Neurochemical Research, Vol. 25, Nos. 9/10, 2000, pp. 1265–1274

Synaptosomes Still Viable after 25 Years of Superfusion* Luca Raiteri1 and Maurizio Raiteri1,2 (Accepted March 6, 2000)

Superfused synaptosomes have been utilized in studies of neurotransmitter release during 25 years. This review summarizes the aspects of neurotransmission that have been and could be successfully investigated with this technique. The major aim of the article is to draw attention on the versatility of superfused synaptosomes and to suggest how the system could be exploited in clarifying several aspects of synaptic neurochemistry including neurotransmitter transport, receptor localization, receptor-receptor interactions, functional aspects of multi-sited receptor complexes, receptor heterogeneity and mechanisms of neurotransmitter exocytosis-endocytosis.

KEY WORDS: Synaptosomes; neurotransmitters; superfusion technique.

It is now 25 years since our ‘simple apparatus for studying the release of neurotransmitters from synaptosomes’ was introduced (1). The original system, here shown in an ancient picture (Fig. 1), consisted of four identical superfusion chambers having at the bottom filter holders of porous glass. Standard synaptosomes were plated as very thin layers on microporous filters and updown superfused. Since then the original apparatus has undergone some modifications; the changes introduced have however been of cosmetic nature (more superfusion chambers, introduction of upper reservoirs to deliver solutions of varying composition into the chambers and of gassing devices etc.), while the core of the superfusion system has remained unchanged. Fig. 2 shows the last version of the 12-chamber apparatus now available on the market.1 Why Superfused Synaptosomes? Distinguishing True Releasers from Reuptake Inhibitors. Some read-

ers may wonder why was an apparatus for the superfusion of synaptosomes originally devised. A quarter of a century ago neurotransmission was thought of as a relatively simple process, made of a few fundamental steps. Among these, release and reuptake of neurotransmitters attracted the interest of many neurochemists and neuropharmacologists. One important problem to solve was that of distinguishing between drugs able to enhance release directly and drugs that could do it indirectly, i.e. by preventing transmitter reuptake (Fig. 3). Although both types of drug would in the end augment the synaptic concentration of the transmitter, their pharmacological and therapeutic effects might not have been the same. We now know that this is indeed the case as, for instance, pure inhibitors of serotonin reuptake are excellent antidepressants, whereas direct serotonin releasers, such as fenfluramine or ecstasy, display completely different biological activities. To solve the problem, we then thought that true releasers could be distinguished from reuptake inhibitors by superfusing

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2

INTRODUCTION

Dipartimento di Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, Viale Cembrano 4, 16148 Genova, Italy. Europe: UGO BASILE, Via G. Borghi 43, Comerio, VA, Italy Fax: ++ 39 0332 745488; E-mail: [email protected] USA and Canada: STOELTING Co, Wood Dale, Il USA; Fax: 630 8609775; E-mail: [email protected]

Address reprint requests to: Maurizio Raiteri, Dipartimento di Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, Viale Cembrano 4-16148 Genova, Italy. Tel: 39 010 3532651; Fax: 39 010 3993360; E-mail: [email protected] * Special issue dedicated to the 25th anniversary of Neurochemical Research.

1265 0364-3190/00/09/1000–1265$18.00/0 © 2000 Plenum Publishing Corporation

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Fig. 3. Reuptake inhibitors vs. true releasers. Pure reuptake inhibitors are unable to increase release from thin layers of synaptosomes updown superfused (see also Fig. 5).

Fig. 1. The prototypic superfusion apparatus. Taken from Raiteri M., Angelini F. and Levi G. (1).

Fig. 4. Carrier-mediated release. Neurotransmitters are physiologically released by vesicular exocytosis. Several transmitters can also be released through the uptake carrier which, under some pharmacological and pathological conditions, can work in the inside-out direction.

Fig. 2. The semi-automated version of the superfusion system now commercially available.

up-down a very thin layer of synaptosomes and monitoring transmitter release in presence of the drug under study. The idea was that the transmitter released spontaneously could have been removed by the superfusion medium quickly enough to escape reuptake. If this were the case, a true releaser, but not a pure reuptake inhibitor, would have increased release. We did not ignore that the process of reuptake immediately followed the release of the transmitter and that to obtain a separation of the two processes would have theoretically

been very difficult; but luckily enough, known uptake blockers could not significantly increase the spontaneous release of the transmitters under study indicating that, in our system, reuptake did not in fact occur. Since then, several drugs have been successfully classified as pure reuptake inhibitors or true releasers using superfused synaptosomes, in various laboratories. Neurotransmitters Can Be Released through Reversal of the Uptake Carrier. The use of superfused synaptosomes made it possible to discover that some neurotransmitters, i.e. those for which a reuptake carrier exists on the presynaptic terminal, can be released not only by vesicular exocytosis but, under some conditions, through the carrier working in the inside-out direction (Fig. 4). We observed for the first time that this mecha-

Superfused Synaptosomes in Studying Transmitter Release nism (termed carrier-mediated release or, more recently, release by transporter reversal) can occur in the CNS with GABA (2,3), glycine (2) and glutamate (4). Subsequently, carrier-mediated release of norepinephrine (5), dopamine (6) and serotonin (7) was shown to take place in superfused synaptosomes. These findings have been confirmed in several laboratories and in various CNS preparations, including in vivo microdialysis (8–34; see, for reviews, references 35–38). All these data lead to conclude that the mechanism of carrier-mediated release certainly plays important roles in the mechanism of action of indirectly acting amines and in some pathological conditions. Indeed, drugs like the amphetamines, fenfluramine and the indirect sympathomimetic amines could not act if carrier-mediated release of biogenic amines could not occur. Recently, the carrier-mediated release of glutamate has been attributed critical importance in some neuropathological conditions, in particular brain ischemia (11,21,28,34,39–42). The Method of Choice to Study Presynaptic Receptors. When the era of presynaptic receptors began, our superfusion technique proved particularly useful in the identification and characterization of receptors that are localized on CNS axon terminals and mediate regulation of transmitter release. We now know that the release of one transmitter can be regulated by several modulators acting at their respective receptors present on the terminals releasing the transmitter. We also know that the release regulation through presynaptic receptors is a general mechanism that involves all the transmitters so far known. Hundreds of papers from many laboratories have described a myriad of presynaptic auto- and heteroreceptors (see, for reviews, references 43–57). Some experts believe that superfused synaptosomes represent the preparation of choice in studies aimed at the identification and characterization of release-regulating presynaptic receptors. It should be stressed that the term presynaptic has an essentially anatomic meaning, i.e. it refers to the localization of the receptor on the presynaptic axon boutons; apart from this, presynaptic receptors do not differ from postsynaptic receptors; they are receptors in every respect and therefore presynaptic receptor regulating release represent an excellent model to study various aspects of receptors in general, including function, transduction mechanisms and pharmacology. The model seems particularly useful in the identification and characterization of pharmacological subtypes of receptors (see, for instance, references 50,58–62). Major Advantages of the Superfusion Technique. There are a number of reasons that make our superfusion technique particularly suitable to investigate some of the many unsolved questions related to presynaptic

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neurotransmission. We will try to clarify some of the peculiar aspects of the technique with the help of the picture shown in Fig. 5. As already mentioned, pure uptake inhibitors do not increase spontaneous release of transmitters, thus demonstrating that reuptake does not occur in our system. Similarly, receptor antagonists have often been shown not to affect spontaneous/evoked transmitter release, indicating that the transmitters released are removed by the superfusion fluid before they can activate presynaptic auto- and heteroreceptors. The transmitter released from one terminal can not activate receptors present on neighbouring terminals (presynaptic heteroreceptors), excluding the possibility of indirect effects. Thus the scenario is that of a thin layer of synaptosomes having their membrane targets (transporters, receptors etc.) virtually free of endogenously released agonists. Each of these targets can however be selectively activated by adding the appropriate ligand, at the desired concentration, to the superfusion medium. The characteristics of the system are such that any effect on the release of one neurotransmitter can be attributed exclusively to an action on the nerve terminal releasing that transmitter. One may therefore conclude that the heterogeneity of synaptosomal preparations, indeed a major drawback under some circumstances, bears relatively little importance when a given target present on one family of nerve endings is investigated. In a sense, the situation is reminiscent of that of recombinant receptors or transporters expressed in heterologous cells, although the advantages of studying the function of a target protein inserted in its natural environment appear obvious.

Fig. 5. Distinctive features of the superfusion technique. Endogenous transmitters/modulators released are immediately removed by the superfusion medium before they can be retaken up by transporters and before they can activate autoreceptors or heteroreceptors present on nerve terminals. Reuptake can therefore not occur and indirect effects are minimized or prevented. During superfusion all the presynaptic targets (transporters, receptors, channels, enzymes, etc.) can be considered virtually free of endogenous ligands; each of these targets can be studied separately by adding the appropriate ligand, at the desired concentration, to the synaptosome thin layer. The effects observed on the release of one transmitter can reasonably be attributed to direct actions at the terminals storing that transmitter.

1268 Understanding Multi-Sited Receptors. Receptor complexes, such as the glutamate receptor of the NMDA type and the GABAA receptor, can be investigated in their native forms, using superfused synaptosomes with some advantages with respect to other experimental approaches. Glutamate and glycine are considered coagonists at NMDA receptors; moreover, NMDA receptors possess various modulatory sites including a polyamine site, a Mg2+ site, a Zn2+ site and so forth. The interactions of these multiple sites can not be easily quantified or, in some cases, clearly identified using more intact CNS tissue preparations, because all the respective ligands are endogenous components that are present in unknown concentrations in the extracellular space. The understanding of these interactions in native receptors can be greatly facilitated using superfused synaptosomes where recognition sites and modulatory sites are virtually free of endogenous ligands. As a first step, it is necessary to establish the presence of releaseregulating NMDA receptors on a given family of axon terminals. For example, noradrenergic terminals in cortex and hippocampus and dopaminergic terminals in the corpus striatum possess NMDA receptors the activation of which brings about increase of norepinephrine and dopamine release, respectively (63–66). Considering the characteristics of the superfusion system discussed above, the possibility to ‘dissect’ each of the multiple sites of complex receptors from which endogenous ligands are washed away, but exogenous ligands can be added as desired, appears obvious. Thus the effect of NMDA on norepinephrine or dopamine release can be directly shown to depend on the presence of glycine (this in slices is often impossible) and the sensitivities of the evoked release to various ligands, for instance to Zn2+, to H+ and to polyamine ligands, can be quantitatively evaluated, so that important information on the subunit composition of the NMDA receptor subtypes involved can be obtained. Studies with neocortex and hippocampal synaptosomes superfused in presence of the HIV-1 envelope protein gp120 show that concentrations of the protein compatible with those presumably present in the brain of AIDS patients can potentiate the effect on norepinephrine release of glutamate acting at NMDA receptors in rat (67) and human (68) brain (see Fig. 6). Experiments with various peptide fragments of gp120 indicate that the protein acts through its V3 loop at NMDA receptors probably by mimicking the natural coagonists glycine/D-serine. Both gp120 and its V3 loop display an impressive potency at the NMDA receptor glycine site and appear to be, in our system, the most potent glycine site agonists known so far (69).

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Fig. 6. The HIV-1 envelope protein gp120 present in the brain of AIDS patients potentiates the release of norepinephrine (NE) evoked by N-methyl-D-aspartate (NMDA) receptor agonists. The characteristics of the superfusion technique (see Fig. 5) permit to conclude that gp120 acts at NMDA receptors sited on noradrenergic nerve terminals by mimicking with impressive potency the natural glutamate coagonist glycine.

Thanks to the possibility of studying presynaptic receptors free of endogenous ligands it has even been possible to identify a NMDA receptor that can be activated by glycine or D-serine only, i.e. in the absence of the glutamate coagonist (70). The release of cholecystokinin or somatostatin from superfused rat neocortex synaptosomes depolarized with K+ (a condition that causes removal of the Mg2+ block from the NMDA channel) can be potentiated by adding to the superfusion medium low micromolar concentrations of glycine or D-serine (Fig. 7). A receptor of the NMDA type is involved because the effect of glycine or D-serine was insensitive to strychnine, but it was blocked by the glycine site selective antagonist 7-Cl-kynurenate and by the channel antagonist MK-801. Endogenous glutamate was expected not to be present because of its quick removal by the superfusion medium; in any case, the effect of glycine or D-serine was totally insensitive to selective antagonists of the glutamate recognition site of the NMDA receptor, like D-AP5 or CGS 19755 or CPP (70) showing that, in this atypical NMDA receptor, the ion channel can be activated by glycine or D-serine only. Furthermore, the effect of glycine or D-serine was highly sensitive to low concentrations of Zn2+ or ifenprodil, suggesting that the receptor contains the three subunits NR1A, NR2A and NR2B (70). Similarly to NMDA receptors, native receptor complexes of the GABAA type can be functionally investigated with thin layers of superfused synaptosomes.

Superfused Synaptosomes in Studying Transmitter Release

Fig. 7. NMDA receptors can mediate cholecystokinin (CCK) or somatostatin (not shown) release. These receptors can be activated not only by glutamate/NMDA but also by glycine or D-serine ‘only’ in absence of any glutamatergic co-agonist. The effect of glycine was blocked by 7-Cl-kynurenic acid (7-Cl-KYNA), antagonist at the glycine site and by MK-801, blocker of the receptor channel; but not by antagonists at the glutamate recognition site of the NMDA receptor, like D-AP5, CGS 19755 and CPP. * p < 0.01 vs. glycine. Taken, in part, from Paudice et al. (70).

The multiple modulatory sites present on these receptors can be dissected out and the function of each can be analyzed by adding selective ligands, alone or in combination, while endogenous ligands (GABA, neurosteroids, Zn2+, benzodiazepines etc.) are washed away. Recent papers describe the characterization of two GABAA receptor subtypes differing in their sensitivity to benzodiazepines (62,71), neurosteroids (72) and Zn2+ ions (73) and the changes in the pharmacological properties due to modifications occurring in some receptor subunits of alcohol non-tolerant rats (71,73). Multiple Transmitter Carriers Can Be Present on One Nerve Terminal. The peculiar properties of the superfusion system have led to the unexpected finding that one nerve terminal can possess two or more neurotransmitter carriers: in particular, the carrier that mediates reuptake of the home-made transmitter (homocarrier) can coexist with high-affinity carriers able to capture other transmitters (heterocarriers). Interestingly, the uptake through the heterocarrier is followed by increased release of the home-made transmitter, apparently by exocytosis, suggesting that the release of one transmitter may be regulated by other transmitters not only by conventional presynaptic heteroreceptors

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but also through heterocarriers (see, for review, reference 74). Whatever the functional significance of heterocarriers, the finding that more than one transporter is certainly present on the same nerve terminal clearly means that neurotransmitter transporters are not cellspecific markers (see reference 75, for discussion). The coexistence of two carriers on the same nerve terminal appears to reflect, in some cases, transmitter coexistence. A few years ago we could observe that glycine released GABA and GABA released glycine from synaptosomal preparations of rat spinal cord. Our hypothesis was that glycine and GABA are co-transmitters in some spinal cord nerve endings (76). In a recent electrophysiological study Jonas et al. (77) showed that glycine and GABA can indeed be co-released from the same spinal cord axon terminal onto glycine and GABAA receptors that coexist on motor-neurons. We are now investigat ing the reciprocal interactions between glycine and GABA in mouse spinal cord synaptosomes, in order to shed light on the mechanisms by which the two transmitters are co-stored in synaptic vesicles and reciprocally affect each other release. Studying Receptor-Receptor Interactions. If addition to the superfusion medium of an agonist selective for one receptor permits to investigate the function of this receptor ‘in isolation’, a second receptor can be activated by adding a selective agonist. If the latter agonist modifies the effect on release of the former, one can conclude that a) the two receptors coexist on the same axon terminal; b) there may be a cross-talk between the two receptors. Receptor-receptor interaction is therefore one of the phenomena that can be studied most successfully with our technique. We previously demonstrated that NMDA and AMPA receptors coexist on the same noradrenergic axon terminal in the rat hippocampus (78). Activation of either receptor can cause release of norepinephrine (64). Furthermore, activation of AMPA receptors exerts a permissive role on that of NMDA receptors since the latter can be activated by glutamate when the medium contains physiological concentrations of Mg2+ ions that normally hinder NMDA receptor activation by NMDA (78). Similar mechanism was found to be present on dopaminergic synaptosomes superfused in presence of AMPA or NMDA or their mixture (66). Very recently we observed that somatostatin receptors also coexist with NMDA receptors on noradrenergic axon terminals of the hippocampus. In presence of Mg2+, NMDA can not release norepinephrine from superfused hippocampal synaptosomes; however somatostatin, inactive on its own, permits norepinephrine release by NMDA in

1270 Mg2+-containing medium. The releasing effect can be blocked by heparin, an antagonist at the inositol trisphosphate receptors that mediate Ca2+ release from the endoplasmic reticulum; also, the effect of NMDA plus somatostatin is sensitive to protein kinase C inhibitors. We propose (see Fig. 8) that somatostatin activates a receptor coupled to the phosphatydilinositol pathway (79), leading to release of Ca2+ and activation of protein kinase C. Phosphorylation of NMDA receptor subunits might then facilitate receptor activation by glutamate through a removal of the Mg2+ block (80). Release Studies with Superfused Synaptosomes from Genetically Modified Mice. Scientists are showing increasing interest in the superfusion technique as they realize that it can indeed help to solve problems that could not be as appropriately approached by other techniques. The recent availability of genetically modified mice lacking a given receptor or receptor subtype has greatly enhanced our understanding of the physiological function of different receptors. It is known that knock-out mice often show modest differences with respect to wild-type mice when examined by in vivo behavioral tests, probably due to compensatory mechanisms. Differences can clearly emerge, however, when comparing one function strictly linked to the activation of one receptor. Autoreceptors regulating the release of dopamine have long been known to be DA2 type (49). However, this receptor type is heterogeneous and comprises three subtypes termed DA2, DA3

Fig. 8. Interaction between NMDA receptors and somatostatin (SRIF) receptors co-localized on axon terminals releasing norepinephrine (see text).

Raiteri and Raiteri and DA4 (for reviews see references 81,82). Although the pharmacological tools available easily permit to exclude that DA autoreceptors belong to the DA4 subtype, whether these receptors are DA2 or DA3 has been controversial. The recent use of knock-out mice has provided one convincing answer. The Glowinski group (83) reported that the evoked release of [3H]dopamine from superfused mouse striatal synaptosomes could not be inhibited by DA2/DA3 mixed type agonists in mice that lack DA2 receptors, suggesting that autoreceptors belong to the DA2 subtype. Similarly, the laboratory of Jean-Pierre Changeux used superfused mouse brain synaptosomes to characterize the nicotinic receptors that mediate stimulation of GABA release. Unequivocal evidence that the receptor in point contains a β2 subunit was obtained in experiments with wild-type, heterozygous and homozygous β2 null mutant mice showing that the nicotine-stimulated GABA release decreased along with the number of copies of the null mutant gene (84). Superfused Synaptosomes as a Model to Study Mechanisms of Exocytosis/Endocytosis. One aspect of neurotransmission in which superfused synaptosomes will probably be of great help in the near future concerns the mechanisms of neurotransmitter release. Exocytosis and endocytosis are emerging as tremendously complex processes in which an increasing number of presynaptic actors appear to be involved. Several proteins (synaptobrevins, syntaxins, SNAP-25, synaptotagmins, synapsins and so on) have come on the stage in recent years and the critical contribution of some to transmitter release has been proposed. The group of Südhof, one of the leading in the field of exocytosis, has recently adopted the synaptosome superfusion technique (85). It was known from electrophysiology that, in mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cyclic AMP; in contrast, CA1region synapses do not exhibit this type of LTP. Lonart et al. (85) show that cyclic AMP can enhance glutamate release from CA3, but not CA1, superfused synaptosomes through multiple actions. Most important, they conclude that “These results agree well with the electrophysiological observations and suggest that the standard procedures used in electrophysiological studies to examine glutamate release (membrane depolarization, Ca2+ influx and hypertonic sucrose) can also be applied to CA1 and CA3 region synaptosomes” (85). The function of presynaptic proteins involved in the processes of exocytosis and endocytosis can be analyzed by using selective probes. Clostridial toxins have been of great help in understanding exocytosis essentially because specific neuronal receptors exist to

Superfused Synaptosomes in Studying Transmitter Release internalize these toxins into intact nerve endings. Several laboratories have shown that release of neurotransmitters from synaptosomes is prevented following preincubation with clostridial toxins able to cleave selectively some presynaptic proteins (86–90). On the other hand, the hypothesized function in exocytosis of other presynaptic proteins has been difficult to verify, particularly in mammalian neurons, because the appropriate probes are often membrane-impermeant. These probes can obviously be injected into squid giant axons, but the results obtained may not always be extrapolated to the mammalian brain. Some groups have approached the problem by permeabilizing mammalian synaptosomes with various agents, including streptolysin-O (91) or by freezing-thawing synaptosomes to create transient openings in the nerve ending membrane (92). The major problem with these techniques is that, although the desired probes, including antibodies (93), can be introduced into synaptosomes and seem to produce the expected effects on transmitter release, cytoplasmic constituents can concomitantly exit from the nerve endings leading to uncontrolled changes in the intraterminal milieu. We believe that these difficulties can be overcome by applying a simple technique originally devised to introduce small membrane-impermeant molecules into synaptosomes (94). We recently found that non-permeant molecules of large size, including pertussis toxin or antibodies, if present during homogenization of brain tissue, can be entrapped within subsequently isolated synaptosomes (95). The entrap-

Fig. 9. Schematic representation of the entrapping technique. Membrane impermeant compounds of different molecular size can be introduced into synaptosomes if present during the homogenization step of the synaptosomal preparation.

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ping technique, illustrated in Fig. 9, has the great advantage that the molecule present in the homogenization medium represents the sole variable; in other words, synaptosomes with the entrapped molecule behave exactly like standard synaptosomes except for the function(s) possibly affected by the compounds introduced during the homogenization. The results we recently obtained with pertussis toxin (PTx) are particularly interesting also considering that studies with PTx and synaptosomes are extremely difficult because the very slow internalization of PTx requires prolonged preincubations that damage the nerve terminals. We showed that intrasynaptosomal concentrations of PTx probably not exceeding 50 pM almost doubled the release of GABA and glutamate

Fig. 10. Pertussis toxin (PTx) entrapped in rat cerebrocortical synaptosomes causes enhancement of 12 mM KCl-evoked GABA and glutamate release and decrease of the (−)baclofen inhibitory actions at GABAB auto- and heteroreceptors. Neocortical tissue homogenization was performed in absence or presence of 5 nM PTx. 12 mM KCl; 12 mM KCl plus 10 µM (−)baclofen; 12 mM KCl plus 100 µM (−)baclofen. From Raiteri et al. (95).

1272 evoked by depolarization with 12 mM KCl (Fig. 10). Moreover, the inhibition of the K+-evoked overflows of GABA and glutamate by (-)baclofen acting, respectively, at GABAB auto- and heteroreceptors was strongly reduced in PTx-treated with respect to control synaptosomes (95). The possibility of preparing synaptosomes that contain membrane-impermeant probes selective for the various presynaptic proteins will certainly help understanding the mechanisms of exocytosis and endocytosis. ACKNOWLEDGMENTS The works discussed in this review were supported by grants from the Italian Ministry of Health “Progetto Nazionale Ricerca AIDS”, Italian MURST, Italian CNR “Target Project on Biotechnology” and MURST/CNR Biotechnology Program L.95/95. The financial support of Telethon—Italy is gratefully acknowledged. We wish to thank Mrs Maura Agate for editorial assistance in preparing the manuscript.

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