaelis-Menten equation
All data were corrected for diffusion and binding by subtracting 0°C control values prior to performing the calculations. Where appropriate, relatedness of the data was determined using the Hewlett-Packard program for the statistic for two means (STAT 1-30A).
RESULTS
Estimation of GABA neurons and total DNA Table 1 gives the data for numbers of GABA neurons in K24 and K3 cultures after 7, 14, and 21 DIV. There was a significant loss of GABA neurons between 7 and 14 DIV in K3 cultures. By 2 1 DIV, GABA neurons, which were all in some stage of degeneration, represented only 1.7% of the total in K24 cultures. Conversely, there was only a small decrease in GABA neurons between 7 and 21 DIV in K24 cultures. However, a n estimated 25,000 total neurons are lost in K24 cultures after 2 1 DIV as compared with about 55,000 total neurons (essentially the whole neuronal population) in K3 cultures over the same time period. Mean values for total cell DNA were found t o be 6.83 f 0.29 pg/K2* culture and 6.72 f 0.29 pg/Ks culture (each based on 51 values). T o determine whether these values were in fact representative of the total cell population, estimates of total cells/K24 culture were made. T h e mean cell density for 49 areas of 4 X lo4 pm2 representing all regions from the edge t o the center of a culture was found t o be 50.6 f 3.1. T h e total surface area covered by cells a t 21 DIV was 8.55 X los pm2. This indicates there were about 1.082 x ]LO6 cells/K24 culture. If there are 6.73 pg DNA/cerebellar cell (McEwen e t al.,
NEURONAL AND NONNEURONAL GABA UPTAKE
601
TABLE 1 Numbers of Labeled Neurons in Cultures Fed with Medium Containing 24 mlM K Ion (KZ4)or 3 mM K Ion (K,) after Various Periods In Vitro" Days in uilro
7
Medium
K*4
4690 3794
K3
i i
498 532
14
21
4298 + 706 400 k 139
3674 i 561 62 + 22
"All values were obtained from a total of 4 cultures ( 5 cultures for K,, 21 DIV) from t w o different experiments, and are expressed as means f SEM. The difference between K,, and K, cultures was not significant a t 7 DIV, b u t was significant a t 1 4 DIV ( p < 0.01) and 21 DIV ( p < 0.001). Also, the decreases in GABA neurons in K, cultures from 7-14 DIV ( p < 0.001) and 14-21 DIV (p < 0.05) were significant. F o r details of labeling and radioautography see Methods.
1972) then 6.83 yg DNA/K24 culture is equivalent to 1.015 X lo6 cells, about 94% of the estimated value. Residual DNA from dead or dying cells should not make a significant contribution to the total DNA, since dead or dying cells detach from the substrate, and most are removed when the culture medium is changed completely during feeding each week. Determination of the total GABA neurons/K24 culture a t 21 DIV permitted calculation of the total GABA neuron DNA/K24 culture. Thus, 3674 GABA neurons/K24 culture X 6.73 pg DNA/cerebellar cell = 0.0247 yg GABA neuron DNA/K24 culture.
T i m e course of ["H]GABA
uptake
There was no significant difference between K24 and K3 cultures in uptake of 4.8 X M [3H]GABA over a 40 min period a t 7 DIV (Table 2). However, a t 14 DIV ( p < 0.05) and 21 DIV ( p I0.05) the differences were significant. A t 21 DIV, the amount of [3H]GABA taken up by GABA neurons was several orders of magnitude greater per cell than that taken up by nonneuronal cells for all time periods sampled (Fig. 1). Nonneuronal uptake was linTABLE 2 Cell: Medium Ratios for Cultures of Various Ages after Increasing Periods of Incubation with 4.8 x lO-'M [3H]GABAa Incubation time (min) Div Medium
7 14 21
K3
K*4 K3 K,, K, K,,
1
5
0 0
0.11 i 0.16i 0.13i 0.38 i 0.10 i 0.24 f
0.01 ? 0.02 0.11 + 0.03 0.03 * 0.01 0.11 + 0.05
10 0.04 0.02 0.06 0.06 0.06
0.03
0.23 i 0.04 0.30 + 0.09 0.17 i 0.04 0.57 i 0.13 0.20 i 0.09 0.53 i 0.17
20 0.32 i 0.23 i 0.36i 1.13 i 0.29 i 0.78 i
40 0.19 0.10 0.11 0.46 0.10 0.20
0.61 2 0.83 i 0.47 t 1.94 + 0.60 i 1.12 i
0.09 0.12 0.18 1.00 0.16 0.20
a Cultures were incubated a t 37°C o n a gyrotary shaker a t 80 rpm, o r a t 0°C with intermittent agitation. The data are presented as lo-* pmollyg DNA: pmol/ml medium ( X + SEM), have been corrected f o r binding and diffusion, and represent t w o experiments ( 4 cultures/point).
LASHER
14
12
v) I D
B
3 W 0 8
a
2
06
04
3
0 2
INCUBATION TIME (MIN.)
Fig. 1. Plots of the time course of 4.8 X lV9M [,'H]GABA uptake by nonneuronal cells in K i cultures (O--O) and CABA neurons in K24 cultures (.--O). For nonneuronal cells, cell: pmol GABA/pg DNA:pmol GABAlml, and for GABA medium ratios (C/M) are presented as neurons C/M values are given as pmol GABA/wg GABA neuron DNA:pmol GABA/ml. The apparent errors for GABA neuron values were not calculated.
ear through 40 min, but GABA neuron uptake was essentially complete by 20 min. At 21 DIV the 0°C control values were 13-3% of the 37°C values flor K24 cultures and 50-5% for K:l cultures as incubation time increased.
Kinetics of [3H]GABAu p t a k e The data indicate, that for any given concentration, GABA neurons in cudtures 21 DIV had an average velocity of uptake several orders of magnitude TABLE 3 Kinetic Constants for t h e Accumulation of [ 3HJ GABA in Cultures 21 DIV a n d in GABA Neurons (GN)a Apparent K , values (10 - 6 M)
Apparent V,,, values (pmollg D N A / m i n )
Culture o r cell type
High affinity uptake
L o w affinity uptake
High affinity uptake
K3 K*4 GN GN/K,
0.291 0.326 0.325 1.1
0 6329 4184 4184
0.058 0.169 28.74 496
L o w affinity uptake 0 833 151.5 x 151.5 x
lo3
lo3
a F o r high affinity uptake, best fits for regression lines were obtained for GABA concentrations of 0.005-1.048 X M (K,,, r 2 = 0.9997), 0.005-1.048 X M (K.,, r2 = 0.9995), a n d 0.005-1.048 X M (GN, r 2 = 0.9998). F o r low affinity uptake, best fits for regression lines were obtained for GABA concentrations of 10-400 x M (K,,, r 2 = 0.9993) a n d 10-400 X lo-' M (GN, r2 = 0.9994). Regression lines were c o m p u t e d using values of l / V vs. l [ S ] (see Fig. 2).
603
NEURONAL AND NONNEURONAL GABA UPTAKE
greater than that of nonneuronal cells. Examination of the kinetic constants for K:3 and K24 cultures and GABA neurons (Table 3), obtained from Lineweaver-Burk plots of 1/P versus l/[S] (Fig. 2 ) , indicated that the apparent K , for GABA neurons was about the same as that for nonneuronal cells for HIGH AFFINITY UPTAKE 8o
0
1
/K3
v 40
30 20
1
v, 0
-50
50
50
lo5x
1O'X
100
1
1 v
200
-50
0
1
50
105x
LOW AFFINITY
1
UPTAKE GN
lo
1 -5
10 X
-5
0
5
10
1
T
-5
0
5
10
20
1
103x
H
Fig. 2. Lineweaver-Burk plots ["HIGABA uptake during a 10 min incubation for K3 and KZ4 cultures 21 DIV. The GABA concentrations used and the "goodness of fit" of the regression lines ( r 2 )are given in the legend of Table 3. At least 4 data points were used in computing each regression line. Only the portion of the regression line near the intersection of the ordinate and abscissa is shown. Actual data points are indicated by 0 or 0. The GABA neuron (GN) values were calculated using the formula given in Methods.
LASHER
604
TABLE 4 Effect of Sodium Deficiency and Ouabain Treatment o n [ 'HIGABA Uptake" i,pm/pg DNA
____
~~
Treatment +Na, 37°C -Na, 37°C +Na, 0°C -0uabain +Ouabain
1457
i
368 * 412 i 2145 -t 1795 i
977 278 139 230 118
2176 481 309 4566 2120
k
i ii
I
905 270 14 606 202
% decrease
K,,
GN
K3
198.8 X l o 3 (227.3 X 1 O ' ) h 31.2 x 103 0 669.5 x 10' 0
-
-
-
75 75 72
78 78 86
84 84 100
K24
K3
-_
GN
-
-
-
16
54
100 ~~
a For
t h e sodium deficiency experiments, NaCl was replaced with choline chloride and the phosphate buffer with 1 0 mM TES. Cultures were preincubated in Na-deficient saline for 40-60 min. The [3H] GABA concentration was 4.4 x lo-' M. and incubation was for 1 5 min. Ouabain (0.2 mM) was added 40 min prior t o addition of t h e ['HIM), and incubation was f o r 1 0 min a t 37°C. +NA and -ouabain GABA (0.098 X values for K, and K,, cultures and GABA neurons are not comparable due t o the different concentrations of [ 3 H ]GABA and incubation times used. h GABA neuron uptake at 37°C after correcting the data for binding and diffusion at 0°C.
the high affinity uptake system. However, the apparent V,,, for GABA neurons was 496 times that for nonneuronal cells. After correcting the data for binding and diffusion, the low affinity uptake system appeared to be minimal or lacking in nonneuronal cells a t GABA concentrations above 4 x M, but had a large capacity and high velocity in GABA neurons.
Effect of sodium deficiency and ouabain o n [3H]GABAu p t a k e For cells in both K3 and K24 cultures, 72-86% of [3H]GABA (4.4 X lop6 M ) uptake appeared to be sodium and temperature dependent (Table 4). Treatment with 0.2 mM ouabain resulted in a significant decrease (54%) in uptake by K24 cultures ( p < 0.02), but had little or no effect on uptake by K3 cultures. Uptake of 9.8 X M ["H]GABA by GABA neurons was totally abolished by ouabain. However, a t a GABA concentration of 0.4 mM, ouabain had no effect on either K3 or K24 cultures. Radioautographic analysis of ["H]GABA accumulation (4.4 X lop6 M, 15 min, 37OC) in K24 cultures in the presence of 4.0 X lop5 M ouabain indicated an absence of label in most GABA neuron processes and a large decrease in label over most cell bodies. DISCUSSION
The ability to calculate data relating t o the properties of GABA neurons from the biochemical difference between K24 and K3 cultures depends on the validity of a number of assumptions. The first assumption is that the biochemical difference is due only to the absence or presence of neurons. The data indicate that culture of rat cerebellums in a medium with 3 m M K + ion results in the death of almost the entire neuronal population after 21 DIV, while neuron survival and differentiation is enhanced in a medium containing
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24 mM K + ion. However, it is apparent from the DNA values that nonneuronal cells, which make up over 90% of the total, replicate at the same rate in both culture conditions. In addition, the biochemical and radioautographic data on the effect of ouabain, suggest that the difference in dpm/kg DNA for K:3 and K24 cultures is due almost entirely to a ouabain sensitive component of GABA neurons. However, the lack of a response to ouabain treatment in K3 cultures is somewhat puzzling, since data from other studies suggest that normal (Henn and Hamberger, 1971) and neoplastic glial cells (Snodgrass and Iversen, 1974) from the CNS have a ouabain sensitive [3H]GABA uptake system, as do glial cells from the peripheral nervous system (Davies and Johnston, 1974). However, in both of the CNS studies there was probably some contamination by neuronal endings or processes. On the other hand, in a study using cultured rat C-6 glioma, ouabain had only a minimal effect on [3H]GABA uptake (Schrier and Thompson, 1974). In addition, there are a number of studies using slices, minces, or explant cultures of vertebrate CNS tissue containing neurons and glial cells which indicate that ouabain treatment resulted in only 26-66% inhibition of ["H]GABA uptake, similar to the 54% inhibition seen in the K24 cultures (Tunnicliff, Cho, Blackwell, Martin, and Wood, 1973; Cho, Tunnicliff, and Martin, 1974; Davidoff and Adair, 1974; Kelly, Luttges, Johnson, and Grove, 1974). Also, fractions of CNS tissue enriched in neuronal membranes showed an increased sensitivity to ouabain inhibition of ["HIGABA uptake (Henn and Hamberger, 1971; Snodgrass, Hedley-White, and Lorenzo, 1973). These data suggest that in tissues containing both glial cells and neurons only part of [3H]GABA uptake is due to the presence of Na-K ATPase, or a t least one sensitive to ouabain. Furthermore, the bulk of ouabain-sensitive [3H]GABA uptake is probably neuronal. A second assumption required to calculate the GABA neuron component is that the number of GABA neurons is in fact equal t o the number of labeled cells (mean value/K24 culture). Light microscope (Lasher, 1974) and electron microscope (Burry and Lasher, 1975) radioautographic evaluation of cultures incubated with ["H]GABA indicates that exogenous GABA is taken up and accumulated rapidly almost entirely by stellate neurons and Purkinje cells. Granule cell neurons and most glial cells do not appear to accumulate GABA rapidly. While it is not yet possible to say either that there is an absolute correspondence between labeled cells and GABA neurons, or that all GABA neurons take up [3H]GABA,the approximation is probably quite close. The third assumption required is that the value of 6.73 pg DNA/cerebellar cell nucleus is a good approximation of total cerebellar cell DNA (McEwen, Plapinger, Wallach, and Magnus, 1972). This is probably true, since cytoplasmic DNA represents a very small part of total cell DNA (Granick and Gibor, 1967), and the average value of DNA/diploid rat cell is about 6.4 pg (e.g., see Sober, 1970) The validity of the calculated values also depends on the assumption that DNA is a good basis for normalizing the data. This assumption is supported by the close correspondence between the value for the mean total cells in a K24 culture estimated from actual cell counts and the value derived from the
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DNA determinations. In addition, the contribution of residual DNA from dead or dying cells to the total DNA values is probably no more than the standard error of the mean for these values. It would appear that the assumptions discussed above are valid, given the extent of present knowledge. It is, therefore, possible to calculate the values for the time course and kinetics of ["H]GABA uptake by GABA neurons. When these calculations are made a number of striking differences are found between GABA neurons and nonneuronal cells. In the study on the time course of uptake, GABA neurons accumulated 300-900 times more ["HJGABA (4.8 X lop9 M ) than nonneuronal cells for any time period up to 20 min. The kinetic studies indicated that GABA neurons also have a highly efficient low affinity uptake system which is not a result of simple diffusion. By substituting the data obtained from the kinetic studies into the Michaelis-Menten equation V = (V,,,) [S]/Km+ [S], it was possible to calculate the relative velocities for the high and low affinity transport of [3H]GABA into K3 cultures and GABA neurons. Thus, for 4.8 X lop9 M [3H]GABA the values were 0.42 pmol/g DNA/min (high) and 0.17 pmollg DNA/min (low) for GABA neurons; and 9.0 X pmol/g DNA/min (high) for nonneuronal cells. These values indicate that 99.8% of the label should be accumulated by GABA neurons; 71% is accumulated by the high affinity uptake system. In addition, it should take all the GABA neurons in one culture 20 min to accumulate 0.21 pmol of GABA. Data from the time course experiments indicate an uptake of 0.16 pmol of GABA/2O min, 76% of the predicted value. As uptake of 4.8 x M ["HIGABA leveled off by 20 min, neuronal metabolism or homoexchange of GABA may be important factors after this time. Also, evaluation of the apparent kinetic constants indicated that for virtually any concentration of exogenous GABA, over 99% of it should be accumulated by GABA neurons, given equal access of all cells to the label. Studies using brain slices, minces, or explant cultures do not indicate separate kinetics for neurons and nonneuronal cells, but it has often been assumed that the constants found are representative of neurons. The data from the present study suggest otherwise. The key to the problem may be equal access of all cells to the label. In slices, minces, or explant cultures, GABA neurons probably make up only a small proportion of the total neuronal population and they are generally within the tissue. Therefore, when label is presented exogenously, it has to diffuse between large numbers of nonneuronal cells before it is accessible to GABA neurons. Thus, the kinetics obtained are more a reflection of nonneuronal cell uptake than GABA neuron uptake. In a companion electron microscope radioautographic study (Burry and Lasher, 1975), where access of all cells to exogenous [3H]GABA was equal, the grain density over neuronal components (presynaptic terminals, perikerya, and processes) was 0.098 f 0.018/p2, while the over glial cells was 0.010 rt 0.004/p2 in K24 cultures 28 DIV incubated 5-15 min in 0.3 or 1 X low6M [3H]GABA. In these cultures, labeled GABA neurons and their synapses comprised about 10% of the total neuronal population. This indicates that
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probably 98% of the label was in fact in GABA neurons, given the specificity of uptake by these cells. This figure is in close agreement with that expected on the basis of kinetic constants. Patterns of label accumulation in numerous other radioautographic studies are also probably a reflection more of the relative uptake velocities of the cells involved than of metabolic turnover, especially in short incubations. This is supported by the observation that in the presence of 1.0 X lop5 M amino-oxyacetic acid, uptake of 0.25 X M [“H]GABA into the rat brain slices was not significantly affected, while GABA-transaminase activity was inhibited by 80% (Snodgrass and Iversen, 1973). Application of the kinetic values for low affinity uptake obtained in the present study on GABA neurons in culture indicate that the reuptake process is sufficiently powerful to reaccumulate all of the GABA estimated to be released per nerve impulse within the time duration of an inhibitory postsynaptic potential. T h e author thanks Althea Atherton, EM1 Nichols, and Sue Fraser for their excellent technical assistance during various phases of this study, Kristine McInvaille for drawing the graphs, and Dr. Floyd Bloom, Dr. Perry Molinoff, and Dr. Leslie Iversen for their critical evaluation of the manuscript and helpful suggestions. This research was supported by the National Institutes of Health Grant NS-09641 from the National Institute of Neurological Diseases and Stroke and by a grant from the Eli Lilly Company. REFERENCES
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