- Email: [email protected]
[3H]GABA binding in the cerebellum of the reeler murine mutant
Neurochem. Int. Vol. 7, No. 1, pp. 37~14, 1985 Printed in Great Britain.All rights reserved
0197-0186/85 $3.00+ 0.00 Copyright © 1985PergamonPress Ltd
[3H]GABA B I N D I N G IN THE CEREBELLUM OF THE REELER M U R I N E M U T A N T NIKOLAOS MATSOKISand THEONY VALCANA Laboratory of Human and Animal Physiology, Department of Biology, University of Patra, Patra, Greece (Received 13 January 1984; accepted 11 April 1984) Abstract--In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [3H]GABA binding have been observed. This study shows decreased affinity of the Na +-independent, high affinityGABA binding component of synaptosomal membranes and an increased affinity of the Na +-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na +-dependent and Na+-independent binding sites was not significantly altered. The decreased affinity of the Na+-independent GABA binding and the increased affinity of the Na+-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.
The reduction of the cerebellar granule cell population which accompanies not only some murine mutations but also neonatal exposure to X-radiation, cytotoxic drugs, or viral infection has been shown to lead to changes in the relative density of interneurons and afferent components, as well as to changes in the concentration of various neurotransmitter substances and the activity of associated enzymes (Hoffer et al., 1971; Valcana et al., 1972, 1974a, b; Crepel and Mariani, 1976; Hudson et al., 1976a, b; Landis et aL, 1975; McBride et al., 1976a, b; Roffler-Tarlov and Sidman, 1978; Sievin et al., 1982; Johnston and Coyler, 1982; Soreq et al., 1982). In the reeler mutant mouse the Purkinje, Golgi II and stellate cells of the cerebellum and the cells of the deep nuclei are spared (Hamburgh, 1960; Sidman el al., 1965; Sidman, 1974; Rakic 1976; Wilson et al., 1981) and this condition is accompanied by a significant increase in ~-aminobutyric acid (GABA) concentration as well as other markers of the GABAergic system (Johnston and Coyler, 1982). However, this GABA enrichment may be due to compensatory changes resulting from the degeneration of the excitatory granule cell input to the Purkinje cell system, a loss that would affect such processes as transmitter transport, transmitter release, presynaptic GABA uptake, and postsynaptic GABA receptor properties (Martin, 1976; Vizi, 1979; Henkart, 1980; Purves and Lichtman, 1980).
The presence of GABA receptors in brain tissue has been established by determining specific [3H]GABA binding to crude synaptic membranes, and such binding has been observed in both the presence and absence of Na ÷ ions. The Na ÷-dependent binding is thought to represent the presynaptic re-uptake receptor system and the Na ÷-independent binding, the postsynaptic receptor system. A decrease in Na ÷-independent GABA binding in the reeler cerebellum has been reported from this and other laboratories (Olsen and Mikoshiba 1978; Matsokis et al., 1982). However, whether these alterations result from changes in receptor binding sites or reflect changes in affinity is not clear. The purpose of this study, therefore, is to elucidate the characteristics of Na ÷-dependent as well as Na ÷-independent GABA binding in control and reeler mice. The study of the GABAergic synapse in the reeler mutant cerebellum, where the cytoarchitectural changes are well known may allow us to relate the anatomical to the biochemical aspects of synaptogenesis which are crucial to our understanding of synapse regulation, development, and function in normal and abnormal brain states.
EXPERIMENTAL PROCEDURES Reeler (rr) and normal (rt, tt) mice (Jackson Lab., U.S.A.) were decapitated at 20-22 days of age and the 37
38
NIKOLAOS MATSOKIS and THEONY VALCANA
cerebellum, the cerebral hemispheres, and mesodiencephalon were removed for immediate use or stored at -20'~C for 10-60 days until utilized for the isolation of synaptic membranes. The synaptosomal membrane fraction was prepared as described by Toffano et al. (1978) and Williams and Risley (1979), with minor modifications. The tissue was homogenized in 5",~ (w/v) of ice cold 0.32 M sucrose using a glass-Teflon homogenizer. The homogenate was centrifuged at 1000g for 20 min. The nuclear pellet was discarded and the supernatant was centrifuged at 48,000g for 20 rain. The pellet was resuspended in 20 vol of distilled H20, homogenized in a glass-glass homogenizer (20 up-and-down strokes) and recentrifuged at 48,000 g for 20 min. The pellet was dispersed again in a glass-glass homogenizer and centrifuged at 8000g for 10min. The mitochondria-free supernatant of this step was centrifuged again at 48,000g for 20 min. The pellet of this centrifugation was resuspended in distilled H20 (50mg wet tissue/ml H20), the resulting membrane preparation is referred to in this work as membrane preparation "A". These membranes were used immediately to determine Na+-dependent GABA binding. For further purification, the membranes, kept at - 2 0 C for 12 24h, were thawed, suspended in 50vol 50mM Tris-citrate buffer (pH 7.1) containing 0.02~0 Triton X-100 and incubated at 37°C for 30 min. This Triton treatment produces the maximal Na+-independent specific GABA binding (Toffano et al., 1978: Williams and Risley, 1979). At the end of the incubation the preparations were centrifuged twice at 48,000g for 20 min, the pellets were dispersed in 50 vol H,O by glass-glass homogenization. This final membrane preparation yielded is referred in this work as membrane preparation "B". In addition to these membrane preparations, nuclear and mitochondrial fractions were isolated in order to compare [3H]GABA binding between these subcellular fractions and the synaptic membranes. The nuclei (1000g pellet) and the mitochondria (8000 g pellet) were washed once with 0.32 M sucrose (50 vol), twice with H20 (50 vol) and then suspended in H20 and analyzed for [3H]GABA binding as described below. Na+-Independent and Na+-dependent [3H]GABA binding was determined according to the methods of Enna et al. (1975) and Toffano et al. (1978) respectively. Samples of homogenates, and the various subcellular fractions, containing 100 500/tg protein, were incubated at 4 C for 15 min in a final volume of 1 ml of 50 mM Tris-citrate buffer, pH 7.1, and various concentrations (5 300 nM) of[3H]GABA, in the absence or presence of 100mM NaCI for the determination of total Na+-independent and Na~-dependent binding respectively. To determine Na+-independent nonspecific binding at each concentration of [3H]GABA, parallel incubations were carried out in the presence of[3H]GABA and 1000 M excess of nonradioactive GABA. To determine Na+-dependent nonspecific binding parallel incubations were carried out in the presence of [3H]GABA and 300 or 1000#m nonradioactive GABA (1000M excess of the highest [3H]GABA concentration used). At the end of the incubation period the assay tubes were centrifuged at 48,000g for 30 min at 4'C. The supernatant was removed and after careful washing of the wall with wet filter paper, the radioactivity of the pellets was determined in a liquid scintillation counter (LS-7000, Beckman) using the following mixture of scintillation fluid: 60 g naphthanok
2g PPO, 0.35g POPOP, 100ml ethylene glycol, 100ml methanol and dioxane to a final volume of 1 1. Specific binding was calculated from the difference between total and nonspecific binding and was analyzed by the Scatchard (1949) method for the determination of the binding constants. The statistical significance of difference between normal and reeler mice was determined by the Student t-test. The protein concentration of the various preparations was determined by the method of Lowry et aL (1951). RESULTS The presentation of our results is in terms of specific binding. In all tissues a n d subfractions studied, the nonspecific binding in the presence and absence of N a ions was not different between normal a n d reeler mice. N a + -Independent [~H]GABA binding
In the reeler m u t a n t the N a ~-independent [3H]GABA binding in the cerebellar synaptosomal m e m b r a n e s is reduced but this binding is unaltered in the c o r r e s p o n d i n g m e m b r a n e s prepared from cerebral hemispheres or mesodiencephalon (Table 1). Our results also reveal that the altered cerebellar binding is unique to the s y n a p t o s o m a l m e m b r a n e s a n d the binding to the cerebellar mitochondrial m e m b r a n e s or crude nuclear fraction is not affected in the reeler (Fig. 1, left). This decreased binding of [3H]GABA is present whether the results are expressed as pmol per mg protein (Table 1) or as pmol per cerebellum (data not s h o w n here). A graphic analysis of a single experiment o f N a + -independent G A B A binding to cerebellar crude synaptosomal m e m b r a n e s " B " is shown in Fig. 2. In the range of 5--250nM of free ligand studied, the kinetic pattern of this and two more experiments revealed two i n d e p e n d e n t binding sites, one of high affinity (Kd, 11 n M ) and low density (Bin,x, = 0.253 p m o l / m g protein) a n d a n o t h e r with 20 times lower affinity (Kd: = 197 n M ) and 4 times higher binding density (Bmax~= 1.073 p m o l / m g protein) (Table 2). The depressed N a ' - i n d e p e n d e n t [3H]GABA binding observed in reeler cerebellar synaptosomal membranes incubated with 25 n M [3H]GABA (Table I; Fig. 1, left) reflects, mainly, changes in the affinity of the first high affinity class of binding sites, because a significant increase in the equilibrium dissociation c o n s t a n t (Kd,) and a non-significant decrease in the maximal binding density (Bronx,) is found. However, the binding c o n s t a n t s of the low affinity binding c o m p o n e n t (Kd2, Bronx:) were not affected in the reeler m u t a t i o n (Table 2). =
[3H]GABA binding in reeler cerebellum
39
Table I. Specific Na+-independent [3H]GABA binding to crude synaptosomal* membranes "B" from various brain regions of normal and reeler mice [3H]GABA binding (pmol/mg protein) Normal Reeler
Cerebellum
Cerebral hemispheres
Mesodiencephalon
0.319 + 0.029t 0.158 + 0.016
0.250 _+0.067 0.254 ___0.075
0.214 _ 0.059 0.218 + 0.061
{50%~}
P < 0.02
*Synaptosomal membranes "B" prepared according to Toffano et al., (1978) were frozen for 1(~18 h and treated with 0.02% Triton X-100 before assayed as described in Experimental Procedures. ?Values represent mean + SE from 4 membrane preparations analyzed in duplicate. Specific Na+-independent binding was taken as the difference between the binding observed at 25 nM [3H]GABA and that observed in the presence of 25 nM [3H]GABA + 25 #M non-radioactive GABA. Numbers in parentheses represent the % change from normal values and P the statistical significance of this change.
Na +-Dependent [3H]GABA binding The Na+-dependent [3H]GABA binding, attributed to a presynaptic uptake system, was studied in cerebellar homogenates from control and reeler mice as well as in various subcellular fractions. Comparison of the Na+-dependent [3H]GABA binding among the fractions examined reveals that the higher binding is associated with the synaptosomal fraction.
O 3 -c
+ N o - independent
No * - dependent
m
Normol
m
Normol
Reeler
[~]
Reeler
-*'~
02
c .5 13o (.9
O1
Nuc
Mit
Syn ("B")
Nu¢
i MIt
Syn ( "A" )
Fig. I. Specific Na+-independent and Na+-dependent [3H]GABA binding to cerebellar cellular subfractions from normal and reeler mice. Bars represent mean _+SE from 445 samples. (Nuc.: nuclei, Mit.: mitochondria, Syn. "B"; synaptosomal membranes treated with Triton X-100, Syn. "A": synaptosomes not treated with Triton X-100. *, P < 0.01, **P < 0.02).
However, considerable binding (50% of that observed in synaptosomes) is found in the nuclear fraction (Fig. l, right). The Na+-dependent binding of [3H]GABA to whole cerebellar homogenates and synaptosomai membranes was higher in the mutant; however, this increase was not observed in the nuclear or the mitochondrial fraction (Fig. 1, right). Na+-Dependent [3H]GABA binding in homogenates from the cerebral hemispheres and mesodiencephalon was not different between normal and reeler mice. This binding, however, increased significantly (66%) in the reeler cerebellar homogenate (Table 3). This increase is reflected mainly in the synaptosomal fraction and was not significantly evident in the other fractions (Fig. l, right). Scatchard analysis of the Na +-dependent [3H]GABA binding to cerebellar homogenates and cerebellar synaptic membranes of type "A" are presented in Figs 3 and 4, respectively. The binding constants estimated from several such experiments are presented in Table 4. These data show 2 binding components, one of high affinity (Kd~ = 127 nM) and low binding density (Bmax, = 5.8 pmol/mg protein) and a second of low affinity (Kd~= 999 riM) and higher density (Bma~2= 28 pmol/mg protein). The affinities of both binding components are similar in cerebellar homogenates and synaptosomal preparations "A", however, the density of both components is much lower in the synaptosomal fraction (Table 4). The increased Na +-dependent GABA binding observed in reeler cerebellar tissue (Table 3, Fig. l, right) reflects an increase in the affinity of the first, low density, component. No significant change however, was found in the low affinity component. The same appears to hold for the increased binding to the synaptosomal membranes (Figs 3, 4; Table 4).
40
NIKOLAOS MATSOKIS and THEONY VALCANA
*--*
NormGI
l
Reeler
F i %
0.6
T
-
20
~ 04 --
i
$
°2i _
Io
o '
~L
c ~ m
s
' ~-I~_.4..
1
l
1oo
200
Free
~
_ 300
(nM)
T
1 0
01
L 02
Bound
l
I
1
I
1
03
04
05
06
07
(pmol/mg
prote~n}
Fig. 2. Scatchard analysis of Na ÷ -independent [3H]GABA binding to crude synaptosomal membrane preparations "B" from cerebellar tissue of normal ( O - - Q ) and reeler ( 0 - - ( 3 ) 20-22-day old mice. Each incubation tube contained 1 ml of buffer and 300-330/~ g of m e m b r a n o u s protein. The abscissa represents the ratio of [3H]GABA bound per m g protein to the concentration of [3H]GABA in the incubation medium. The ordinate represents bound [3H]GABA per m g protein. The error bars indicate the standard deviation of duplicate determinations. The insert depicts the relationship between the binding and free [3H]GABA concentration in the incubation medium.
DISCUSSION
The cerebellum has a high density of GABA receptors and the binding characteristics of these receptors have been well established. In the absence of Na ions, Triton X-100-treated synaptic membranes show an heterogeneous population of binding sites: a high affinity component with a Kd of 16 nM and Bmax of 1.8 pmol/mg protein and a low affinity
component with a Kd of l l l - 1 3 0 n M and Bmax of 5.3pmol/mg protein. Our results on Na+-inde pendent GABA binding (Table 2) are in full agreement with these reported values. In the presence of Na ÷ ions synaptic membranes not treated with Triton X-100 show one binding component with Kd= 1.2nM and Bmax= 3 0 p m o l / m g protein (see Enna et al., 1975; Wong and Borng, 1977; Toffano et al., 1978; Ticku, 1979; Teichberg, 1980). In analyzing
Table 2. Binding constants of Na+-independent [3H]GABA binding to cerebellar crude synaptosomal membranes "B" from normal and reeler mice Na+-independent [3H]GABA binding Kd~ (nM)
nmaxn ( pmol ~ \mg ProteinJ
Kd2 (nM)
Bmax~ ( pmol ~ \mg ProteinJ
11 _ 0.5t 0.253 4- 0.057 196.7 _+2.9 1.073 + 0.107 17 __.1.1 0.180 ± 0.017 234.3 4- 23.4 0.830 + 0.025 P < 0.05 tValues represent means SE from three synaptosomal membrane "B" preparations assayed in duplicate. The binding constants (Kd, Bmax)were estimated from graphic representation of the binding data according to Scatchard (1949). An example of one such experiment is shown in Fig. 2.
Normal Reeler
[3H]GABA binding in reeler cerebellum
41
Table 3. SpecificNa +-dependent [3H]GABAbinding to crude synaptosomes (membranes "A")* isolated from various brain regions of normal and reeler mice. [3H]GABA binding (pmol/mg protein) Cerebellum Cerebral hemispheres Mesodiencephalon Normal 0.17 _+0.013 0.132 + 0.030 0.102 +_0.011 Reeler 0.296 _ 0.011 0.137 _+0.023 0.099 _+0.017 (66%1') P < 0.05 *Synaptosomal membranes "A" were prepared from fresh tissue according to the method of Toffano 0978) and assayed in duplicate under the incubation conditions described in Experimental Procedures. Specific binding was taken as the differencebetween the binding observed in the presence of 25 nM [3H]GABA and that observed in the presence of 25nM [~H]GABA and 25/~M nonradioactive GABA. tValues represent the mean _ SE of four synaptosomal preparations assayed in duplicate, numbers in parentheses the 70 change from normal values and P the statistical significance of this change.
o u r N a + - d e p e n d e n t binding data, deviations from M i c h a e l i s - M e n t e n kinetics were consistently observed at low ligand concentrations, particularly in the reeler. These results suggest the existence o f a high affinity, low density b i n d i n g site in a d d i t i o n to the low affinity site reported in the literature. The presence o f this binding site while only suggestive in the control animals (binding c o n s t a n t s reported in Table 4 are only approximations), is very p r o m i n a n t in the reeler
o--o
m u t a n t s . The affinity o f these binding sites is similar in b o t h h o m o g e n a t e s a n d s y n a p t o s o m a l p r e p a r a t i o n s ( " A " ) b u t the binding density o f s y n a p t o s o m a l prepa r a t i o n s is lower t h a n t h a t f o u n d in total cerebellar homogenates. This finding m a y be related to the fact t h a t a considerable N a + - d e p e n d e n t binding (50% o f synaptosomes) is f o u n d in the nuclear fraction (Fig. 1, right), a n o b s e r v a t i o n indicating t h a t the nuclear fraction m a y c o n t a i n synaptic terminals rich in
Normol c
%
o-.--o Reeler
I0
--
~
f;
A
T
m ,,,-
~-
4
ee (n
7 rn 2
I
1
I
I
I
I
2
4
(5
8
10
t2
Bound ( p m o l / m g protein)
Fig. 3. Scatchard analysis of Na +-dependent [3H]GABA binding to cerebellar homogenates from normal ( O - - O ) and reeler ( O - - © ) 20-22-day old mice. Each incubation tube contained 1 ml of buffer and l 1 8 # g protein for normal or 99#g protein for reeler mice. The ordinate represents the amount of [3H]GABA bound per mg of protein, and the abeissa the ratio of the [3H]GABA bound per/rag protein to the concentration of [3H]GABA in the incubation medium. The error bars indicate the standard deviation of duplicate determinations. The insert illustrates the relationship between the binding and free [3H]GABA concentration.
42
NIKOLAOS MATSOKIS and THEONY VALCANA
6
~o 5
25
%
c~ 4 E
2O CL
v 15
2
g
eJ-
0 4O L~_
5
k~,,,~
-t@ -
500 Free (nM)
~000
~'[
~0
0
2 4 6 Bound ( pmol/mg profe~n)
8
Fig. 4. Scatchard analysis of Na*-dependent [3H]GABA binding to crude cerebellar synaptosomes (membranes " A " ) from normal ( O - - O ) and reeler ( O - - © ) 20-22-day old
mice. The membranes were prepared and incubated as described under Experimental Procedures. See legend to Fig. 3 for explanation of the abscissa, ordinate, insert, and error bars. Na+-dependent binding. The presence of GABA binding in the cerebellar nuclear fraction has been previously reported by Kingsbury et al. (1980) and has been related to the presence of mossy fiber glomeruli that sediment in this fraction. The main questions raised in this study were whether the previously reported alteration in cerebellar GABA binding in the reeler mouse (Olsen and Mikoshiba, 1978; Matsokis et al., 1982) reflected changes in the affinity or the number of binding
sites, and whether both Nat-independent and Na +-dependent GABA binding were similarly affected. Our results show that the Na+-independent and Na +-dependent binding components of the reeler cerebellum are affected differently. That is, the Na ÷-independent GABA binding decreases while the Na +-dependent binding increases. These changes are due to an increase and a decrease of the equilibrium dissociation constants of the high affinity components of the Na + -independent and Na +-dependent binding systems, respectively (Tables 2, 4). The changes in affinity of GABA binding observed in this study may occur in the Purkinje/deep nuclei cell synapses, the mossy fiber/granule cell synapses or the heterologous mossy fiber/Purkinje cell synapses and could reflect, as in other experimental colfditions, alterations in feedback regulatory mechanisms wherein the sensitivity of neuroceptors changes when the supply of the neurotransmitter is altered by interruption of the afferent fibers or after drug treatment (Cannon and Rosenbleuth, 1949; Reisine et al., 1977; Lloyd and Davidson, 1979; Cross and Waddington, 1981; Biggio et al., 1981; Tamminga, 1981). With respect to the GABA receptor system, for example, it is known that the affinity of the Na +-independent GABA binding is increased in Huntington's Chorea (Lloyd and Davidson, 1979). Similarly the number of high affinity binding sites increases in the rat substantia nigra following striatal lesions with kainic acid (Waddington and Cross, 1979). Our unpublished data and those of Slevin et al. (1982) indicate that denervation hypersensitivity may also characterize the glutaminergic synapse in the reeler cerebellum, since an increase in the density of glutamic acid binding sites is found in this condition. The decreased affinity of the Na+-independent GABA binding and the increased affinity of the Na+-dependent binding, evidenced only in the
Table 4. Binding constants of Na ÷-dependent [~HIGABA binding to cerebellar homogenate and membrane "A'" preparations from normal and reeler mice Homogenate Bm~~'
Kd' (nM)
( \
pmol ~ /
Membranes "A" B~
~
Kd2 (nM)
(_ pmol ~ \mg Protein/
Kd '
(nM)
Bin,"~ (pmol ] \mg Protein/
Kd: (nM)
B,,,~ (pmol ~ \mg Protein/'
i27-+ 8t 5.8_+0.2 999_+8i 28+_I.I 162_+9 /[7_+0.2 968_+98 8_+1.2 (3) (3) (3) (3) (4) 14) (4) (4) 84_+4 4+0.9 943 _+97 31 +0.8 76 2 913 10 Reeler (3) (3) (3) (3) II) II) (1) (I) P < 0.05 The binding constants (Kd, Bmax)were estimated from graphic representation of the binding data according to Scatchard (1949). *The values represent means + SE of a number of binding experiments shown in parentheses. In each experiment five cerebellar from normal or reeler mice were used for each homogenate preparation and 5-15 for each membrane "A" preparation. Each preparation was assayed in duplicate as described in Experimental Procedures. Normal
[3H]GABA binding in reeler cerebellum cerebellum of the reeler brain, m a y be interpreted to indicate, respectively, h y p o - a n d hypersensitivity o f the postsynaptic a n d presynaptic elements the cerebellum G A B A e r g i c synapses, induced by the depressed excitatory granule cell i n p u t a n d / o r the increased mossy fiber c o n t a c t with the ectopic Purkinje cells. REFERENCES Biggio G., Corda M. G., Concas A. and Gessa G. L. (1981) Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra. Brain Res. 220, 334-349. Cannon W. B. and Rosenbleuth A. (1949) The Supersensitivity of Denervated Structure. Macmillan, New York. Crepel F. and Mariani J. (1976) Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the weaver mutant mouse. J. Neurobiol. 7, 579-582. Cross A. J. and Waddington J. L. (1981) Substantia nigra GABA receptors in Huntington's disease. J. Neurochem. 37, 321-324. Enna S. J. and Snyder S. H. (1975) Properties of GABA receptor binding in rat brain synaptic membrane fractions. Brain Res. 100, 81-97. Hamburgh M. (1960) Observations on the neuropathology of "reeler", a neurobiological mutation in mice. Experientia 16, 460-461. Henkart M. (1980) Identification and function of intracellular calcium stores in axons and cell bodies of neurons. Fedn. Proc. 39, 2783-2789, Hoffer B., Siggins G. R. and Bloom F. E. (1971) Studies on NE-containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to NE and related substances administered by microiontophoresis. Brain Res. 25, 523-534. Hudson D. B., Valcana T., Bean G. L. and Timiras P. S. (1976a) Glutamic acid: a strong candidate as the neurotransmitter of the cerebellar granule cell. Neurochem. Res. 1, 73-81. Hudson D. B., Valcana T. and Timiras P. S. (1976b) Monoamine metabolism in the developing rat brain and effects of ionizing radiation. Brain Res. 114, 471-479. Johnston M. V. and Coyle J. T. (1982) Cytotoxic lesions and the development of transmitter systems. Trends Neurosci. 5, 153-156. Kingsbury A. E., Wilkin G. P., Patel A. J. and Balhzs R. (1980) Distribution of GABA receptors in the rat cerebellum. J. Neurochem. 35, 739-742. Landis S. C., Shoemaker W. J., Schlumpf M. and Bloom F. E. (1975) Catecholamines in mutant mouse cerebellum: fluorescence microscopic and chemical studies. Brain Res. 93, 253-266. Lloyd K. J. and Davidson L. (1979) 3H-GABA binding in brain from Huntington's chorea patients: Altered regulation by phospholipids. Science 205, 1147-1149. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Martin D. L. (1976) Carrier--mediated transport and removal of GABA from synaptic regions. In: GABA in Nervous System Function (Roberts E., Chase T. N. and Tower D. B., eds), pp. 347-486. Raven Press, New York.
43
Matsokis N., Argentaki M. and Valcana T. (1982) Na +-dependent and Na +-independent 3H-GABA binding in Reeler Cerebellar dysgenesis. In: Third International Meeting of the International Society for Developmental Neuroscience. (Abstract) p. 155. McBride W. J., Aprison M. H. and Kusano K. (1976a) Contents of several amino acids in the cerebellum brain stem and cerebrum of the "staggerer", "weaver" and "nervous" neurological mutant mice. J. Neurochem. 26, 867-870. McBride W. J., Nadi N. S., Altman J. and Aprison M. H. (1976b) Effect of selective doses of X-irradiation on the levels of several amino acids in the cerebellum of the rat. Neurochem. Res. 1, 141-152. Olsen R. W. and Mikoshiba K. (1978) Localization of GABA receptor binding in the mammalian cerebellum high levels in granule layer and depletion in agranular cerebella of mutant mice. J. Neurochem. 30, 1633-1636. Purves D. and Lichtman J. W. 0980) Elimination of synapses in the developing nervous system. Science 210, 153-157. Rakic P. (1976) Synaptic specificity in cerebellar cortex: study of anomalous circuits induced by single gene mutations in mice, Cold Spring Harbor Syrup. quant. Biol. 40, 333-346. Reisine T. D., Fields J. Z. and Yamamura K. I. (1977) Neurotransmitter receptor alterations in Parkinson's disease. Life Sci. 21, 335-343. Roffier-Tarlov S. and Sidman R. L. (1978) Concentrations of glutamic acid in cerebellar cortex and deep nuclei of normal mice and weaver, staggerer and nervous mutants. Brain Res. 142, 269-283. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Sidman R. L., Green M. C. and Appel S. H. (1965) Catalog of the Neurological Mutants o f the Mouse. Harvard University Press, Cambridge, MA. Sidman R. L. (1974) Interaction among developing mammalian brain cells in the cell surface. In: Proceedings o f the International Society o f Developmental Biologists (Moscona A. A., ed.), pp. 222-253. Wiley, New York. Slevin J. T., Johnston M. V. and Coyle J. T. (1982) Methylazoxy-methanol acetate ablation of mouse cerebellar granule cells: Effects on synaptic neurochemistry. Devl. Neurosci. 5, 3-12. Soreq H., Gurwitz D., Eliyahu D. and Sokolovsky M. (1982) Altered ontogenesis of muscarinic receptors in agranular cerebellar cortex. J. Neurochem. 39, 756-763. Tamminga C. A. (1981) Tardive dyskinesia and the dopamine receptors. In: Neuroreceptors (Usdin E., Bunney W. E. and Davis J. M., eds), pp. 231-240. Wiley, New York. Teichberg V. I. (1980) Amino acid receptors. In: Cellular Receptors of Hormones and Neurotransmitters (Schnister D. and Levitzki A., eds), pp. 369-395. Wiley, New York. Ticku M. K. (1979) Differences in GABA receptor sensitivity in inbred strains of mice. J. Neurochem. 33, 1135-1138. Toffano G., Guidotti A. and Costa E. (1978) Purification of an endogenous protein inhibitor of the high affinity binding of GABA to synaptic membranes of rat brain. Proc. natn. Acad. Sci., U.S.A. 75, 4024-4028. Valcana T., Hudson D. and Timiras P. S. (1972) Effects of X-irradiation on the content of amino acids in the developing rat cerebellum. J. Neurochem. 19, 2229-2232.
44
NIKOLAOS MATSOKIS and THEONY VALCANA
Valcana T., Liao C. and Timiras P. S. (1974a) Effects of X-irradiation on the subcellular distribution of cholinergic enzymes in the developing rat cerebellum. Brain Res. 73, 105-120. Valcana T., Liao C. and Timiras P. S. (1974b) Effects of X-radiation on the development of the cholinergic system of the rat brain. II. Investigation of alterations in acetycholine content. Envir. Physiol. Biochem. 4, 58-63. Vizi E. S. (1979) Presynaptic modulation of neurochemical transmission. Prog. Neurobiol. 12, 181-290. Waddin~ton J. L. and Cross A. J. (1978) Denervation supersensitivity in the striatonigral GABA pathway. Nature 276, 618-620.
Williams M. and Risley E. A. (1979) Characterization of the binding of 3H-muscimol, a potent GABA agonist, to rat brain synaptosomal membranes using a filtration assay. J. Neurochem. 32, 713-718. Wilson L., Sotelo C. and Caviness V. S. Jr (1981) Heterologous synapses upon Purkinje cells in the cerebellum of the reeler mutant mouse: an experimental light and electron microscopy study. Brain Res. 213, 63-82. Wong D. T. and Borng J. S. (1977) Na+-independent binding of GABA to the Triton X-100 treated synaptic membranes from cerebellum of rat brain. Life Sci. 20, 445-452.
0197-0186/85 $3.00+ 0.00 Copyright © 1985PergamonPress Ltd
[3H]GABA B I N D I N G IN THE CEREBELLUM OF THE REELER M U R I N E M U T A N T NIKOLAOS MATSOKISand THEONY VALCANA Laboratory of Human and Animal Physiology, Department of Biology, University of Patra, Patra, Greece (Received 13 January 1984; accepted 11 April 1984) Abstract--In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [3H]GABA binding have been observed. This study shows decreased affinity of the Na +-independent, high affinityGABA binding component of synaptosomal membranes and an increased affinity of the Na +-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na +-dependent and Na+-independent binding sites was not significantly altered. The decreased affinity of the Na+-independent GABA binding and the increased affinity of the Na+-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.
The reduction of the cerebellar granule cell population which accompanies not only some murine mutations but also neonatal exposure to X-radiation, cytotoxic drugs, or viral infection has been shown to lead to changes in the relative density of interneurons and afferent components, as well as to changes in the concentration of various neurotransmitter substances and the activity of associated enzymes (Hoffer et al., 1971; Valcana et al., 1972, 1974a, b; Crepel and Mariani, 1976; Hudson et al., 1976a, b; Landis et aL, 1975; McBride et al., 1976a, b; Roffler-Tarlov and Sidman, 1978; Sievin et al., 1982; Johnston and Coyler, 1982; Soreq et al., 1982). In the reeler mutant mouse the Purkinje, Golgi II and stellate cells of the cerebellum and the cells of the deep nuclei are spared (Hamburgh, 1960; Sidman el al., 1965; Sidman, 1974; Rakic 1976; Wilson et al., 1981) and this condition is accompanied by a significant increase in ~-aminobutyric acid (GABA) concentration as well as other markers of the GABAergic system (Johnston and Coyler, 1982). However, this GABA enrichment may be due to compensatory changes resulting from the degeneration of the excitatory granule cell input to the Purkinje cell system, a loss that would affect such processes as transmitter transport, transmitter release, presynaptic GABA uptake, and postsynaptic GABA receptor properties (Martin, 1976; Vizi, 1979; Henkart, 1980; Purves and Lichtman, 1980).
The presence of GABA receptors in brain tissue has been established by determining specific [3H]GABA binding to crude synaptic membranes, and such binding has been observed in both the presence and absence of Na ÷ ions. The Na ÷-dependent binding is thought to represent the presynaptic re-uptake receptor system and the Na ÷-independent binding, the postsynaptic receptor system. A decrease in Na ÷-independent GABA binding in the reeler cerebellum has been reported from this and other laboratories (Olsen and Mikoshiba 1978; Matsokis et al., 1982). However, whether these alterations result from changes in receptor binding sites or reflect changes in affinity is not clear. The purpose of this study, therefore, is to elucidate the characteristics of Na ÷-dependent as well as Na ÷-independent GABA binding in control and reeler mice. The study of the GABAergic synapse in the reeler mutant cerebellum, where the cytoarchitectural changes are well known may allow us to relate the anatomical to the biochemical aspects of synaptogenesis which are crucial to our understanding of synapse regulation, development, and function in normal and abnormal brain states.
EXPERIMENTAL PROCEDURES Reeler (rr) and normal (rt, tt) mice (Jackson Lab., U.S.A.) were decapitated at 20-22 days of age and the 37
38
NIKOLAOS MATSOKIS and THEONY VALCANA
cerebellum, the cerebral hemispheres, and mesodiencephalon were removed for immediate use or stored at -20'~C for 10-60 days until utilized for the isolation of synaptic membranes. The synaptosomal membrane fraction was prepared as described by Toffano et al. (1978) and Williams and Risley (1979), with minor modifications. The tissue was homogenized in 5",~ (w/v) of ice cold 0.32 M sucrose using a glass-Teflon homogenizer. The homogenate was centrifuged at 1000g for 20 min. The nuclear pellet was discarded and the supernatant was centrifuged at 48,000g for 20 rain. The pellet was resuspended in 20 vol of distilled H20, homogenized in a glass-glass homogenizer (20 up-and-down strokes) and recentrifuged at 48,000 g for 20 min. The pellet was dispersed again in a glass-glass homogenizer and centrifuged at 8000g for 10min. The mitochondria-free supernatant of this step was centrifuged again at 48,000g for 20 min. The pellet of this centrifugation was resuspended in distilled H20 (50mg wet tissue/ml H20), the resulting membrane preparation is referred to in this work as membrane preparation "A". These membranes were used immediately to determine Na+-dependent GABA binding. For further purification, the membranes, kept at - 2 0 C for 12 24h, were thawed, suspended in 50vol 50mM Tris-citrate buffer (pH 7.1) containing 0.02~0 Triton X-100 and incubated at 37°C for 30 min. This Triton treatment produces the maximal Na+-independent specific GABA binding (Toffano et al., 1978: Williams and Risley, 1979). At the end of the incubation the preparations were centrifuged twice at 48,000g for 20 min, the pellets were dispersed in 50 vol H,O by glass-glass homogenization. This final membrane preparation yielded is referred in this work as membrane preparation "B". In addition to these membrane preparations, nuclear and mitochondrial fractions were isolated in order to compare [3H]GABA binding between these subcellular fractions and the synaptic membranes. The nuclei (1000g pellet) and the mitochondria (8000 g pellet) were washed once with 0.32 M sucrose (50 vol), twice with H20 (50 vol) and then suspended in H20 and analyzed for [3H]GABA binding as described below. Na+-Independent and Na+-dependent [3H]GABA binding was determined according to the methods of Enna et al. (1975) and Toffano et al. (1978) respectively. Samples of homogenates, and the various subcellular fractions, containing 100 500/tg protein, were incubated at 4 C for 15 min in a final volume of 1 ml of 50 mM Tris-citrate buffer, pH 7.1, and various concentrations (5 300 nM) of[3H]GABA, in the absence or presence of 100mM NaCI for the determination of total Na+-independent and Na~-dependent binding respectively. To determine Na+-independent nonspecific binding at each concentration of [3H]GABA, parallel incubations were carried out in the presence of[3H]GABA and 1000 M excess of nonradioactive GABA. To determine Na+-dependent nonspecific binding parallel incubations were carried out in the presence of [3H]GABA and 300 or 1000#m nonradioactive GABA (1000M excess of the highest [3H]GABA concentration used). At the end of the incubation period the assay tubes were centrifuged at 48,000g for 30 min at 4'C. The supernatant was removed and after careful washing of the wall with wet filter paper, the radioactivity of the pellets was determined in a liquid scintillation counter (LS-7000, Beckman) using the following mixture of scintillation fluid: 60 g naphthanok
2g PPO, 0.35g POPOP, 100ml ethylene glycol, 100ml methanol and dioxane to a final volume of 1 1. Specific binding was calculated from the difference between total and nonspecific binding and was analyzed by the Scatchard (1949) method for the determination of the binding constants. The statistical significance of difference between normal and reeler mice was determined by the Student t-test. The protein concentration of the various preparations was determined by the method of Lowry et aL (1951). RESULTS The presentation of our results is in terms of specific binding. In all tissues a n d subfractions studied, the nonspecific binding in the presence and absence of N a ions was not different between normal a n d reeler mice. N a + -Independent [~H]GABA binding
In the reeler m u t a n t the N a ~-independent [3H]GABA binding in the cerebellar synaptosomal m e m b r a n e s is reduced but this binding is unaltered in the c o r r e s p o n d i n g m e m b r a n e s prepared from cerebral hemispheres or mesodiencephalon (Table 1). Our results also reveal that the altered cerebellar binding is unique to the s y n a p t o s o m a l m e m b r a n e s a n d the binding to the cerebellar mitochondrial m e m b r a n e s or crude nuclear fraction is not affected in the reeler (Fig. 1, left). This decreased binding of [3H]GABA is present whether the results are expressed as pmol per mg protein (Table 1) or as pmol per cerebellum (data not s h o w n here). A graphic analysis of a single experiment o f N a + -independent G A B A binding to cerebellar crude synaptosomal m e m b r a n e s " B " is shown in Fig. 2. In the range of 5--250nM of free ligand studied, the kinetic pattern of this and two more experiments revealed two i n d e p e n d e n t binding sites, one of high affinity (Kd, 11 n M ) and low density (Bin,x, = 0.253 p m o l / m g protein) a n d a n o t h e r with 20 times lower affinity (Kd: = 197 n M ) and 4 times higher binding density (Bmax~= 1.073 p m o l / m g protein) (Table 2). The depressed N a ' - i n d e p e n d e n t [3H]GABA binding observed in reeler cerebellar synaptosomal membranes incubated with 25 n M [3H]GABA (Table I; Fig. 1, left) reflects, mainly, changes in the affinity of the first high affinity class of binding sites, because a significant increase in the equilibrium dissociation c o n s t a n t (Kd,) and a non-significant decrease in the maximal binding density (Bronx,) is found. However, the binding c o n s t a n t s of the low affinity binding c o m p o n e n t (Kd2, Bronx:) were not affected in the reeler m u t a t i o n (Table 2). =
[3H]GABA binding in reeler cerebellum
39
Table I. Specific Na+-independent [3H]GABA binding to crude synaptosomal* membranes "B" from various brain regions of normal and reeler mice [3H]GABA binding (pmol/mg protein) Normal Reeler
Cerebellum
Cerebral hemispheres
Mesodiencephalon
0.319 + 0.029t 0.158 + 0.016
0.250 _+0.067 0.254 ___0.075
0.214 _ 0.059 0.218 + 0.061
{50%~}
P < 0.02
*Synaptosomal membranes "B" prepared according to Toffano et al., (1978) were frozen for 1(~18 h and treated with 0.02% Triton X-100 before assayed as described in Experimental Procedures. ?Values represent mean + SE from 4 membrane preparations analyzed in duplicate. Specific Na+-independent binding was taken as the difference between the binding observed at 25 nM [3H]GABA and that observed in the presence of 25 nM [3H]GABA + 25 #M non-radioactive GABA. Numbers in parentheses represent the % change from normal values and P the statistical significance of this change.
Na +-Dependent [3H]GABA binding The Na+-dependent [3H]GABA binding, attributed to a presynaptic uptake system, was studied in cerebellar homogenates from control and reeler mice as well as in various subcellular fractions. Comparison of the Na+-dependent [3H]GABA binding among the fractions examined reveals that the higher binding is associated with the synaptosomal fraction.
O 3 -c
+ N o - independent
No * - dependent
m
Normol
m
Normol
Reeler
[~]
Reeler
-*'~
02
c .5 13o (.9
O1
Nuc
Mit
Syn ("B")
Nu¢
i MIt
Syn ( "A" )
Fig. I. Specific Na+-independent and Na+-dependent [3H]GABA binding to cerebellar cellular subfractions from normal and reeler mice. Bars represent mean _+SE from 445 samples. (Nuc.: nuclei, Mit.: mitochondria, Syn. "B"; synaptosomal membranes treated with Triton X-100, Syn. "A": synaptosomes not treated with Triton X-100. *, P < 0.01, **P < 0.02).
However, considerable binding (50% of that observed in synaptosomes) is found in the nuclear fraction (Fig. l, right). The Na+-dependent binding of [3H]GABA to whole cerebellar homogenates and synaptosomai membranes was higher in the mutant; however, this increase was not observed in the nuclear or the mitochondrial fraction (Fig. 1, right). Na+-Dependent [3H]GABA binding in homogenates from the cerebral hemispheres and mesodiencephalon was not different between normal and reeler mice. This binding, however, increased significantly (66%) in the reeler cerebellar homogenate (Table 3). This increase is reflected mainly in the synaptosomal fraction and was not significantly evident in the other fractions (Fig. l, right). Scatchard analysis of the Na +-dependent [3H]GABA binding to cerebellar homogenates and cerebellar synaptic membranes of type "A" are presented in Figs 3 and 4, respectively. The binding constants estimated from several such experiments are presented in Table 4. These data show 2 binding components, one of high affinity (Kd~ = 127 nM) and low binding density (Bmax, = 5.8 pmol/mg protein) and a second of low affinity (Kd~= 999 riM) and higher density (Bma~2= 28 pmol/mg protein). The affinities of both binding components are similar in cerebellar homogenates and synaptosomal preparations "A", however, the density of both components is much lower in the synaptosomal fraction (Table 4). The increased Na +-dependent GABA binding observed in reeler cerebellar tissue (Table 3, Fig. l, right) reflects an increase in the affinity of the first, low density, component. No significant change however, was found in the low affinity component. The same appears to hold for the increased binding to the synaptosomal membranes (Figs 3, 4; Table 4).
40
NIKOLAOS MATSOKIS and THEONY VALCANA
*--*
NormGI
l
Reeler
F i %
0.6
T
-
20
~ 04 --
i
$
°2i _
Io
o '
~L
c ~ m
s
' ~-I~_.4..
1
l
1oo
200
Free
~
_ 300
(nM)
T
1 0
01
L 02
Bound
l
I
1
I
1
03
04
05
06
07
(pmol/mg
prote~n}
Fig. 2. Scatchard analysis of Na ÷ -independent [3H]GABA binding to crude synaptosomal membrane preparations "B" from cerebellar tissue of normal ( O - - Q ) and reeler ( 0 - - ( 3 ) 20-22-day old mice. Each incubation tube contained 1 ml of buffer and 300-330/~ g of m e m b r a n o u s protein. The abscissa represents the ratio of [3H]GABA bound per m g protein to the concentration of [3H]GABA in the incubation medium. The ordinate represents bound [3H]GABA per m g protein. The error bars indicate the standard deviation of duplicate determinations. The insert depicts the relationship between the binding and free [3H]GABA concentration in the incubation medium.
DISCUSSION
The cerebellum has a high density of GABA receptors and the binding characteristics of these receptors have been well established. In the absence of Na ions, Triton X-100-treated synaptic membranes show an heterogeneous population of binding sites: a high affinity component with a Kd of 16 nM and Bmax of 1.8 pmol/mg protein and a low affinity
component with a Kd of l l l - 1 3 0 n M and Bmax of 5.3pmol/mg protein. Our results on Na+-inde pendent GABA binding (Table 2) are in full agreement with these reported values. In the presence of Na ÷ ions synaptic membranes not treated with Triton X-100 show one binding component with Kd= 1.2nM and Bmax= 3 0 p m o l / m g protein (see Enna et al., 1975; Wong and Borng, 1977; Toffano et al., 1978; Ticku, 1979; Teichberg, 1980). In analyzing
Table 2. Binding constants of Na+-independent [3H]GABA binding to cerebellar crude synaptosomal membranes "B" from normal and reeler mice Na+-independent [3H]GABA binding Kd~ (nM)
nmaxn ( pmol ~ \mg ProteinJ
Kd2 (nM)
Bmax~ ( pmol ~ \mg ProteinJ
11 _ 0.5t 0.253 4- 0.057 196.7 _+2.9 1.073 + 0.107 17 __.1.1 0.180 ± 0.017 234.3 4- 23.4 0.830 + 0.025 P < 0.05 tValues represent means SE from three synaptosomal membrane "B" preparations assayed in duplicate. The binding constants (Kd, Bmax)were estimated from graphic representation of the binding data according to Scatchard (1949). An example of one such experiment is shown in Fig. 2.
Normal Reeler
[3H]GABA binding in reeler cerebellum
41
Table 3. SpecificNa +-dependent [3H]GABAbinding to crude synaptosomes (membranes "A")* isolated from various brain regions of normal and reeler mice. [3H]GABA binding (pmol/mg protein) Cerebellum Cerebral hemispheres Mesodiencephalon Normal 0.17 _+0.013 0.132 + 0.030 0.102 +_0.011 Reeler 0.296 _ 0.011 0.137 _+0.023 0.099 _+0.017 (66%1') P < 0.05 *Synaptosomal membranes "A" were prepared from fresh tissue according to the method of Toffano 0978) and assayed in duplicate under the incubation conditions described in Experimental Procedures. Specific binding was taken as the differencebetween the binding observed in the presence of 25 nM [3H]GABA and that observed in the presence of 25nM [~H]GABA and 25/~M nonradioactive GABA. tValues represent the mean _ SE of four synaptosomal preparations assayed in duplicate, numbers in parentheses the 70 change from normal values and P the statistical significance of this change.
o u r N a + - d e p e n d e n t binding data, deviations from M i c h a e l i s - M e n t e n kinetics were consistently observed at low ligand concentrations, particularly in the reeler. These results suggest the existence o f a high affinity, low density b i n d i n g site in a d d i t i o n to the low affinity site reported in the literature. The presence o f this binding site while only suggestive in the control animals (binding c o n s t a n t s reported in Table 4 are only approximations), is very p r o m i n a n t in the reeler
o--o
m u t a n t s . The affinity o f these binding sites is similar in b o t h h o m o g e n a t e s a n d s y n a p t o s o m a l p r e p a r a t i o n s ( " A " ) b u t the binding density o f s y n a p t o s o m a l prepa r a t i o n s is lower t h a n t h a t f o u n d in total cerebellar homogenates. This finding m a y be related to the fact t h a t a considerable N a + - d e p e n d e n t binding (50% o f synaptosomes) is f o u n d in the nuclear fraction (Fig. 1, right), a n o b s e r v a t i o n indicating t h a t the nuclear fraction m a y c o n t a i n synaptic terminals rich in
Normol c
%
o-.--o Reeler
I0
--
~
f;
A
T
m ,,,-
~-
4
ee (n
7 rn 2
I
1
I
I
I
I
2
4
(5
8
10
t2
Bound ( p m o l / m g protein)
Fig. 3. Scatchard analysis of Na +-dependent [3H]GABA binding to cerebellar homogenates from normal ( O - - O ) and reeler ( O - - © ) 20-22-day old mice. Each incubation tube contained 1 ml of buffer and l 1 8 # g protein for normal or 99#g protein for reeler mice. The ordinate represents the amount of [3H]GABA bound per mg of protein, and the abeissa the ratio of the [3H]GABA bound per/rag protein to the concentration of [3H]GABA in the incubation medium. The error bars indicate the standard deviation of duplicate determinations. The insert illustrates the relationship between the binding and free [3H]GABA concentration.
42
NIKOLAOS MATSOKIS and THEONY VALCANA
6
~o 5
25
%
c~ 4 E
2O CL
v 15
2
g
eJ-
0 4O L~_
5
k~,,,~
-t@ -
500 Free (nM)
~000
~'[
~0
0
2 4 6 Bound ( pmol/mg profe~n)
8
Fig. 4. Scatchard analysis of Na*-dependent [3H]GABA binding to crude cerebellar synaptosomes (membranes " A " ) from normal ( O - - O ) and reeler ( O - - © ) 20-22-day old
mice. The membranes were prepared and incubated as described under Experimental Procedures. See legend to Fig. 3 for explanation of the abscissa, ordinate, insert, and error bars. Na+-dependent binding. The presence of GABA binding in the cerebellar nuclear fraction has been previously reported by Kingsbury et al. (1980) and has been related to the presence of mossy fiber glomeruli that sediment in this fraction. The main questions raised in this study were whether the previously reported alteration in cerebellar GABA binding in the reeler mouse (Olsen and Mikoshiba, 1978; Matsokis et al., 1982) reflected changes in the affinity or the number of binding
sites, and whether both Nat-independent and Na +-dependent GABA binding were similarly affected. Our results show that the Na+-independent and Na +-dependent binding components of the reeler cerebellum are affected differently. That is, the Na ÷-independent GABA binding decreases while the Na +-dependent binding increases. These changes are due to an increase and a decrease of the equilibrium dissociation constants of the high affinity components of the Na + -independent and Na +-dependent binding systems, respectively (Tables 2, 4). The changes in affinity of GABA binding observed in this study may occur in the Purkinje/deep nuclei cell synapses, the mossy fiber/granule cell synapses or the heterologous mossy fiber/Purkinje cell synapses and could reflect, as in other experimental colfditions, alterations in feedback regulatory mechanisms wherein the sensitivity of neuroceptors changes when the supply of the neurotransmitter is altered by interruption of the afferent fibers or after drug treatment (Cannon and Rosenbleuth, 1949; Reisine et al., 1977; Lloyd and Davidson, 1979; Cross and Waddington, 1981; Biggio et al., 1981; Tamminga, 1981). With respect to the GABA receptor system, for example, it is known that the affinity of the Na +-independent GABA binding is increased in Huntington's Chorea (Lloyd and Davidson, 1979). Similarly the number of high affinity binding sites increases in the rat substantia nigra following striatal lesions with kainic acid (Waddington and Cross, 1979). Our unpublished data and those of Slevin et al. (1982) indicate that denervation hypersensitivity may also characterize the glutaminergic synapse in the reeler cerebellum, since an increase in the density of glutamic acid binding sites is found in this condition. The decreased affinity of the Na+-independent GABA binding and the increased affinity of the Na+-dependent binding, evidenced only in the
Table 4. Binding constants of Na ÷-dependent [~HIGABA binding to cerebellar homogenate and membrane "A'" preparations from normal and reeler mice Homogenate Bm~~'
Kd' (nM)
( \
pmol ~ /
Membranes "A" B~
~
Kd2 (nM)
(_ pmol ~ \mg Protein/
Kd '
(nM)
Bin,"~ (pmol ] \mg Protein/
Kd: (nM)
B,,,~ (pmol ~ \mg Protein/'
i27-+ 8t 5.8_+0.2 999_+8i 28+_I.I 162_+9 /[7_+0.2 968_+98 8_+1.2 (3) (3) (3) (3) (4) 14) (4) (4) 84_+4 4+0.9 943 _+97 31 +0.8 76 2 913 10 Reeler (3) (3) (3) (3) II) II) (1) (I) P < 0.05 The binding constants (Kd, Bmax)were estimated from graphic representation of the binding data according to Scatchard (1949). *The values represent means + SE of a number of binding experiments shown in parentheses. In each experiment five cerebellar from normal or reeler mice were used for each homogenate preparation and 5-15 for each membrane "A" preparation. Each preparation was assayed in duplicate as described in Experimental Procedures. Normal
[3H]GABA binding in reeler cerebellum cerebellum of the reeler brain, m a y be interpreted to indicate, respectively, h y p o - a n d hypersensitivity o f the postsynaptic a n d presynaptic elements the cerebellum G A B A e r g i c synapses, induced by the depressed excitatory granule cell i n p u t a n d / o r the increased mossy fiber c o n t a c t with the ectopic Purkinje cells. REFERENCES Biggio G., Corda M. G., Concas A. and Gessa G. L. (1981) Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra. Brain Res. 220, 334-349. Cannon W. B. and Rosenbleuth A. (1949) The Supersensitivity of Denervated Structure. Macmillan, New York. Crepel F. and Mariani J. (1976) Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the weaver mutant mouse. J. Neurobiol. 7, 579-582. Cross A. J. and Waddington J. L. (1981) Substantia nigra GABA receptors in Huntington's disease. J. Neurochem. 37, 321-324. Enna S. J. and Snyder S. H. (1975) Properties of GABA receptor binding in rat brain synaptic membrane fractions. Brain Res. 100, 81-97. Hamburgh M. (1960) Observations on the neuropathology of "reeler", a neurobiological mutation in mice. Experientia 16, 460-461. Henkart M. (1980) Identification and function of intracellular calcium stores in axons and cell bodies of neurons. Fedn. Proc. 39, 2783-2789, Hoffer B., Siggins G. R. and Bloom F. E. (1971) Studies on NE-containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to NE and related substances administered by microiontophoresis. Brain Res. 25, 523-534. Hudson D. B., Valcana T., Bean G. L. and Timiras P. S. (1976a) Glutamic acid: a strong candidate as the neurotransmitter of the cerebellar granule cell. Neurochem. Res. 1, 73-81. Hudson D. B., Valcana T. and Timiras P. S. (1976b) Monoamine metabolism in the developing rat brain and effects of ionizing radiation. Brain Res. 114, 471-479. Johnston M. V. and Coyle J. T. (1982) Cytotoxic lesions and the development of transmitter systems. Trends Neurosci. 5, 153-156. Kingsbury A. E., Wilkin G. P., Patel A. J. and Balhzs R. (1980) Distribution of GABA receptors in the rat cerebellum. J. Neurochem. 35, 739-742. Landis S. C., Shoemaker W. J., Schlumpf M. and Bloom F. E. (1975) Catecholamines in mutant mouse cerebellum: fluorescence microscopic and chemical studies. Brain Res. 93, 253-266. Lloyd K. J. and Davidson L. (1979) 3H-GABA binding in brain from Huntington's chorea patients: Altered regulation by phospholipids. Science 205, 1147-1149. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Martin D. L. (1976) Carrier--mediated transport and removal of GABA from synaptic regions. In: GABA in Nervous System Function (Roberts E., Chase T. N. and Tower D. B., eds), pp. 347-486. Raven Press, New York.
43
Matsokis N., Argentaki M. and Valcana T. (1982) Na +-dependent and Na +-independent 3H-GABA binding in Reeler Cerebellar dysgenesis. In: Third International Meeting of the International Society for Developmental Neuroscience. (Abstract) p. 155. McBride W. J., Aprison M. H. and Kusano K. (1976a) Contents of several amino acids in the cerebellum brain stem and cerebrum of the "staggerer", "weaver" and "nervous" neurological mutant mice. J. Neurochem. 26, 867-870. McBride W. J., Nadi N. S., Altman J. and Aprison M. H. (1976b) Effect of selective doses of X-irradiation on the levels of several amino acids in the cerebellum of the rat. Neurochem. Res. 1, 141-152. Olsen R. W. and Mikoshiba K. (1978) Localization of GABA receptor binding in the mammalian cerebellum high levels in granule layer and depletion in agranular cerebella of mutant mice. J. Neurochem. 30, 1633-1636. Purves D. and Lichtman J. W. 0980) Elimination of synapses in the developing nervous system. Science 210, 153-157. Rakic P. (1976) Synaptic specificity in cerebellar cortex: study of anomalous circuits induced by single gene mutations in mice, Cold Spring Harbor Syrup. quant. Biol. 40, 333-346. Reisine T. D., Fields J. Z. and Yamamura K. I. (1977) Neurotransmitter receptor alterations in Parkinson's disease. Life Sci. 21, 335-343. Roffier-Tarlov S. and Sidman R. L. (1978) Concentrations of glutamic acid in cerebellar cortex and deep nuclei of normal mice and weaver, staggerer and nervous mutants. Brain Res. 142, 269-283. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Sidman R. L., Green M. C. and Appel S. H. (1965) Catalog of the Neurological Mutants o f the Mouse. Harvard University Press, Cambridge, MA. Sidman R. L. (1974) Interaction among developing mammalian brain cells in the cell surface. In: Proceedings o f the International Society o f Developmental Biologists (Moscona A. A., ed.), pp. 222-253. Wiley, New York. Slevin J. T., Johnston M. V. and Coyle J. T. (1982) Methylazoxy-methanol acetate ablation of mouse cerebellar granule cells: Effects on synaptic neurochemistry. Devl. Neurosci. 5, 3-12. Soreq H., Gurwitz D., Eliyahu D. and Sokolovsky M. (1982) Altered ontogenesis of muscarinic receptors in agranular cerebellar cortex. J. Neurochem. 39, 756-763. Tamminga C. A. (1981) Tardive dyskinesia and the dopamine receptors. In: Neuroreceptors (Usdin E., Bunney W. E. and Davis J. M., eds), pp. 231-240. Wiley, New York. Teichberg V. I. (1980) Amino acid receptors. In: Cellular Receptors of Hormones and Neurotransmitters (Schnister D. and Levitzki A., eds), pp. 369-395. Wiley, New York. Ticku M. K. (1979) Differences in GABA receptor sensitivity in inbred strains of mice. J. Neurochem. 33, 1135-1138. Toffano G., Guidotti A. and Costa E. (1978) Purification of an endogenous protein inhibitor of the high affinity binding of GABA to synaptic membranes of rat brain. Proc. natn. Acad. Sci., U.S.A. 75, 4024-4028. Valcana T., Hudson D. and Timiras P. S. (1972) Effects of X-irradiation on the content of amino acids in the developing rat cerebellum. J. Neurochem. 19, 2229-2232.
44
NIKOLAOS MATSOKIS and THEONY VALCANA
Valcana T., Liao C. and Timiras P. S. (1974a) Effects of X-irradiation on the subcellular distribution of cholinergic enzymes in the developing rat cerebellum. Brain Res. 73, 105-120. Valcana T., Liao C. and Timiras P. S. (1974b) Effects of X-radiation on the development of the cholinergic system of the rat brain. II. Investigation of alterations in acetycholine content. Envir. Physiol. Biochem. 4, 58-63. Vizi E. S. (1979) Presynaptic modulation of neurochemical transmission. Prog. Neurobiol. 12, 181-290. Waddin~ton J. L. and Cross A. J. (1978) Denervation supersensitivity in the striatonigral GABA pathway. Nature 276, 618-620.
Williams M. and Risley E. A. (1979) Characterization of the binding of 3H-muscimol, a potent GABA agonist, to rat brain synaptosomal membranes using a filtration assay. J. Neurochem. 32, 713-718. Wilson L., Sotelo C. and Caviness V. S. Jr (1981) Heterologous synapses upon Purkinje cells in the cerebellum of the reeler mutant mouse: an experimental light and electron microscopy study. Brain Res. 213, 63-82. Wong D. T. and Borng J. S. (1977) Na+-independent binding of GABA to the Triton X-100 treated synaptic membranes from cerebellum of rat brain. Life Sci. 20, 445-452.