Population Of Synaptic Vesicles Research Articles

Immunoisolation of GABA-Specific Synaptic Vesicles Defines a Functionally Distinct Subset of Synaptic Vesicles

Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.

Calcium-dependent Oligomerization of Synaptotagmins I and II: SYNAPTOTAGMINS I AND II ARE LOCALIZED ON THE SAME SYNAPTIC VESICLE AND HETERODIMERIZE IN THE PRESENCE OF CALCIUM

Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 +/- 4 microM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.

Synaptic Vesicle Populations in Saccular Hair Cells Reconstructed by Electron Tomography

We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 +/- 65 nm diameter; mean +/- SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by approximately 20-nm-long filaments, at an average density of 55% of close-packed (376 +/- 133 vesicles per SB). These vesicles could support approximately 900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+ depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mM Ca2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which ( approximately 32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most approximately 70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.

Thy-1 is a component common to multiple populations of synaptic vesicles.

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

Caenorhabditis elegans rab-3Mutant Synapses Exhibit Impaired Function and Are Partially Depleted of Vesicles

Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

The Vesicular Monoamine Transporter VMAT2 and Vesicular Acetylcholine Transporter VAChT Are Sorted to Separate Vesicle Populations in PC12 Cells

Publisher Summary Vesicular transporters of transmitters are excellent markers for functional neuroanatomy and for potential identification of vesicular contents at the subcellular level. In this chapter, the distribution of two vesicular monoamine transporters (VMAT1 and VMAT2), a vesicular acetylcholine transporter (VAChT), a transmembrane transporter (SV2), and two other proteins have been examined. VMAT1 is endogenous in PC12 cells, and it is abundant on LDCVs. This location of VMAT1 is consistent with the fact that LDCVs take up norepinephrine in PC12 cells. On the otherhand, VMAT2 is not endogenous in PC12 cells. In the chapter VMAT2 has been expressed in PC12 cells under a constitutive viral promoter to see if it would be sorted to secretory vesicles. In the transfected PC12 cells, VMAT2 is preferentially localized on the LDCVs and not on the SSVs. The lack of a prominent population of VMAT2-positive synaptic vesicles in these PC12 cells is in contrast to the distribution of endogenous VAMT2 in central and peripheral adrenergic neurons. In these adrenergic neurons in vivo, VMAT2 is localized on LDCVs and on a distinct population of catecholamine-containing synaptic vesicles that are termed small dense-core vesicles (SDCVs). The chapter also analyzes subcellular distributions of three other SSV components, synaptophysin, SV2, and VAChT. Proteins destined for cholinergic SSVs may arrive via LDCVs or constitutive vesicles and recycle through the early endosomes, where they are concentrated for inclusion in the SSV membrane. Proteins in this category include SV2, VAChT, and synaptophysin. VMAT2, the neuronal VMAT in LDCVs and SDCVs of noradrenergic neurons, is not targeted to cholinergic SSVs when expressed in PC12 cells. Thus, SDCVs of noradrenergic neurons may represent a distinct population of synaptic vesicles that is biosynthetically separate from cholinergic SSVs in PC12 cells.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

Pathological changes induced by an acidic phospholipase A 2 from Ophiophagus hannah venom on heart and skeletal muscle of mice after systemic injection

An acidic phospholipase A 2 (OHV A-PLA 2) isolated from the venom of the king cobra ( Ophiophagus hannah) was tested for its ability to cause pathological changes to myocardium, skeletal muscle and cardiac ganglia. White mice were injected intravenously with dose of 8 mg/kg or 4 mg/kg of OHV A-PLA 2 and tissue samples were taken at 6 or 24 hr. Light microscopic examination failed to show significant changes in cardiac muscle and ganglia. Skeletal muscle showed myofibre degeneration and necrosis. Electron microscopic study revealed myodegeneration in cardiac and skeletal muscles, and reduction in synaptic vesicle population of preganglionic nerve terminals in cardiac ganglia. Ultrastructural changes in tissues were dose related. The lower dose (4 mg/kg) of OHV A-PLA 2 produced mild myocardial changes, the myofilaments were intact but contracted, and the A band and I band were skewed. OHV A-PLA 2 caused myocardial degeneration at a higher dose of 8 mg/kg. The changes included dissolution of actin and myosin filaments, dilatation and disorganization of sarcoplasmic reticulum and degeneration of mitochondria. The skeletal muscle lesions were more severe than the myocardial changes. Some of the myofibrils were severely disorganized and lack typical striated appearance, sarcomeres disrupted, most of mitochondria were vesiculated and destroyed.

Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd(2+)- or K(+)-induced ACH release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd2+ in Ca(2+)-free medium for a period of 3 h, (2) 25 or 40 mM K+ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluorescein-conjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated alpha-bungarotoxin. Unstimulated and K(+)-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd2+ in Ca(2+)-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K(+)-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd2+ stimulation in Ca(2+)-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd2+ stimulation in Ca(2+)-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K+ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.

Chapter 21: Storage and release of acetylcholine in a sympathetic ganglion

This chapter discusses the behavior of releasable transmitter, if it is stored in at least two compartments: one, a readily releasable store, provides ACh immediately available for secretion and the other contains transmitter that awaits mobilization to sites of release. This idea was formulated by Birks and MacIntosh, on the basis of their kinetic analysis of the rate of ACh released from a sympathetic ganglion stimulated through its nerve trunk. Their analysis of the release of pre-formed ACh yielded a bi-exponential relationship, and they concluded that some 15% of releasable ACh was discharged with a faster time-constant than the less readily releasable store. Since this pioneering analysis by Birks and MacIntosh, a variety of evidence has been presented supporting the idea that the stored ACh in cholinergic terminals behaves as if it is heterogeneous with respect to release kinetics and to ACh turnover. Morphology of nerve terminals has shown a population of synaptic vesicles closely associated with sites of release, the active zone, whereas other vesicles clearly require mobilization to bring them to the proximity of docking sites at the active zone. Thus, the concept of transmitter mobilization from reserve to readily releasable stores of ACh has become established. But the mechanisms that regulate this process have not been well explored. This chapter presents three examples of factors that appear to interact or influence this process of mobilization.

Compartmentalization of monoaminergic synaptic vesicles in the storage and release of neurotransmitter.

Monoaminergic nerves are characterized by the presence of a population of small synaptic vesicles (40-60 nm in diameter) containing a few large vesicles (80-90 nm in diameter). Thus, although both types of vesicles contain monoamines, the small vesicles must be considered as the organoid responsible for the storage and release of the neurotransmitter, whereas the large ones possibly are involved in the modulation of the process. The small vesicles are electron-lucent or have an osmiophilic electron-dense core that is always linked to the vesicle membrane. Considering morphological and histochemical evidence under different experimental conditions, we proposed the existence of two compartments in the small vesicles: the core and the matrix, corresponding respectively to the electron-dense core and the electron-lucent space between the core and the vesicle membrane in osmium tetroxide fixations. The sizes of both compartments are inversely related, i.e., the smaller the core, the larger the matrix and vice versa. The core even disappears, giving way to a small electron-lucent vesicle made exclusively by the matrix. Thus, the matrix is a constant component of the vesicle, whereas the core is a transient one. Each compartment has a different pool of amine: a loosely bound, easily releasable pool in the matrix and a tightly bound, more resistant pool in the core. These two pools subserve, respectively, a tonic or phasic release of the neurotransmitter, correlated with a tonic or phasic stimulation of the receptor. The core may be considered as a storage or reserve pool. Experimental evidence from our laboratory supports the concept that different mechanisms are operative in both compartments in the release of the neurotransmitter. For instance, a Ca2(+)-independent release would be primarily concerned with the neurotransmitter contained in the matrix, and a Ca2(+)-dependent efflux would be primarily related with the neurotransmitter stored in the core. However, it still must be established that a simple relationship exists between each kind of stimulus and each vesicle compartment, rather than both compartments being integrated in a dynamic functional unit.

Activity-dependent recruitment of endogenous glutamate for exocytosis

The Ca 2+-dependent release of the neurotransmitter glutamate from purified nerve terminals (synaptosomes) of the rat hippocampus was studied in a rapid perfusion apparatus. The response of the terminals was investigated with respect to the kinitics and duration of the release of endogenous glutamate upon brief and sustained stimulation and upon repetitive stimulation. The synaptosomes were stimulated by sustained chemical depolarization (0.5–3 min 30 mM K +). The cellular levels of glutamate, free Ca 2+ and ATP in the nerve terminals were measured. The Ca 2+-dependent release of glutamate showed an immediate elevation upon K +-depolarization. When the stimulation was maintained, a prolonged phase of glutamate release was observed. After 3 min, the Ca 2+-dependent release stopped, although K +-depolarization was still effective. When synaptosomes were stimulated again after a relatively short stimulation period (30 s), the second response was similar to the previous one. After a longer stimulation period, maintained until termination of release, the second response did not show the immediate initial elevation of Ca 2+-dependent glutamate release. Only 30 s after stimulation the release developed with a time profile comparable to the first response. This initial lack of response was not due to low cytosolic levels of glutamate or ATP or to changes in cellular Ca 2+-buffering. It can be concluded that the capacity to release glutamate after brief depolarizations is fully restored during the repolarization period. However, if stimulation periods are of long duration (until termination of release), this capacity is no longer fully restored, especially with respect to a fast component of release. New glutamate is recruited only during the subsequent depolarization and with a delay. This points towards both activity- and time-dependent mechanisms. We hypothesize that different populations of synaptic vesicles are involved in the biphasic character of Ca 2+-dependent glutamate release and in the activity- and time-dependent recruitment of glutamate: a “fusion ready” population released during a brief stimulation period and a “supplementary” population released during a sustained stimulation period.

A morphometric analysis of isolated Torpedo electric organ synaptic vesicles following stimulation

The electric organ of Torpedo has been stimulated with 1800 pulses at 0.1 Hz to produce biochemical and morphological heterogeneity of its synaptic vesicle population. This was verified by biochemical and morphometric analyses of the synaptic vesicle population isolated by sucrose density gradient zonal separation following stimulation. Biochemical or metabolic heterogeneity was verified using 2 established criteria: the appearance of a second peak of acetylcholine (ACh) in denser fractions of the zonal gradient and a corresponding overlapping peak of incorporated radiolabelled ACh. Morphologic heterogeneity was deduced by the presence in this second peak of a subclass of synaptic vesicles having a mean diameter of 68 nm i.e., a diameter 20–25% smaller than the 90 nm subclass that represents the most prominent subclass of the intact terminal population. Despite having satisfied these 3 criteria, functionally relevant heterogeneity cannot be assumed. One reason is due to our failure to recover the 90 nm subclass of vesicle which provides the physical basis to explain the 2 ACh peaks along the gradient. Because of this, the point is raised whether the stimulation-induced ACh peak is not merely an artifact due to inadequate sampling. On the other hand, radioactive labelling of the ACh pool provides a more convincing demonstration of the existence of 2 metabolically different subclasses. We conclude that morphological heterogeneity of the ACh vesicle population has never been established and that metabolic heterogeneity, as it has been studied to date, pertains to a single-sized subclass population of vesicles measuring 68 nm in diameter.

Neuropeptide Y-like immunoreactivity in neurons of the solitary tract nuclei: vesicular localization and synaptic input from GABAergic terminals

The ultrastructural localization of neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the medial nuclei of the solitary tracts (mNTS) of adult rat brain. Peroxidase-antiperoxidase (PAP) reaction product was localized extensively to the central lumen of large (100–150 nm), dense-core vesicles. The labeled vesicles were seen in axon terminals of untreated, control animals and in perikarya and dendrites of rats receiving intraventricular injections of colchicine 24 h prior to sacrifice. The labeled terminals were of two types. The first type contained numerous small, clear vesicles that were rimmed with peroxidase product and 1–6 large, densecore vesicles that were labeled throughout their central lumen. The second type contained a more homogeneous population of labeled large, dense-core vesicles. Axon terminals showing NPY-LI formed wither asymmetric synapses with unlabeled dendrites or were without recognized junctions. Within labeled terminals, as well as within perikarya and dendrites, the majority of the dense-core vesicles were located near non-synaptic portions of the plasmalemma that were heavily ensheathed with glial processes. Only a few unlabeled terminals penetrated the glial investments to form synaptic contacts on soma or dendrites containing NPY-LI. These synaptic contacts were of both symmetric and asymmetric types. Combined immunoperoxidase labeling for glutamic acid decarboxylase and immunogold labeling for NPY further established that at least some of the terminals forming symmetric junctions on the NPY-immunoreactive dendrites were GABAergic. These results provide ultrastructural evidence that in the mNTS, NPY-LI is localized principally to large dense-vesicles within neurons whose output is partially regulated by GABA. The preferential distribution of the labeled vesicles along non-synaptic, glial-invested portions of the plasmalemma suggests that neuronal NPY may modulate the activity of nearby astrocytes. Additionally, the localization of NPY-LI in terminals containing a mixed population of synaptic vesicles and forming asymmetric axodendritic junctions suggests that NPY and/or coexisting transmitter may also exert certain known hypotensive effects by excitation of local intrinsic or projection neurons in this brain region.

A morphometric analysis of synaptic vesicle distributions

Synaptic vesicle populations have been morphometrically analyzed for size and density. Populations composed of a single size class of vesicles are represented by normal (Gaussian) or positive (log-normal)skew histograms. Populations with multiple size classes generate negative (left) skew distributions. Fixatives containing aldehydes differentially affect these distribution patterns but vesicles are able to withstand tonic effects over a wide range. Reader bias' contribute the most error in the data-collecting process. But despite this, the sizing of vesicle populations can be accomplished with great accuracy. Vesicle density computations, on the other hand, vary over a wide range and are of less value for comparative purposes.

Morphology and central synaptic connections of the efferent neurons innervating the crayfish hindgut

The distribution, morphology and synaptic connections of the hindgut efferent neurons in the last (sixth) abdominal ganglion of the crayfish, Orconectes limosus, have been investigated using light and electron microscopy in conjunction with retrograde cobalt/nickel and HRP labeling through the intestinal nerve. The hindgut efferent neurons occur singly and in clusters, and are unipolar. Their axonal projections are uniform and consist of a thick primary neurite with typical lateral projections and limited arborization of varicose fibers in the ganglionic neuropil. They also send lower order axon processes to the ganglionic neural sheath, where they arborize profusely, forming a network of varicose fibers. The majority of the efferent neurons project to the anterior part of the hindgut. HRP-labeled axon profiles are found in both pre- and postsynaptic position in the neuropil of the ganglion. HRP-labeled axon profiles also establish pre- and postsynaptic contacts in the intestinal nerve root. All hindgut efferent terminals contain similar synaptic vesicle populations: ovoid agranular vesicles (50–60 nm) and a few large granular vesicles (100–200 nm). It is suggested that the hindgut efferent neurons in the last abdominal ganglion are involved in: (1) innervation of the hindgut; (2) central integrative processes; (3) “en route” synaptic modification of efferent and afferent signals in the intestinal nerve; (4) neurohumoral modulation of peripheral physiological processes.

Ultrastructural features of dopamine axon terminals in the anteromedial and the suprarhinal cortex of adult rat

The ultrastructural features and synaptic relationships of dopamine (DA) axon terminals were examined in the prefrontal cortex of adult rat after immunocytochemical staining with a highly specific polyclonal antiserum directed against DA-glutaraldehyde-lysylprotein conjugate (donated by M. Geffard). Single and serial ultrathin sections were obtained from the deep layers of the anteromedial and the suprarhinal DA fields. The DA axon terminals from both regions averaged 0.7 μm in diameter, contained a mixed population of small, round and clear synaptic vesicles associated with a few larger dense-cored or fully immunostained vesicles, and frequently exhibited synaptic contacts which were exclusively made on dendritic shafts and spines. These synapses were mostly of the symmetrical type (80%) and were more often seen on dendritic shafts than spines, particularly in the suprarhinal (89%) compared with the anteromedial cortex (62%). As estimated either by stereological extrapolation from single sections or by direct observation in serial sections, the synaptic incidence of these DA varicosities was significantly greater in the anteromedial than suprarhinal DA field. In the longest series of thin sections, a junctional complex could be observed on 93% of the DA varicosities from the anteromedial cortex but only on 56% in the suprarhinal cortex. Such an inter-regional disparity in the relational characteristics of the DA input will need to be taken into account in elucidating the role and properties of this monoamine in cerebral cortex.

Ultrastructural identification of synaptic terminals from the axon of type 3 interneurons in the cat lateral geniculate nucleus.

Synaptic terminals from the axons of type 3 neurons in the A-laminae of the cat LGN impregnated with the Golgi gold-toning procedure were examined at light and electron microscopic levels. The axons were identified by their somatic origin, thin diameter, and, in one of these cells, by dense undercoating beneath the axolemma, which is a known characteristic of the axon initial segment. The axon of one of the analyzed cells was profusely branched and extended throughout most of lamina A within the dendritic domains of the cell, and both types of processes were oriented along projection lines in LGN. This suggests that the dendrites and axons of type 3 cells receive inputs and exert effects, of probably inhibitory nature, within restricted retinotopic regions of LGN. The vast majority of the axon terminals of these cells were distributed in series along axonal branches. In one of the type 3 cells, however, a dense cluster of terminals arising from a secondary axonal branch was observed. Ultrastructurally, the analyzed synaptic terminals of the type 3 cells contained flattened or pleomorphic synaptic vesicles, dark mitochondria, and established synapses that appeared to be of symmetrical type when the membranes were perpendicularly cut. On the basis of these characteristics these terminals are classified as F boutons, following Guillery's (Z. Zellforsch. 96:1-38, '69), nomenclature. The postsynaptic elements to the axon terminals were dendrites of small to medium size, which received "en passant" synaptic contacts in extraglomerular regions of the geniculate neuropil by the terminals distributed in series. The axon terminals located in clusters, however, made synapses with dendrites in glomerular regions of the neuropil, where they were not seen postsynaptic to retinal or other types of terminals. This is in contrast to the postsynaptic nature of F2 boutons in the same glomeruli, which have been identified as dendritic appendages of the GABA positive type 3 neurons in the cat LGN (Montero: J. Comp. Neurol. 254:228-245, '86). On the other hand, the axonal F terminals differ from F1 boutons in terms of synaptic relations and ultrastructure, since the latter have been shown to be presynaptic to F2s and somata and to contain crowded populations of flat synaptic vesicles which give them a characteristic dark appearance. Terminals equivalent to F1 boutons have been shown to originate from perigeniculate cells in the rat LGN. From these observations it is suggested that the geniculate GABAergic interneurons support two morphologically and functionally different type of inhibitory terminals synapsing the dendrites of relay cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix.

The three populations of synaptic vesicles in electromotor nerve terminals were analysed quantitatively. Empty vesicles (VP0), fully charged vesicles (VP1) and charged but smaller VP2-type vesicles are present in approximately equal amounts in the nerve terminal. The populations show differences in the kinetics of in vitro uptake of acetylcholine, ATP and Ca2+. VP0 and VP2 accumulate acetylcholine and ATP but no Ca2+, whereas VP1 shows negligible acetylcholine and ATP but high Ca2+ uptake. Thus the expression of uptake properties of this secretory organelle depend on the stage it has reached in its life cycle and might constitute a signal for processing. VP2 was found to contain much less core proteoglycan than VP0 and VP1 indicating that part of it has been lost by exocytosis. In synaptic extracellular matrix containing fractions an antigen is detectable that cross-reacts with an antiserum against the vesicle proteoglycan. This material elutes upon gel filtration in a position similar to a smaller form of proteoglycan found in vesicles. We conclude that the electromotor nerve terminal releases a proteoglycan by the regulated secretory pathway that is deposited in the extracellular matrix. It might have a function in keeping pre- and postsynaptic structures in alignment constituting a transsynaptic signal. Based on the findings described, a model of the vesicles' life-cycle is discussed, whereby the VP2 population is the major source of quantal release of acetylcholine.

Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals.

In the nucleus raphe dorsalis of the cat, an electron microscopic immunocytochemistry method was used to identify the fine structure of serotoninergic dendritic profiles and axon terminals analyzed in serial sections. Two classes of serotoninergic dendrites were distinguished in the nucleus. The first class was constituted by conventional serotonin (5-HT) dendrites that were contacted by unlabeled axon terminals containing differing populations of synaptic vesicles. The second class consisted of serotoninergic dendrites that contained vesicles in their dendritic shafts. Such 5-HT dendrites were further subdivided into two groups according to their synaptic contacts. In some 5-HT vesicle-containing dendrites, the vesicles were densely packed in small clusters and were associated with a well-defined synaptic specialization. These dendrites were classified as serotoninergic presynaptic dendrites and established synaptic contacts with unlabeled and labeled dendrites and were contacted by unlabeled axon terminals. In other 5-HT vesicle-containing dendrites, extensive serial section examination showed that the vesicles could be observed near the membrane but were never found to be associated with any synaptic membrane specialization. Serotoninergic axon terminals that were presumed to be recurrent collaterals of 5-HT neurons were present in the nucleus. Some of them were observed in synaptic contact with dendrites or dendritic protrusions whereas others did not exhibit synaptic specializations. The existence of serotoninergic dendrodendritic synaptic contacts and axon terminals suggests direct local interactions between serotoninergic neurons within the nucleus raphe dorsalis.