Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decar☐ylase activity in control and degranulated rat hippocampus

BRAIN RES~RCH ELSEVIER

Brain Research 644 (1994) 313-321

Research Report

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus Philippe Taupin *, Yezekiel Ben-Ari, Marie-Paule Roisin INSERM U 29, 123 Bd de Port-Royal, 75014 Paris, France (Accepted 18 January 1994)

Abstract

Using discontinuous density gradient centrifugation in isotonic Percoli sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippoeampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K + depolarization. After irradiation, compared to the control values, the release of glutamate andGABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers. Key words: Hippocampus; Mossy fiber; Synaptosome; Irradiation; Amino acid release; Glutamate decarboxylase

1. Introduction

The hippocampal mossy fiber (MF) synapse is the link in the trisynaptic excitatory circuit, between the mossy fiber terminals of dentate granule cells and CA3 pyramidal cells of A m m o n ' s horn [7,24]. In the hippocampus, glutamate is the neurotransmitter of the pyramidal cells and granule cells [1,13,14,40,54] whereas G A B A is considered as the neurotransmitter of the inhibitory interneurones [52]. Recently, G A B A immunoreactivity at the ultrastructural level was found in hippocampal mossy fiber axons and in terminals of the granule cells of the monkey and rat [50]. This confirmed a previous report from a light microscopy study showing G A B A immunoreactivity in the rat stratum

* Corresponding author. Reprint request: M.P. Roisin. Fax: (33) (1) 46 34 16 56. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 1 3 7 - 2

lucidum [40,63]. However, very few G A D immunoreactivity was detected at light microscopy level in the CA3 field of the rat and monkey hippocampus [4,37] and no G A D immunoreactivity was detected at the ultrastructural level in the MF terminals [23]. The colocalization of inhibitory (GABA) and excitatory (glutamate) neurotransmitters in an excitatory synapse raised the possibility of their corelease. Corelease of glutamate and G A B A from a same population of synaptosomes has been already described for GABAergic cortical synaptosomes, in response to depolarizing stimulation [17]. The control of CA3 hippocampal neural activity would appear to be more complex than its modulation by a release of G A B A from interneurones [2,10,23]. We are interested in the biochemical investigation of the M F synapses that are implicated in memory processing and sprouting induced by seizures [6,42,44]. For this purpose we have previously established a procedure for the preparation of M F synaptosomes on

314

P. Taupin et al. /Brain Research 644 119941 313-321

a three-step Percoll gradient [57]. The aim of the present study was to characterize the amino acid re]ease and content in MF and others hippocampal synaptosomes. In addition we determined the subcellular distribution of the G A D activity, the G A B A synthesizing enzyme. Since neonatal irradiation of the hippocampal region destroys granule cells and MF terminals [5,43], this study was performed from degranulated hippocampus (IRR) and compared with CNT values to identify neurotransmitters in the MF terminals. A preliminary report of this work has been presented [56].

2. Materials and methods 2.1. Dt'granulation procedure Newborn male Wistar rats (postnatal day P0) were irradiated at 6 Gy (600 rads) with a cobalt bomb [43]. The 7 rays were collimated for irradiation of the two hippocampal regions. The rats were sacrifled 2 months after irradiation. The experimental protocols were approved by the French Ethical Committee (statement no. 04223).

solution to ensure that release of amino acids had returned to basal levels (samples 13 181. In order to measure the Ca 2' independent basal release and the Ca -~~ independent K + evoked release, the same experiments were performed in parallel with Ca 2+ free superfusion Krebs medium containing 3.3 mM Mg('l 2 and 11.5 mM EGTA. Samples 8-13 were pooled to determine the amino acid K" evoked release. Superfusate samples were deproteinized by boiling for 5 rain at 100°C and stored at 211°Cuntil amino acid analysis.

2.4. HPLC amino acid determination Collected samples were centrifuged for 5 min at 12,000× g and the content of amino acids determined by high performance liquid chromatography (HPLC) using a derivatization procedure with ortho-phthaldialdehyde [47]. A 4.6× 75 mm column packed with ultrasphere XL 3 /x ODS (Beckman) was used for the separation of the derivatizated amino acids with a mobile phase consisting of 50 mM sodium acetate (pH 7)/methanol/tetrahydrofuran, 80: 19:1, v / v and a gradient 50 mM sodium acetate (pH 7)/methanol, 20:80, v/v. The derivatizated amino acids were detected by fluorescence (excitation 330 nm, emission 418 nm). Values for the amino acids were determined on the basis of external standards with values quantified by the peak area processed with a Shimadzu CRBA integrator.

2.5. Amino acid content 2.2. Synaptosomes preparation Synaptosomes were prepared from hippocampus (0.6 g wet weight) of adult control rats (CNT) or adult rats after neonatal irradiation (1RR), 12 and 18 respectively, as previously described [57]. A 10% ( w / v ) homogenate, prepared in 0.32 M sucrose, 5 mM Tris, 1 mM MgCI2, pH 7.4, was centrifuged at 1,000× g for 10 min. The pellet, which contains the MF synaptosomes [30], was washed once by resuspension in the homogenization buffer and recentrifuged as above. The resuspended pellet (P) and pooled supernatants (S) were layered on three-steps gradients, 10%, 15% and 23% Percoll, in 0.32 M sucrose, 1 mM MgCI 2 (pH 7.4) (2 ml of P or S suspension/2.3 ml each of 10-15-23% Percoll). The gradients were centrifuged at w2t 2.13x 10 v rad2/s, 21,500 rpm in a 70.1 Ti rotor (32,500× g, 7 min, 4°C). Synaptosomal fractions PII and SII were collected from the 10-15% Percoll interface and Pill and SIII from the 15-23~ interface. The synaptosomes isolation was carried out at 4°C and took approximately 1 h. Fractions PII and PIII have been characterized as enriched in MF synaptosomes, whereas fractions SII and Sill have been characterized as enriched in small synaptosomt:s.

2.3. Super]usion of ~:vnaptosomal subcellular fractions Release of amino acids was studied using the superfusion method as previously described [20]. Briefly 1 ml of sephadex G 1[) placed into polypropylene Econocolumns (Biorad) was equilibrated in Krebs medium (composition in mM: 126 NaC1, 3.5 KC1, 2 CaCI 2, 1.3 MgCI2, 1.2 N a H z P O 4, 25 NaHCO3, 10 glucose), saturated with a mixture of 95% 0 2 / 5 % CO 2 at 37°C. One mg of each synaptosomal fraction was layered over the sephadex G 10 columns. Synaptosomes were then continuously superfused, for 30 min, with an oxygenated Krebs medium at a flow rate 0.5 m l / m i n and the superfusate was discarded. This procedure was repeated for 6 min and each I min superfusate fraction was collected for estimation of the basal amino acid release (samples 1--6). Release was then stimulated by superfusion for 6 min in Krebs solution containing 50 mM KCI (with simultaneous equimolar reduction of NaCI). Samples 7-12 were collected. Synaptosomes were then superfused in normal Krebs

Samples of each synaptosomal fraction were deproteinized with 0.6 M HCIO 4 for 10 min at 4°C and centrifuged at 12,000x g for 10 min. Supernatants were neutralized by 2.4 M KHCO 3 and centrifuged again. Samples of the subsequent supernatant were taken for the determination of total amino acid content by HPLC analysis.

2.6. GAD (EC 4.1.1.15.) assay micro method The GAD activity was assayed through the fl)rmation of [14C]CO, from L [1-14C] glutamate (53 mCi/mmol; Amersham) according to the method of Moskal and Bazu [36], with some modifications. The incubation mixture, in the presence or in the absence of 500 p,M pyridoxal 5'-phosphate (PLP), in total volume of 30/zl, containing 25 mM sodium phosphate buffer pH 7.2, 25 mM /3 mercaptoethanol, 0.2~ Triton X-100 and sample fractions ( 10-50 /zg of protein), was preincubated for 15 min at 37°C in a 1.5 ml Eppendorf tube. The enzyme reaction was started with 5 mM of L [l-v~C] glutamic acid (0.05 /zCi/assay). A strip of Whatman 3 MM paper (4x2.5 mm) containing 20 p,l of 1 M hyamine hydroxide was placed inside a polypropylene tube which was then introduced into the prepared Eppendorf tube. The system was closed and incubated for 1 hr at 37°C. The reaction was stopped by injecting 200 /zl of 2 N H2SO ~ with a syringe through the cap into the mixture reaction. The tube was then closed with parafilm paper, post-incubated for 1 h at 37°C and 15 min at 4°C. The strips of paper were transferred into vials and the radioactivity was measured by liquid scintillation counting using a Beckman model LS60001C.

2. Z Other procedures Electron microscopy was performed as previously described [57]. Protein content was determined by the Bradford assay [9], using y globulin as standard. For samples containing Percoll, protein determination was carried out after 2 min of incubation [62]. Statistical analysis of the data was estimated using the Student's two-tailed. non-paired t-test. A value of p < 0.05 was considered to be significant.

P. Taupm et al../" Brain Research 044 (1994) 313-32!

3. Results

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W h e n the crude n u c l e a r pellet (P) a n d the supern a t a n t (S) fractions from the rat h i p p o c a m p u s were layered on a t h r e e - s t e p d i s c o n t i n u o u s Percoll gradient, 8 fractions were o b t a i n e d : fractions P I - P 1 V a n d fractions S I - S I V , which are derived from P and S respectively. F r a c t i o n s PII a n d P I I I are e n r i c h e d in M F synaptosomes, whereas small s y n a p t o s o m e s are mainly recovered in SII a n d SIII. Mossy fiber s y n a p t o s o m e s have b e e n c h a r a c t e r i z e d by electron microscopy a n d m a r k e r s such as d y n o r p h i n [11,35] a n d zinc [3,15,16,21, 28,29]; fraction PIII c o n t a i n s 75% of the well-preserved M F s y n a p t o s o m e s a n d a higher e n r i c h m e n t in dynorphin a n d zinc t h a n PII fraction (see [57] for f u r t h e r details). Fig. 1 r e p r e s e n t s a typical M F s y n a p t o s o m e recovered in the fraction PIII. This s y n a p t o s o m e exhibits all the characteristics of the M F e n d i n g in situ: a large size (larger t h a n 2 / z m ) , a complex morphology, a high density of synaptic vesicles a n d m u l t i p l e synapses. 3.1. P o t a s s i u m synaptosomes

evoked

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T h e release of a m i n o acids, u n d e r basal c o n d i t i o n s a n d K + evoked in the p r e s e n c e a n d a b s e n c e of Ca 2÷, was p e r f o r m e d , from h i p p o c a m p a l s y n a p t o s o m a l fractions to distinguish t r a n s m i t t e r pools from n o n t r a n s m i t t e r pools of a m i n o acids (Fig. 2). U n d e r basal conditions (3 m M K+), there was a stable release of differ-

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Fig. 2. Basal (K + 3 mM) and K ~ evoked release (K ~ 50 mM) of glutamate, G A B A and aspartate, in the presence and absence of Ca~+. Hippocampal synaptosomes of control hippocampus were prepared as described in Materials and methods. Fractions PII and PIII (enriched in mossy fiber synaptosomes) and fractions Sll and Sill (in small synaptosomes) were incubated at 37°C in oxygenated Krebs medium in presence of 2 mM calcium and in absence of calcium with EGTA 0.5 mM. Basal values (K ÷ 3 mM)were the means + S.E.M. of 6 samples (1-6) collected from 4 independent experiments. Depolarization (K ÷ 50 mM) was for 6 min and measured in the pooled samples (8-13), see Materials and methods. Values expressed in nmot/mg protein/6 min are the means + S.E.M. for 4 independent experiments. * P < 0.01, K + evoked release in the presence of Ca2 ~ significantly greater than in absence of Ca2+.

Fig. 1. Electron micrograph of a mossy fiber synaptosome in the fraction Pill. SSV, small spherical vesicles; m, mitochondria; sp, spines. Scale bar = 1 izm (× 31,500).

e n t a m i n o acids. D e p o l a r i z a t i o n in the p r e s e n c e of Ca 2+ with 50 m M K + caused a significant increase over basal values in aspartate, g l u t a m a t e a n d G A B A release from the h i p p o c a m p a l s y n a p t o s o m a l fractions. T h e K + evoked release of g l u t a m a t e was g r e a t e r t h a n that of G A B A a n d aspartate. C o n c e n t r a t i o n s of all a m i n o acids r e t u r n e d to control levels in the first

P. Taupin et al. /Brain Research 644 (1994) 313-321

316

Table 1 Amino acid release expressed as percentage of the total content in fractions enriched in synaptosomes from control hippocampus

3.2. Effect of irradiation on potassium et;oked release of glutamate, GABA and aspartate

Fraction

The release of glutamate, GABA and aspartate from synaptosomes of I R R hippocampus was determined in the presence and absence of Ca 2+ and compared with synaptosomes of CNT hippocampus. In comparison with CNT values, an increase of the basal effiux of the amino acids was observed after irradiation. As observed for control fractions, the K + evoked release of glutamate, GABA and aspartate was partially Ca 2+ dependent (data not shown). Since after neonatal irradiation the weight and the protein content of the adult hippocampus were decreased by 34% and 20%, respectively, the data for amino acid release were expressed in nmol per whole hippocampus (Fig. 3). After irradiation, there was no significant change in the release of glutamate and GABA in fractions PII, SII and SIII. In contrast in PIII fraction, the release of glutamate and GABA was significantly decreased by 57.5 and 73.9%, respectively. A significant increase in the release relative to the CNT values was observed for aspartate in PII (1.6 fold).

PII Pill SII Sill

K + evoked release/content (%) Glutamate

GABA

Aspartate

4.6 (48.2 ± 2.5) 4.8 (26.1 ± 6.1) 3.6 (43.5 ± 7.1) 6.0(27.9±6.1)

10.8 (5.9± 1.1) 7.6(5.2±0.9) 6.8(4.4±0.8) 5.9 (5.4± 1.0)

3.7 (25.6±3.2) 2.9 (20.1 k 3.6) 2.9 (23.9± 4.5) 3.6(20.8±3.8)

The amino acid release was evoked by 50 mM K + for 6 rain/rag protein, in the presence of 2 mM Ca 2+ and the basal values were subtracted, Number in parenthesis correspond to the total content of amino acid in each fraction tested and expressed in n m o l / m g protein. The data expressed as percentage of the total content were the mean ± S.E.M. of 4 independent experiments.

sample after wash of the high K + medium (samples 9-13, data not shown). Deleting Ca 2+ from the superfusion medium reduced the K + evoked effiux of the three amino acids, aspartate, glutamate; however, the K + evoked release of aspartate from PII! and SII was reduced to only 10% when Ca 2+ was removed from the superfusion medium. Evoked release of glutamate, G A B A and aspartate were also estimated as percentage of total amino acid content of synaptosomal fractions. After a 6 min depolarization of synaptosomal fractions, relatively small proportions of the total glutamate, G A B A and aspartate were recovered in the superfusion medium (3.6-6%, 5.9-10.8%, 2.9-3.7%, respectively) (Table 1). There was no significant change in the release of other amino acids such as serine, glycine, alanine or tyrosine associated with the high K +, even though substantial content of these compounds were present (Table 2). Similar results were obtained with the other synaptosomal fractions SII and SIII (data not shown).

3.3. MF synaptosomes store glutamate, GABA and aspartate Amino acid content was determined to explain the difference observed between the evoked release which decreased only in PIII fraction after irradiation. As observed for CNT hippocampus, fraction PII showed the highest level of glutamate, GABA and aspartate (Fig. 4). After irradiation, a slight decrease of glutamate content was observed in the fractions PII, SII and SIII (30, 18, and 24%, respectively) whereas content of GABA and aspartate were not affected. In contrast, the fraction PIII exhibited a significant decrease in

Table 2 Amino acid content and effect of 50 mM K + on amino acid release from fractions enriched in rat hippocampal mossy fiber synaptosomes Amino acid content ( n m o l / m g protein)

Amino acid release ( p m o l / m g protein) Calcium 2 mM

Calcium free (EGTA 0.5 raM)

K + 3mM

K + 50mM

K- 3raM

K +50mM

447.8 353.4 549.6 59.7

+ 48.8 ± 92.1 ± 19.8 + 12.3

513.3 414.1 977.8 90.2

± ± ± ±

227.4 408.9 259.6 29.6

± 39.4 _+ 49.9 + 19.0 ± 20.6

252.4 378.5 357.1 29.6

558 439 234 69

± 150 ± 75 ± 52 + 15

445 622 516 115

± 50 ± 157 ±204 ± 43

384 358 305 61

± 34 ± 92 ±84 ± t7

Fraction Pll serine glycine alanine tyrosine

14.4 ± 22.7 ± 3.5 ± 0.7 ±

5.0 9.0 0.8 0.1

146.6 73.6 57.1 33.4

± 172.9 + 63.1 _+ 41.5 + 59.6

Fraction Pill serine glycine alanine tyrosine

24.5 ± 8.6 22.7 + 9.0 2.2±0.8 0.7 ± 0.1

408 + 578 ± 340+ 79 +

The amino acid release was evoked by 50 mM K + for 6 m i n / m g protein, see legend to Fig. 1 for experimental procedure.

112 136 116 25

P. Taupin et al. / Brain Research 644 (1994) 313-321

glutamate, GABA and aspartate content as compared with CNT hippocampus (62, 52 and 44%, respectively).

3.4. GAD activity distribution across the Percoll gradient from control and irradiated hippocampus Since the brain contains two forms of GAD, which differ in their interaction with the cofactor PLP, the activity of GAD was measured in the absence or presence of an excess of PLP in all the gradient fractions [19,31]. Addition of PLP increased the activity by 2 fold in the h o m o g e n a t e as well as in fractions P I I I , SII a n d

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Fig. 4. 4 . ' Total tal amino ar~ino acid aci content in synaptosomal fractions either from control co "ol or irradiated irradia hippocampus. Data were expressed in nmol per Values were the means + S.E.M. for 4 pe whole chote hippocampus. IJppoca independent pen at experiments. exp, ~rimenl * P < 0.05, significantly different than corresponding CNT values.

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Fraction Fig. 3. Comparison of amino acid release from fractions enriched in hippocampal synaptosomes of control or irradiated hippocampus. The amino acid release was evoked by 50 mM K + for 6 min, in the presence of 2 mM Ca 2+. Values of different experimental conditions expressed as nmol/whole hippocampus/6 min are the means± S.E.M. for 4 independent experiments. * P < 0.05, K + evoked release from irradiated hippocampus (IRR) significantly different than that from control (CNT) hippocampus.

Sill and by 3 fold in fraction PII (data not shown). In CNT hippocampus, one of the fraction enriched in MF synaptosomes (PII) exhibited the highest enrichment of total GAD activity (4.5 times higher than the homogenate), whereas no enrichment of GAD activity was found in the other fraction enriched in MF synaptosomes (fraction PIII). In the gradient S, the fractions SI and SII were enriched in GAD activity (1.87 and 2 times higher than the homogenate respectively) (Fig. 5A). When the data were expressed per hippocampus, both fractions enriched in MF synaptosomes (PII and Pill) and in small synaptosomes (SII and Sill), reassembled 85% of the GAD activity, against 15% in fractions PI and SI (Fig. 5B). GAD activity was mainly associated with fractions collected at the interface 1015% Percoll (PII 59% and SII 17%). We did not observe after irradiation any change in the distribution

318

P. Taupin et al. /Brain Research 644 (1994) 313-321

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Fraction Fig. 5. Distribution of GAD activity on Percoll gradients from control and irradiated hippocampus. The enzyme activity was measured in the presence of PLP, Data were given as the means + S.E.M. of 4 independent experiments. A: results expressed in m o l / m g protein * P < 0.05, significantly greater than corresponding values of the homogenate. B: data were expressed in tool per whole hippocampus, the difference between all control fractions (CNT) and irradiated fractions (IRR) was not statistically significant.

of the total G A D activity compared to C N T hippocampus (Fig. 5A and B).

4. D i s c u s s i o n

4.1. Metabolically L,iable M F synaptosomes release glutamate and GABA The procedure of Percoll gradient centrifugation is suitable for separation of small amounts of metabolically active synaptosomes from nerve terminals [27,46, 60]. The results of this study demonstrated that the two hippocampal fractions enriched in MF synaptosomes, isolated by Percoll gradient centrifugation, are able to release endogenous neurotransmitter from stores upon K + induced depolarization indicating the viability [8,18,27,46,61] of the MF synaptosomes. Terrian et al. [59] showed that hippocampal MF synaptosomal preparation obtained in a subcellular fraction (P3) releases glutamate in a Ca 2+ dependent manner when depolarized, whereas fractions further purified by sucrose gradient did not.

The release of amino acids was performed by the superfusion technique. Among the seven amino acids tested, high K + increased significantly the release of only aspartate, glutamate and GABA. From all the fractions enriched in synaptosomes, glutamate was the dominant neurotransmitter released. The evoked release of glutamate and G A B A was partially Ca 2 + dependent. Aspartate release from the fraction SII enriched in small synaptosomes was not Ca 2+ dependent, whereas the release of aspartate from the fractions PII and SIII was partially Ca 2+ dependent. The partial Ca 2+ dependent release of aspartate has been observed from hippocampal slices [47,55] and the release of aspartate in a Ca 2+ independent manner has been observed from cerebellar synaptosomal preparation; it is suggested that the aspartatergic endings are lost during the synaptosomal preparation procedure [39]. Thus the procedure applied here seems particularly suitable for hippocampal aspartatergic synaptosome preparation. The lack of Ca 2+ dependent release of aspartate observed in fraction PIII, enriched in MF synaptosomes, reinforces the hypothesis that glutamate, rather than aspartate, is the excitatory amino acid transmitter of the MF terminals, as previously observed [14,58]. Although transmitter release can occur in the absence of external Ca 2 +, it is common to assume that Ca 2+ coming from intraterminal storage sites is still required [32,48,51]. Moreover the reversal of the plasma m e m b r a n e uptake carriers of glutamate, aspartate and G A B A during high K ~ induced depolarization is also involved in the Ca :+ independent release of glutamate, aspartatc and G A B A [38,41]. The Ca 2+ dependent release most likely reflects a release from pools of amino acid transmitter in nerve endings and not from glia nor cell body structures [26]. Indeed, the fractions PI and SI, which are enriched in membranous elements (e.g. gliosomes) exhibit no Ca:* dependent release of aspartate and glutamate (data not shown). After irradiation, as compared with CNT values, under depolarization, the release of glutamate and G A B A was significantly reduced only in PIll fraction, 58 and 74%, respectively. These results suggest that in PIII fraction, a large part of the released glutamate and G A B A originated well from MF synaptosomes, since hippocampal neonatal irradiation destroys 90% of the granule cells and their MF [43]. Part of the remaining released glutamate came probably from other glutamatergic small synaptosomes present in PlII. Thus, our data suggest that G A B A could be coreleased with glutamate from the MF synapses. Whereas in Pll fraction also enriched in MF synaptosomes, the release of glutamate and G A B A was not reduced after irradiation. However, these data do not eliminate the possibility that glutamate and G A B A are the transmitters of the MF synaptosomes in the fraction PI1. The gluta-

f{ Tatq)m el al. / Brain Re.search 644 (1994) 313 321

mate release from small glutamatergic synaptosomes and G A B A release from synaptosomes of inhibitory interneurons may be so high enough to mask a decrease occurring with the loss of M F terminals. In support of this contention, using morphological and biochemical criteria, we have previously demonstrated that the PIII fraction is higher enriched in large MF synaptosomes than PII fraction [57]. 4.2. M F s y n a p t o s o m e s store glutamate, partate

GABA

a n d as-

Analysis of amino acid content showed that glutamate is the most abundant amino acid in all hippocampal synaptosomal fractions, followed by aspartate and glycine. G A B A was present at intermediate level and tyrosine at low levels. Release of 3 - 1 0 % of the total glutamate, G A B A and aspartate contents were induced by a 6 min depolarization. After irradiation, as compared to C N T values, the content of glutamate, aspartate and G A B A was decreased by 62, 44 and 52%, respectively; the reduction of these amino acids correlate with the loss of mossy fibre terminals. These results suggest that in P I I I fraction, a large part of the glutamate and G A B A originated well from MF synaptosomes, indicating that these amino acids could be colocalised in the M F synaptosomes. This study confirms with biochemical technique the presence of glutamate and G A B A in MF terminals, which has been previously detected by immunoreactivity at the ultrastructural level [50,54].

~!';

On one hand, small synaptosomes storing G A B A and containing G A D were mainly distributed ill the less dense layer of the Percoll gradient (l(/- 15% interface): they probably originated from GABAergic interneurones [22,53], since there is no difference in G A B A release and content between CNT and I R R hippocampus. These results were in agreement with electrophysiological works showing that neonatal irradiation of the hippocampus destroys granule cells and mossy fibers leaving intact the great majority of interneurones without affecting the GABAergic inhibitory responses [25]. On the other hand MF synaptosomes storing G A B A were mainly distributed in the denser layer (15-23% interface), as revealed by the decrease of release and content of G A B A after irradiation. However G A B A present in MF terminals does not seem to originate from in situ synthesis. This raises the possibility that GABA: (i) is synthesized by an alternative enzyme pathway [33], (ii) may originate from an uptake mechanism [23]; thus without depleting the transmitter pools of glutamate. The coexistence of an inhibitory and excitatory neurotransmitters in an excitatory synapse is a paradox. The possible functional role of G A B A in the MF remains to be determined. Note: a paper has been recently published in .I. N e u r o c h e m . showing a correlation between the decrease of glutamate release from fractions enriched in synaptosomes (fraction P3) and the destruction of the dentate granule cells by colchicine treatment [12].

Acknowledgements 4.3. M F endings do n o t contain G A D

GABAergic neurons are characterized by the presence of glutamic acid decarboxylase, the G A B A synthesizing enzyme [34,37,49]. We did not observe any enrichment of G A D activity in the fraction PIII. Furthermore only a small amount of G A D was measured in this fraction and represented 5% of the total activity. These data suggest that MF synaptosomes do not contain G A D or contain low levels of this enzyme or inactivated G A D . Indeed zinc, which is known to inhibit G A D activity [45,64], is highly enriched in MF terminals [3,15,16,21,28,29]. Furthermore, after irradiation, we did not observe any difference in the activity of G A D assayed in the different synaptosomat fractions. This was particulary interesting in fraction P i l l where a decrease of both G A B A release and G A B A store were detected after irradiation. These biochemical results reinforce previous work showing that there was no G A D immunoreactivity at the electron microscopy level in the MF synapse [23]. This study demonstrated that two populations of synaptosomes storing and releasing G A B A in the hippocampus can be separated by the Percoll procedure.

We thank D. Djabira for technical assistance. The research was supported by the I N S E R M and Ph. T. was financially supported by the company S E R V t E R .

Abbreviations MF CNT IRR GABA GAD

mossyfiber normaladult hippocampus adult hippocampus irradiated at neonatal stage y-aminobutyric acid L-glutamic1-decarboxylase(EC 4.1.1.15.)

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