Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.     

Hyperforin depletes synaptic vesicles content and induces compartmental redistribution of nerve ending monoamines

Hyperforin, a phloroglucinol derivative found in Hypericum perforatum (St. John's wort) extracts has antidepressant properties in depressed patients. Hyperforin has a unique pharmacological profile and it inhibits uptake of biogenic monoamines as well as amino acid transmitters. We have recently showed that the monoamines uptake inhibition exerted by hyperforin is related to its ability to dissipate the pH gradient across the synaptic vesicle membrane thereby interfering with vesicular monoamines storage. In the present study we demonstrate that hyperforin induces dose-dependent efflux of preloaded [3H]5HT and [3H]DA from rat brain slices. Moreover, we show that hyperforin attenuates depolarization- dependent release of monoamines, while increasing monoamine release by amphetamine or fenfluramine. It is also demonstrated that preincubation of brain slices with reserpine is associated with dose- dependent blunting of efflux due to hyperforin. Our data indicate that hyperforin-induced efflux of [3H]5HT and [3H]DA reflect elevated cytoplasmic concentrations of the two monoamines secondary to the depletion of the synaptic vesicle content and the compartmental redistribution of nerve ending monoamines.     

Ultrastructural changes in granule cell somata and mossy fibers of the rat hippocampus during picrotoxin-induced convulsions

Summary Ultrastructural changes in hippocampal granule cells, mossy fibers and mossy fiber boutons were examined following the administration of picrotoxin in adult rats. Generalized seizures occurred within 5–10 min after the intraperitoneal injection of picrotoxin. The electron-microscopic examination of hippocampal tissues from rats that had been perfused with fixative during the seizure revealed that the large dense-core vesicles increased in number and accumulated on the presynaptic membranes of mossy fiber boutons; some of these vesicles appeared to be fused with the membranes, and omega-shaped exocytotic profiles were frequently seen. Furthermore, greatly increased numbers of coated vesicles (60–90 nm in diameter) were observed on the maturing faces of Golgi fields of granule cells. Thus, our study not only indicates an increased incidence of exocytosis of large dense-core vesicles during picrotoxin-induced seizures, but also suggests that these vesicles are replaced in excess from the perikaryon of the granule cell.     

Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines

The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.     

IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca(2+)-dependent, evoked release of glutamate, GABA and serotonin

Synaptic vesicles in the nerve terminal play a pivotal role in neurotransmission. Neurotransmitter accumulation into synaptic vesicles is catalyzed by distinct vesicular transporters, harnessing an electrochemical proton gradient generated by V-type proton-pump ATPase. However, little is known about regulation of the transmitter pool size, particularly in regard to amino acid neurotransmitters. We previously provided evidence for the existence of a potent endogenous inhibitory protein factor (IPF), which causes reduction of glutamate and GABA accumulation into isolated, purified synaptic vesicles. In this study we demonstrate that IPF is concentrated most in the synaptosomal cytosol fraction and that, when introduced into the synaptosome, it leads to a decrease in calcium-dependent exocytotic (but not calcium-independent) release of glutamate in a concentration-dependent manner. In contrast, alpha-fodrin (non-erythroid spectrin), which is structurally related to IPF and thought to serve as the precursor for IPF, is devoid of such inhibitory activity. Intrasynaptosomal IPF also caused reduction in exocytotic release of GABA and the monoamine neurotransmitter serotonin. Whether IPF affects vesicular storage of multiple neurotransmitters in vivo would depend upon the localization of IPF. These results raise the possibility that IPF may modulate synaptic transmission by acting as a quantal size regulator of one or more neurotransmitters.     

Non-lipid osmiophilic granules in the presynaptic neuroplasma of the boutons

Summary Brain, spinal cord and peripheral (sensory and sympathetic) ganglia of cats and rats have been fixed in Susa, imbedded and impregnated on slides with a mixture of osmium tetroxide and egg albumen solution.This method produced small (about one micron) black granules in the boutons around the multipolar nerve cells in the cord and in the medulla. The granules were absent around the other nerve cells in the cortex (pyramidal cells, etc.) and in the peripheral ganglia.These osmiophilic granules may be clusters of synaptic vesicles containing transmitter substance. If the clusters are large enough, as in the boutons, they are visible under the light microscope, if they are smaller;, as in the smaller synaptic knobs, they remain invisible.If the osmiophilic granules are clusters of vesicles containing the transmitter substance, this substance might be an acidic amino acid.     

Synapses in the rat substantia nigra

The composition and organization of the input to the rat substantia nigra were studied with the electron microscope. Four distinct types of synaptic boutons were described. The first contained small (381 A), clear synaptic vesicles. The second type contained the small, clear vesicles and several large, dense-core vesicles. The third ending contained large, dense-core vesicles and larger (581 A) clear vesicles. The fourth ending, found on the axon hillock and other terminal boutons, contained slightly elongated, clear synaptic vesicles. The presence of these four boutons was discussed in light of the known afferent input and neurochemical composition of the substantia nigra.     

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGlu_u that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

This study uses several high-resolution imaging techniques to investigate the structural correlates of presynaptic potentiation at hippocampal mossy fiber boutons, observing an increase in release sites and in release synchronicity accompanied by synaptic vesicle dispersion in the terminal and accumulation at release sites, but no modulation of the distance between calcium channel and release sites.     

The first luminal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense-Core and Somatodendritic Clathrin-Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.     

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butyric acid (GABAA) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 microM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 microM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.     

Elektronenmikroskopische Untersuchungen am Ammonshorn

Zusammenfassung Vergleichende phasenkontrast- und elektronenmikroskopische Untersuchungen der Moosfaserschicht des Ammonshorns des Kaninchens ergaben:Im Phasenkonstrastbild hebt sich die Moosfaserschicht besonders deutlich ab. Der infrapyramidale Trakt der Moosfasern ist fast ebenso lang wie der suprapyramidale Trakt.In der Moosfaserschicht sind nach einer Nachfixierung mit OsO₄ die apikalen und basalen Dendriten der Pyramidenzellen nahezu vollständig von kontraststärkeren Strukturen umgeben als im übrigen Gewebe. Diese sind elektronenmikroskopisch eindeutig als die synaptischen Endformationen der Moosfasern zu identifizieren. Die synaptischen Endformationen der Moosfasern enthalten größere Mengen dicht gelagerter synaptischer Vesikel, außerdem Vesikel, die ein osmiophiles Zentrum besitzen. Die Befunde werden im Zusammenhang mit dem verstärkten Zinkgehalt der Moosfaserschicht diskutiert.

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Summary Comparative phase- and electron microscopical investigations have been carried out on the hippocampal layer of mossy fibres of the rabbit.The phase contrast microscope allows a particularly distinct demonstration of the layer of mossy fibres. The infrapyramidal tract of the mossy fibres is of approximately the same length as the suprapyramidal tract.After postfixation with OsO₄ it is seen that the apical and basal dendrites of pyramidal cells in the mossy fibre layer are almost entirely surrounded by darker structures, which are more clearly definable than in the surrounding tissue. Electron microscopically these structures could definitely be identified as the boutons of the mossy fibres. The synaptic endings of the mossy fibres contain larger masses of more densely packed synaptic vesicles and also a moderate number of vesicles, which include an osmiophilic centre. Our results are discussed with regard to the increased zinc content of hippocampal mossy fiber layer.

     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type.Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing densecore vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50–100 nm), vesicles with a highly electron-dense core (60–140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neuro-secretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Zinc-iodine-osmium impregnation of giant synaptosomes obtained from the hippocampal formation of the rabbit]

The fraction of giant synaptosomes from the r. inferior of the rabbit hippocampus was studied using impregnation with zinc iodide-osmium tetroxide (XIO) reagent and electron microscopy. In this fraction, light and dark synaptosomes were observed. The reaction product was found in the clear-centered synaptic vesicles (200-400 A) as electron-dense structures of different forms and small osmiophilic particles on the vesicular membranes. Dense-cored vesicles and postsynaptic structures were not revealed with ZIO-reagent. The structures revealed with ZIO-reagent in the giant synaptosomes of the hippocampus are supposedly related to stroage of the neurotransmitter-glutamate.     

Synaptic connections of substance P-immunoreactive nerve terminals in the substantia nigra of the rat

Summary A monoclonal antibody that recognises the C-terminal part of substance P was used to study immunoreactive structures in the substantia nigra by the unlabeled antibody, peroxidase-antiperoxidase procedure. Immunoreactivity was present in nerve fibres in all parts of the substantia nigra, particularly in the pars reticulata and pars lateralis. Electron microscopically two types of bouton immunoreactive for substance P were found: Type 1 contained large electron-lucent vesicles, occasional large granulated vesicles and formed symmetrical synapses with dendrites. Type 2 boutons contained smaller, round electron-lucent vesicles, many large granular vesicles and formed asymmetrical synapses (having prominent postjunctional dense bodies) with dendrites and perikarya.Immunoreactive fibres with varicosities that had been identified light microscopically were studied in serial sections in the electron microscope. Each identified varicosity contained synaptic vesicles and formed a single synapse. An individual fibre formed boutons of only one kind (type 1 or type 2) and could form multiple synapses with the same neuron. Thus, an identified fibre in the pars compacta had eight varicosities, each of which was in synaptic contacts (type 2) with the dendrites or soma of the same neuron.The results are consistent with the concept that substance P is a synaptic transmitter in the substantia nigra and indicate that neurons in this region may receive a significant input from substance P-containing afferents, and that there are at least two types of such afferent fibres.