Journal of Neuroscience Methods, 38 (1991) 51-62 ~? 1991 Elsevier Science Publishers B.V. 0165-0270/91/$03.50
51
NSM 01237
An improved method for the preparation of rat cerebellar glomeruli F. Viennot, J.-C, Artault, G. Tholey, J. De Barry and G. G o m b o s Centre de Neurochimie du CNRS and INSERM U.44, 5 Rue Blaise Pascal, F-67084 Strasbourg Cedex (France) (Received 18 September 1990) (Revised version received 29 December 1990 and 1 March 1991) (Accepted 4 March 1991)
Key words: Glomeruli; Cerebellum; Choline; AAT; GDH Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransminer receptors.
Introduction
In cerebellum, afferent mossy fibers make synapses with granule cell dendrites in the granular layer of the folium. The mossy fiber terminals and the granule cell dendrites are associated in a polysynaptic complex called glomerulus which is formed of a mossy fiber giant terminal, called the rosette, surrounded by several granule cell dendritic digits. At the periphery of this structure, Golgi neuron terminals also make synapses with granule cell dendrites; the whole structure is wrapped in astrocytic processes. Most of the mossy fibers use excitatory amino acids as neurotransmitters (Pumain et al., 1979; Freeman et al., 1983), but few of them are cholinergic (IsraEl and Whittaker, 1965; HajEs et al., 1974; Balfizs et al., 1975) or serotoninergic (Chan-Palay, 1977), while Golgi neurons are mostly GABAergic (Eccles et al., 1967;
Correspondence: Fran~oise Viennot, CNRS, 5 Rue Blaise Pascal, F-67084 Strasbourg Cedex, France. Tel.: (88) 614848; Fax: (88) 612908.
Hamori and Szentagothai, 1980: Triller et al., 1987). Thus, in this single polysynaptic complex, excitatory and inhibitory inputs coexist. Considering the quasi-homogeneity of the presynaptic terminals from the mossy fibers and from Golgi neurons and their coexistence in this structure, a glomerular preparation should be a better model for the study of glutamatergic and GABAergic neurotransmission than classical heterogeneous synaptosomal preparations; also the subclasses of glutamate and GABA receptors present in glomerular synapses can be determined (Viennot et al., 1991). A subcellular fractionation method using relatively mild tissue fragmentation techniques has indeed been developed and glomerular structures with preserved morphological characteristics were obtained (Haj6s et al., 1974, 1975; Tapia et al., 1974; Wilkin et al., 1974). Glomeruli prepared by this method appeared however to be contaminated by other synaptosomes. Further centrifugations, which prolonged the procedure and reduced yields were needed to reduce this contamination (Balfizs et al., 1975). More rapid methods for bulk preparation of glomeruli (Hamberger et al., 1975,
52 1976; Terrian et al., 1985) have been reported but they lack biochemical characterization. Here we describe a new method, somewhat shorter and simpler than some of the previously described methods and with a detailed biochemical characterization of the fractions obtained.
Material and methods
284C51 was from Wellcome (Paris, France), isoOMPA from Koch Light (Sochibo, Boulogne/ Seine, France) and the Spurr resin was from Taab Laboratories (Reading, U.K.). The filter holders (3 cm diameter) were from Sartorius (Palaiseau, France), pyruvate, G D H , a-ketoglutarate, NAD. N A D H and ADP were from Boehringer Mannheim (Mannheim, F.R.G.) and pyrydoxal phosphate was from Merck (Darmstadt, F.R.G.).
Chemicals
Subcellutar fractionation
Butyrylthiocholine, acetylthiocholine, bovine serum albumin, calf thymus D N A and yeast RNA were purchased from Sigma (St. Louis, MO); BW
Thirty adult rats (Wistar strain inbred in our laboratory, approx. 200 g body weight) were killed by decapitation. The cerebella were rapidly re-
Flow sheet
f o r the p r e p a r a t i o n
(T) Tris-HCI
= Total
10 mM,
x
x g
(3 % w/v)
glomeruli
in
0.32 M, MgCI 2 I mM,
pH 7.4
; 10 m i n
2
- -
Pellet -
homogenate
Sucrose
> 900
of cerebellar
Supernatant
Pellet
1
resuspended
Pool of the 3 supernatants = S in 20 % F i c o l l - s u c r o s e
H2
Ficoll concentration (g / ml) . . . . . . . . . .
5%
M3
" i
M2
g
~
MI
120 m i n
~
GI - ~
I~I
G2 ~
~
- - - >
10 % 80000
> Myelin-like f r a c t i o n (S)
15 % m >
....... 25 %
> Glomerular f r a c t i o n (G) Nuclear fraction
Fig. 1. Subcellularfractionation scheme.
(P)
53 moved, pooled and finely minced with scissors in a petri dish on ice. They were homogenized in a Dounce homogenizer (10 u p - d o w n strokes with a pestle of 76-152 ~tm clearance, then 10 more strokes with a pestle of 25-76 ffm clearance) in 30 ml buffer (0.32 M sucrose, 1 mM MgC12, 10 mM Tris-HC1, pH 7.4). The homogenate was collected in a 50-ml syringe and then forced through a nylon filter (150/~m mesh diameter) mounted in a plastic filter holder. Another 50-ml syringe attached on the other side of the filter collected the filtrate by aspiration (see Fig. 1). The same procedure was repeated through a series of nylon filters (130, 80 and 50 ffm), the filtrate was transfered to a graduated cylinder and the volume was then adjusted to 100 ml. This fraction was called fraction T (T = total homogenate). The whole procedure is summarized in Fig. 1. Fraction T was centrifuged at 900 x g for 10 rain (Sorvall RC-5B centrifuge, SA-600 rotor). The pellet was washed twice by resuspending the pellet (by manual shaking) in the same buffer and the centrifugation repeated. The corresponding supernatants were pooled in the so-called fraction S. This fraction S contained the cytosol, most of the synaptosomes, smooth and rough endoplasmic reticulum and other ergastoplasmic membranes, free mitochondria, plasma membranes, myelin and axons. The pellet which contained myelinated axons, nuclei, red blood cells, glomeruli and some cell perikarya was suspended in 30 ml of 20% Ficoll/0.32 M sucrose (fraction H2 = homogenate No. 2). 10 ml of fraction H2 were layered onto 10 ml 25% Ficoll in a buffer cushion, and three 10-ml layers of 15, 10 and 5% Ficoll, respectively, all containing 0.32 M sucrose, were then layered sequentially on top of fraction H2. Centrifugation in a swinging rotor (SW 25.2 Beckman) at 80 000 x g for 2 h allowed both floatation and sedimentation of subcellular particles: 5 bands and one pellet (P) were obtained. The bands were as follows: those at the 2 5 % - 20% Ficoll and 20-15% Ficoll interfaces were called G1 and G2; those at the 15-10% Ficoll, 10-5% Ficoll and through the 5% Ficoll layer were called M1, M2 and M3, respectively. Fractions were collected by using a peristaltic pump and monitored with a UV detector (260/310 nm). Aliquots of each band were immediately
processed for electron microscopy, others were stored frozen at - 8 0 ° C for subsequent biochemical assay.
Electron microscopy Aliquots of each band were pipetted in an Eppendorf tube and then centrifuged at 12 400 x g for 15 min in a Beckman Microfuge to form a 1-mm thick pellet (Cotman and Flansburg, 1970). The pellets were fixed in 1% glutaraldehyde, 0.32 M sucrose and post-fixed in 2% OsO 4, 0.2 M cacodylate buffer, p H 7.2. The osmicated pellets were consecutively dehydrated and then embedded in Spurr resin. Thin sections were cut parallel to the axis of sedimentation in order to control possible heterogeneous distribution of subcellular particles within each fraction due to their stratification in the pellet. The sections were then stained with uranyl acetate and lead citrate. Biochemical assays All measures were made in triplicate and all the results (means of n separate measures with n > 3) were expressed per mg protein (Table I) or on a per cerebellum basis (Table II). Proteins were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. D N A was assayed by the method of Burton (1956) using calf thymus DNA as a standard and RNA was measured at 260 and 280 nm using yeast RNA as a standard. Enzymatic assays Either frozen-thawed or freshly prepared aliquots of each fraction were used. Lactate dehydrogenase (LDH, EC 1.1.1.27) was assayed at 25°C by the method of Johnson (1960). Choline acetyltransferase (CHAT, EC 2.3.1.6) was assayed at 38°C by the method of McCaman and Hunt (1965) as modified by Goldberg and McCaman (1967). Acetylcholine esterase (ACHE, EC 3.1.1.7) was assayed at 30°C by the method of Ellman et al. (1961) with acetylthiocholine as substrate and iso-OMPA as butyryl choline esterase inhibitor. Butyryl choline esterase (BuChE, EC 3.1.1.8) was assayed by the same method but using BW 284C51 in the assay medium as acetylcholine esterase inhibitor. Glutamine synthetase (GS, EC 6.3.1.2)
54
Fig. 2. F o r legend, see previous page.
55
was assayed by the method of Berl (1966) for 15 min at 37°C using ),-glutamylhydroxamate as a standard. Aspartate aminotransferase (AAT, EC 2.6.1.1) was assayed by the method of Karmen (1955) for 5 min at 25°C. Glutamate dehydrogenase (GDH, EC 1.4.1.3) was assayed at 25°C by the method of Graham and Aprison (1969).
Results and discussion Electron microscopy
We first examined at the ultrastructural level each of the 5 bands collected after gradient centrifugation and the pellet. Since we did not observe any difference between fractions M1, M2 and M3, or between fractions G1 and G2, we pooled these fractions in fractions M and G, respectively, which were subsequently used for biochemical measurements. Fraction P consisted of red blood cells, cell nuclei, cell debris and, as shown in Fig. 2A, by granule cell bodies. Fraction M comprised packed and unpacked myelin fragments, myelinated axons, microsomal vesicles and synaptosomes (Fig. 2B). Fraction G (Figs. 2C-D, 3A-B and 4) essentially contained giant nerve endings rich in mitochondria and pleomorphic synaptic vesicles (Fig. 3A-B) which are two characteristics of the mossy fiber rosette. These rosettes were surrounded by numerous dendritic digits from granule cells (Fig. 2D and 4). In some cases these dendrites were in contact with small nerve terminals corresponding to Golgi neuron terminals (Fig. 3A-C). In fraction G, the mitochondria of the rosette appeared reasonably preserved (limited swelling). These glomeruli apparently were in a good state of preservation (Figs. 2, 3) and low magnification micrographs (Fig. 4) gave an indica-
tion of the homogeneity and purity of this glomerular fraction but these criteria were not sufficient and purity was assessed by using biochemical markers. Biochemical measurements
Protein recovery in fractions H2 + S was complete, in fact, it was 108.92 ± 5.36% (data not shown). In the same way the overall recovery of the fractions G + M + P (obtained after the gradient centrifugation step) represented 97.77 _+ 1.64% of the H2 fraction layered onto the Ficoll gradient (data not shown). These values indicated that no appreciable protein loss occurred during the preparation. The protein content in fraction G represented about 11% of total cerebellar proteins. L D H is a soluble enzyme present in the cytoplasm of all cells. The activity of this cytosolic enzyme should indicate how much cytoplasm is contained in each fraction. As expected, LDH activity was the highest (79.12 _+ 6.08%) in fraction S (which contained cytosol and most synaptosomes) but it was low in fraction G (2.2%) and much lower than the relative protein content in fraction G (enrichment = 0.51). Most probably the low L D H activity reflects a leakage of L D H and other cytosolic components from nerve terminals a n d / o r dendrites during gradient centrifugation. However, this leakage seems to be limited to the mossy fiber rosette and granule cell dendrites since GAD, another soluble enzyme is highly enriched in fraction G (Viennot et al., unpublished) indicating that the Golgi neuron terminals, which contain GAD, did not loose much of their cytosolic content. In fact the giant mossy fiber terminal appears to be very sensitive to osmotic conditions (Hamberger et al., 1976) while Golgi neuron terminals are particularly resistent (Csillag et al., 1982).
Fig. 2, Electron micrographs showing different fractions collected after gradient centrifugation. Calibration bars = 0.5 ~m. A: fraction P; note granule cell bodies with their characteristic cytoplasmic rim. B: fraction M; note the presence of myelin (packed or unpacked), a myelinated axon, various vesicular profiles and some synaptosomes (arrows). C and D: fraction G; note the giant nerve terminal characterized by terminals rich in synaptic vesicles (arrow heads) and attached dendritic profiles (arrows). Most of the mitochondria appear slightly swollen and some of them are electron dense, which m e a n s that they undergo unavoidable weak cytoplasmic modifications during the preparation.
56
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8
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o
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+1 +1 +1 +1 +~ +1 +1 +1 +1
.= Z
,..a ,..a ¢)
ta.
.-&
+1 +1 +1 +1 +1 ~
+1
51
NSM 01237
An improved method for the preparation of rat cerebellar glomeruli F. Viennot, J.-C, Artault, G. Tholey, J. De Barry and G. G o m b o s Centre de Neurochimie du CNRS and INSERM U.44, 5 Rue Blaise Pascal, F-67084 Strasbourg Cedex (France) (Received 18 September 1990) (Revised version received 29 December 1990 and 1 March 1991) (Accepted 4 March 1991)
Key words: Glomeruli; Cerebellum; Choline; AAT; GDH Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransminer receptors.
Introduction
In cerebellum, afferent mossy fibers make synapses with granule cell dendrites in the granular layer of the folium. The mossy fiber terminals and the granule cell dendrites are associated in a polysynaptic complex called glomerulus which is formed of a mossy fiber giant terminal, called the rosette, surrounded by several granule cell dendritic digits. At the periphery of this structure, Golgi neuron terminals also make synapses with granule cell dendrites; the whole structure is wrapped in astrocytic processes. Most of the mossy fibers use excitatory amino acids as neurotransmitters (Pumain et al., 1979; Freeman et al., 1983), but few of them are cholinergic (IsraEl and Whittaker, 1965; HajEs et al., 1974; Balfizs et al., 1975) or serotoninergic (Chan-Palay, 1977), while Golgi neurons are mostly GABAergic (Eccles et al., 1967;
Correspondence: Fran~oise Viennot, CNRS, 5 Rue Blaise Pascal, F-67084 Strasbourg Cedex, France. Tel.: (88) 614848; Fax: (88) 612908.
Hamori and Szentagothai, 1980: Triller et al., 1987). Thus, in this single polysynaptic complex, excitatory and inhibitory inputs coexist. Considering the quasi-homogeneity of the presynaptic terminals from the mossy fibers and from Golgi neurons and their coexistence in this structure, a glomerular preparation should be a better model for the study of glutamatergic and GABAergic neurotransmission than classical heterogeneous synaptosomal preparations; also the subclasses of glutamate and GABA receptors present in glomerular synapses can be determined (Viennot et al., 1991). A subcellular fractionation method using relatively mild tissue fragmentation techniques has indeed been developed and glomerular structures with preserved morphological characteristics were obtained (Haj6s et al., 1974, 1975; Tapia et al., 1974; Wilkin et al., 1974). Glomeruli prepared by this method appeared however to be contaminated by other synaptosomes. Further centrifugations, which prolonged the procedure and reduced yields were needed to reduce this contamination (Balfizs et al., 1975). More rapid methods for bulk preparation of glomeruli (Hamberger et al., 1975,
52 1976; Terrian et al., 1985) have been reported but they lack biochemical characterization. Here we describe a new method, somewhat shorter and simpler than some of the previously described methods and with a detailed biochemical characterization of the fractions obtained.
Material and methods
284C51 was from Wellcome (Paris, France), isoOMPA from Koch Light (Sochibo, Boulogne/ Seine, France) and the Spurr resin was from Taab Laboratories (Reading, U.K.). The filter holders (3 cm diameter) were from Sartorius (Palaiseau, France), pyruvate, G D H , a-ketoglutarate, NAD. N A D H and ADP were from Boehringer Mannheim (Mannheim, F.R.G.) and pyrydoxal phosphate was from Merck (Darmstadt, F.R.G.).
Chemicals
Subcellutar fractionation
Butyrylthiocholine, acetylthiocholine, bovine serum albumin, calf thymus D N A and yeast RNA were purchased from Sigma (St. Louis, MO); BW
Thirty adult rats (Wistar strain inbred in our laboratory, approx. 200 g body weight) were killed by decapitation. The cerebella were rapidly re-
Flow sheet
f o r the p r e p a r a t i o n
(T) Tris-HCI
= Total
10 mM,
x
x g
(3 % w/v)
glomeruli
in
0.32 M, MgCI 2 I mM,
pH 7.4
; 10 m i n
2
- -
Pellet -
homogenate
Sucrose
> 900
of cerebellar
Supernatant
Pellet
1
resuspended
Pool of the 3 supernatants = S in 20 % F i c o l l - s u c r o s e
H2
Ficoll concentration (g / ml) . . . . . . . . . .
5%
M3
" i
M2
g
~
MI
120 m i n
~
GI - ~
I~I
G2 ~
~
- - - >
10 % 80000
> Myelin-like f r a c t i o n (S)
15 % m >
....... 25 %
> Glomerular f r a c t i o n (G) Nuclear fraction
Fig. 1. Subcellularfractionation scheme.
(P)
53 moved, pooled and finely minced with scissors in a petri dish on ice. They were homogenized in a Dounce homogenizer (10 u p - d o w n strokes with a pestle of 76-152 ~tm clearance, then 10 more strokes with a pestle of 25-76 ffm clearance) in 30 ml buffer (0.32 M sucrose, 1 mM MgC12, 10 mM Tris-HC1, pH 7.4). The homogenate was collected in a 50-ml syringe and then forced through a nylon filter (150/~m mesh diameter) mounted in a plastic filter holder. Another 50-ml syringe attached on the other side of the filter collected the filtrate by aspiration (see Fig. 1). The same procedure was repeated through a series of nylon filters (130, 80 and 50 ffm), the filtrate was transfered to a graduated cylinder and the volume was then adjusted to 100 ml. This fraction was called fraction T (T = total homogenate). The whole procedure is summarized in Fig. 1. Fraction T was centrifuged at 900 x g for 10 rain (Sorvall RC-5B centrifuge, SA-600 rotor). The pellet was washed twice by resuspending the pellet (by manual shaking) in the same buffer and the centrifugation repeated. The corresponding supernatants were pooled in the so-called fraction S. This fraction S contained the cytosol, most of the synaptosomes, smooth and rough endoplasmic reticulum and other ergastoplasmic membranes, free mitochondria, plasma membranes, myelin and axons. The pellet which contained myelinated axons, nuclei, red blood cells, glomeruli and some cell perikarya was suspended in 30 ml of 20% Ficoll/0.32 M sucrose (fraction H2 = homogenate No. 2). 10 ml of fraction H2 were layered onto 10 ml 25% Ficoll in a buffer cushion, and three 10-ml layers of 15, 10 and 5% Ficoll, respectively, all containing 0.32 M sucrose, were then layered sequentially on top of fraction H2. Centrifugation in a swinging rotor (SW 25.2 Beckman) at 80 000 x g for 2 h allowed both floatation and sedimentation of subcellular particles: 5 bands and one pellet (P) were obtained. The bands were as follows: those at the 2 5 % - 20% Ficoll and 20-15% Ficoll interfaces were called G1 and G2; those at the 15-10% Ficoll, 10-5% Ficoll and through the 5% Ficoll layer were called M1, M2 and M3, respectively. Fractions were collected by using a peristaltic pump and monitored with a UV detector (260/310 nm). Aliquots of each band were immediately
processed for electron microscopy, others were stored frozen at - 8 0 ° C for subsequent biochemical assay.
Electron microscopy Aliquots of each band were pipetted in an Eppendorf tube and then centrifuged at 12 400 x g for 15 min in a Beckman Microfuge to form a 1-mm thick pellet (Cotman and Flansburg, 1970). The pellets were fixed in 1% glutaraldehyde, 0.32 M sucrose and post-fixed in 2% OsO 4, 0.2 M cacodylate buffer, p H 7.2. The osmicated pellets were consecutively dehydrated and then embedded in Spurr resin. Thin sections were cut parallel to the axis of sedimentation in order to control possible heterogeneous distribution of subcellular particles within each fraction due to their stratification in the pellet. The sections were then stained with uranyl acetate and lead citrate. Biochemical assays All measures were made in triplicate and all the results (means of n separate measures with n > 3) were expressed per mg protein (Table I) or on a per cerebellum basis (Table II). Proteins were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. D N A was assayed by the method of Burton (1956) using calf thymus DNA as a standard and RNA was measured at 260 and 280 nm using yeast RNA as a standard. Enzymatic assays Either frozen-thawed or freshly prepared aliquots of each fraction were used. Lactate dehydrogenase (LDH, EC 1.1.1.27) was assayed at 25°C by the method of Johnson (1960). Choline acetyltransferase (CHAT, EC 2.3.1.6) was assayed at 38°C by the method of McCaman and Hunt (1965) as modified by Goldberg and McCaman (1967). Acetylcholine esterase (ACHE, EC 3.1.1.7) was assayed at 30°C by the method of Ellman et al. (1961) with acetylthiocholine as substrate and iso-OMPA as butyryl choline esterase inhibitor. Butyryl choline esterase (BuChE, EC 3.1.1.8) was assayed by the same method but using BW 284C51 in the assay medium as acetylcholine esterase inhibitor. Glutamine synthetase (GS, EC 6.3.1.2)
54
Fig. 2. F o r legend, see previous page.
55
was assayed by the method of Berl (1966) for 15 min at 37°C using ),-glutamylhydroxamate as a standard. Aspartate aminotransferase (AAT, EC 2.6.1.1) was assayed by the method of Karmen (1955) for 5 min at 25°C. Glutamate dehydrogenase (GDH, EC 1.4.1.3) was assayed at 25°C by the method of Graham and Aprison (1969).
Results and discussion Electron microscopy
We first examined at the ultrastructural level each of the 5 bands collected after gradient centrifugation and the pellet. Since we did not observe any difference between fractions M1, M2 and M3, or between fractions G1 and G2, we pooled these fractions in fractions M and G, respectively, which were subsequently used for biochemical measurements. Fraction P consisted of red blood cells, cell nuclei, cell debris and, as shown in Fig. 2A, by granule cell bodies. Fraction M comprised packed and unpacked myelin fragments, myelinated axons, microsomal vesicles and synaptosomes (Fig. 2B). Fraction G (Figs. 2C-D, 3A-B and 4) essentially contained giant nerve endings rich in mitochondria and pleomorphic synaptic vesicles (Fig. 3A-B) which are two characteristics of the mossy fiber rosette. These rosettes were surrounded by numerous dendritic digits from granule cells (Fig. 2D and 4). In some cases these dendrites were in contact with small nerve terminals corresponding to Golgi neuron terminals (Fig. 3A-C). In fraction G, the mitochondria of the rosette appeared reasonably preserved (limited swelling). These glomeruli apparently were in a good state of preservation (Figs. 2, 3) and low magnification micrographs (Fig. 4) gave an indica-
tion of the homogeneity and purity of this glomerular fraction but these criteria were not sufficient and purity was assessed by using biochemical markers. Biochemical measurements
Protein recovery in fractions H2 + S was complete, in fact, it was 108.92 ± 5.36% (data not shown). In the same way the overall recovery of the fractions G + M + P (obtained after the gradient centrifugation step) represented 97.77 _+ 1.64% of the H2 fraction layered onto the Ficoll gradient (data not shown). These values indicated that no appreciable protein loss occurred during the preparation. The protein content in fraction G represented about 11% of total cerebellar proteins. L D H is a soluble enzyme present in the cytoplasm of all cells. The activity of this cytosolic enzyme should indicate how much cytoplasm is contained in each fraction. As expected, LDH activity was the highest (79.12 _+ 6.08%) in fraction S (which contained cytosol and most synaptosomes) but it was low in fraction G (2.2%) and much lower than the relative protein content in fraction G (enrichment = 0.51). Most probably the low L D H activity reflects a leakage of L D H and other cytosolic components from nerve terminals a n d / o r dendrites during gradient centrifugation. However, this leakage seems to be limited to the mossy fiber rosette and granule cell dendrites since GAD, another soluble enzyme is highly enriched in fraction G (Viennot et al., unpublished) indicating that the Golgi neuron terminals, which contain GAD, did not loose much of their cytosolic content. In fact the giant mossy fiber terminal appears to be very sensitive to osmotic conditions (Hamberger et al., 1976) while Golgi neuron terminals are particularly resistent (Csillag et al., 1982).
Fig. 2, Electron micrographs showing different fractions collected after gradient centrifugation. Calibration bars = 0.5 ~m. A: fraction P; note granule cell bodies with their characteristic cytoplasmic rim. B: fraction M; note the presence of myelin (packed or unpacked), a myelinated axon, various vesicular profiles and some synaptosomes (arrows). C and D: fraction G; note the giant nerve terminal characterized by terminals rich in synaptic vesicles (arrow heads) and attached dendritic profiles (arrows). Most of the mitochondria appear slightly swollen and some of them are electron dense, which m e a n s that they undergo unavoidable weak cytoplasmic modifications during the preparation.
56
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.-&
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