Synapsin regulates vesicle organization and activity-dependent recycling at Drosophila motor boutons

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. We investigated synapsin function in mediating synaptic activity during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin(−) boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin (+) boutons, while in synapsin (−) boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin(−) but not in synapsin(+) boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K⁺ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation.     

An autoradiographic identification of Purkinje axon terminals in the cat

Summary The Purkinje axon boutons terminating in nuclei fastigii and interpositus in the cat have been identified after injection of³H leucine into the cerebellar cortex overlying the nuclei. The animals survived from 4–48 h after injection of the isotope. Semithin and ultrathin sections were coated and exposed for 3 and 14 weeks, respectively. The electron micrographs showed labelling over myelinated axons down to a diameter of 0.8 m, and over boutons.Fifty labelled boutons were used for identification of the shape of their synaptic vesicles. The statistical analysis including the test for skewness showed that 39 boutons (78%) fall in one group. Most of the synaptic vesicles in this group are elliptical (ration from 11.1 – 11.7). Slightly ovoid vesicles (ration up to 11.3) are frequent, but flattened vesicles (ration above 11.7) are relatively few in this group of boutons. 8% of the boutons have a rather homogeneous vesicle population (prevalence of round vesicles, ratio 11).Synaptic specializations of Gray's type II or of an intermediate type were found in the boutons belonging to the first group (78% of the boutons). Specializations of Gray's type I were found in the other bouton groups (8% of the boutons).     

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglionin vivo following stimulation-induced exhaustion

Summary Cat superior cervical ganglia (SCG) were tetanically stimulatedin vivo at 30–100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 m^–2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 m^–2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.     

A long-lasting potentiation of transmitter release related to an increase in transmitter stores in a sympathetic ganglion

1. High frequency preganglionic nerve stimulation increases the acetylcholine (ACh) stores of the cat superior cervical ganglion. The increase reaches a maximum 20 min following 60 min conditioning stimulation at 20/s. The effect of this conditioning on ACh release in ganglia perfused with plasma and test stimulated at 4 or 5/s has been studied, and the relationship of ACh stores to ACh release in conditioned ganglia determined.

2. The rate of ACh release in response to test stimulation at 5/s for 15 min, starting 20 min following conditioning, was 174% of the rate found in unconditioned ganglia. The ACh stores of the conditioned ganglia at the end of the test were calculated to be 173% of the control ganglion stores.

3. When test stimulation at 4/s was started 5 min following conditioning, the rate of ACh release showed a variable pattern of increase and decrease over a 75 min period. The mean peak rate of release was about 150% of the control rate, and the duration of potentiation was about 75 min.

4. When conditioned, unperfused ganglia were tested by stimulation at 4/s the ACh stores were found to increase and decrease in parallel with the increase and decrease in ACh release rates found in the perfusion experiments.

5. It was found also that the magnitude of the increase in ACh stores and ACh release was related to the amount of ACh in the ganglionic stores at the onset of conditioning, being greater for the ganglia with the smaller initial stores.

6. It is concluded that the potentiation of ACh release in ganglia conditioned in this way is directly related to the accompanying increase in ACh stores.

7. The possible significance of alterations of ACh stores and ACh release as a mechanism of modulatory control of ganglionic transmission is discussed.

     

Some effects of preganglionic nerve stimulation on synaptic vesicle populations in the rat superior cervical ganglion

1. The ;local vesicle population', namely the population of synaptic vesicles lying within a zone 0.25 mum wide adjacent to the presynaptic membrane and expressed as ;vesicles mum(-2)' has been estimated in rat superior cervical ganglia maintained in vitro for 1 h. Ganglia were either unstimulated or stimulated tetanically during the last few minutes of the in vitro period.2. Individual local vesicle populations at synapses in groups of ganglia which had received the same treatment showed an approximately normal frequency distribution.3. The technique of stimulation did not permit fixation during tetanus, but the mean local vesicle population in ganglia fixed a few seconds after 10 Hz tetani of up to 10 min duration were significantly (P < 0.001) higher than the unstimulated control value of 124.0 +/- S.E. 3.2. Ganglia fixed a few seconds after 3 min tetani at 10 Hz had a mean local vesicle population of 155.7 +/- S.E. 5.7, rising slightly but not significantly to 165.0 +/- S.E. 4.3 at 1 min and 163.1 +/- S.E. 5.2 at 2 min after tetanus. Thereafter the mean local vesicle population fell slowly, reaching control level between 10 and 20 min after tetanus.4. In terms of the vesicle hypothesis, the observations suggest that post-tetanic potentiation might arise from a closer clustering of vesicles about release areas resulting in an increase in fractional release. It is suggested that stimulation increased the local vesicle population by promoting vesicle mobilization rather than by altering the size or shape of the presynaptic terminals. The ;mobilizing agent' might be intracellular Ca(2+).5. The results appear to be consistent with those of other workers where experimental conditions and the region in which vesicles were counted were comparable.6. Alternative interpretations for the findings, not involving the vesicle hypothesis, are considered.     

Local vesicle populations in rat superior cervical ganglia and the vesicle hypothesis

Summary The local vesicle population (LVP), namely the population of synaptic vesicles lying in a zone 0.25 m wide adjacent to the presynaptic membrane, has been measured at synapses of rat superior cervical ganglia fixed for electron microscopy following experiments performed during a 60 min periodin vitro. The mean LVP of 78 synapses in three unstimulated control ganglia was 122.0 ± s.e. 5·9 vesicles m^-2. Ganglia fixed 1 min after 3 min 10 Hz tetani of the preganglionic nerve had a significantly (P<0.001) higher mean LVP (158.0 ± s.e. 5·0). Replacement of the Ca²⁺ of the bathing medium by Mg²⁺ for the final 30 min did not affect the mean LVP of unstimulated ganglia but prevented the increase following preganglionic stimulation. When a bathing medium with Mg²⁺ replaced by Ca²⁺, or when a drug such as caffeine, morphine or adrenaline was present for the final 30 min of the experimental period, preganglionic nerve stimulation reduced the mean LVP below the mean value in paired unstimulated ganglia.The findings are discussed in the light of recent ideas of transmitter physiology and it is concluded that they are compatible with the concept that synaptic vesicles contain, or are associated with, the synaptic transmitter.     

Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP

Efficient vesicle membrane recycling at presynaptic terminals is pivotal for preventing depletion and maintaining high firing rates in neuronal networks. We used a new approach, based on the combination of spectrally different optical probes, to investigate how stimulation determines the fate of synaptic vesicles after endocytosis. We found that in the small central synapses of rat hippocampal neurones low frequency stimulation (40 action potentials at 2 Hz) targets vesicles preferentially to vesicle pools that were kinetically faster. Vesicles taken up during endocytosis triggered by high frequency stimulation (400 action potentials, 20 Hz) were also placed in the back of the release queue. We performed a spatial analysis of the recycled vesicles in living hippocampal boutons using two spectrally different FM-dyes (FM1-43 and FM5-95). By using these consecutively, vesicles endocytosed by either stimulation protocol were labelled with a different colour. This revealed that the kinetic arrangement was also reflected in the spatial organization of vesicles within the bouton. Next, we identified the postsynaptic site of the active zone by transfecting the neurones with postsynaptic density protein PSD-95-CFP. The data from these triple colour experiments suggest that retrieval after low frequency stimulation keeps vesicles in a more confined region closer to the active zone as identified by PSD-95-CFP expression at the postsynaptic site.     

5'-triphosphate recycles independently of acetylcholine in cholinergic synaptic vesicles.

The effect of hemicholinium-3 (HC-3) on vesicular contents in acetylcholine (ACh) and 5-triphosphate (ATP) and the vesicular incorporation of 14C-label derived from [14C]choline ,nd 3H-label derived from [3H]adenosine was investigated after low frequency stimulation (with a subsequent rest period) of the Torpedo electric organ. HC-3 (100 microM) caused an increased depletion of vesicular ACh and blocked the incorporation of 14C-label whereas contents in vesicular ATP and 3H-incorporation were identical with and without HC-3. HC-3 also blocked the recovery of electrical response of the tissue after stimulation but did not cause a change in vesicle numbers. The result suggest that synaptic vesicles continue to recycle ATP in the absence of recycling of ACh and that vesicular uptake and storage of the two components are not coupled to each other.     

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

Observations on the fine structure of the substantia nigra in the cat

Summary A light and electron microscopical investigation has been undertaken of the substantia nigra in the normal cat. The pars reticulata partly contains the arborization of dendrites whose cell bodies are located in the so-called pars compacta. There is a considerable overlap of the dendritic fields in the rostrocaudal direction, while the dendritic fields are very restricted in the mediolateral extension of the substantia nigra. The secondary and all subsequent branches of the dendrites of nigral cells are for considerable distances completely covered by boutons. Only few boutons contact the cell bodies. Three types of boutons are distinguished in the substantia nigra in the cat. Type I, about 90 % of the total, is of the terminal type, contains pleomorphic vesicles and establishes symmetrical synapses with nigral cell soma, dendritic trunks and spines. The type II bouton (about 10 % of the total number) is most commonly of the terminal type, contains spherical vesicles and establishes asymmetrical synapses with cell bodies and dendritic trunks of nigral cells. The type III bouton (about 2 % of the boutons) is always of the en passage type, contains pleomorphic vesicles and establishes symmetrical contacts with dendrites. All boutons in the cat's substantia nigra contain several large (700–1200 Å) dense core vesicles. Occasional axo-axonic contacts between type I and type III boutons are observed. Type I bouton is invariably presynaptic to the other.The findings are discussed in relation to some relevant problems.On leave of absence from the Anatomical Institute of the Medical Faculty, Charles' University in Prague, with an IBRO grant nr. E. 29.99-1.We gratefully acknowledge the valuable technical assistance of Mrs. J.L. Vaaland and the skilful help by Mrs. B.E. Branil in the preparation of the microphotographs.     

Desensitization in denervated mouse muscles

In denervated mouse soleus (DSOL) muscle preparations washed in Na methanesulfonate solutions containing 30 mmol · l^–1 K⁺, bath-applied ACh (55 mol · l^–1) caused the resting potential to decrease from about –36 to –3.2 mV within 2–4s; the potential remained stable or became slightly more negative when ACh was applied for 1–2 min. Two components of the membrane current change (ACh current) were found in DSOL when using a point voltage clamp, and initial current component declining to 1/2 the initial amplitude in 11–15 s (desensitization) and a steady late current comprising 16–47% of the maximum ACh current. Membrane conductance (in s · cm^–2) was 0.35 in the absence of ACh, 20.3 at the peak of the initial current, and 1.57 during late current. The late current saturated a 0.55 to 5.5 mol · l^–1 ACh, whereas the initial current required 55 or more mol · l^–1 ACh to saturate. The null (reversal) potential was 6–13 mV more positive for the late current than for the initial current. The late current was masked when Na⁺ was replaced by Tris⁺, sucrose, or K⁺. An initial and a late current could also be distinguished in non-denervated endplates. The late current was more sensitive to ACh than the initial current, but the null potenital was more negative than that for the initial current in endplates. In denervated membrane, the half time of desensitization was increased when Mg²⁺ was replaced by Ca²⁺ but the changes were on the average less than 15% the control values. It was concluded that desensitizing and non-desensitizing receptors may exist in extrajunctional membranes of denervated muscles and in endplates, the two being attached to different ionic channels.     

Chance or design? Some specific considerations concerning synaptic boutons in cat visual cortex

To understand the rules by which axons lay down their synaptic boutons we analyzed the linear bouton distributions in 39 neurons (23 spiny, 13 smooth) and 3 thalamic axons, which were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The variation of the total number of boutons and the total axonal length was large (789–7912 boutons, 12–126 mm). The overall linear bouton density for smooth cells was higher than that of spiny cells and thalamic afferents (mean ± sd, 110 ± 21 and 78 ± 27 boutons per mm of axonal length). The distribution of boutons varied according to their location on the tree. Distal axon collaterals (first and second order segments in Horton-Strahler ordering) of smooth neurons had a 3.5 times higher, spiny cells and thalamic afferents a 2 times higher bouton density compared to the higher order (more proximal) segments. The distribution of interbouton intervals was positively skewed and similar for cells of the same type. In most cases a -distribution fitted well, but the distributions had a tendency to have a heavier tail. To a first approximation these bouton distributions are consistent with both diffuse and specific models of interneuronal connections. Quite simple rules can explain these distributions and the connections between the different classes of neurons.     

Ventral horn synaptology in the rat

Summary The synaptology of the normal ventral horn of the rat was studied. Presynaptic boutons were classified as S (spherical vesicles), F (flattened vesicles), and G (predominance of 700–1200 Å granular vesicles). In addition, Cf, Cs, M, and T synaptic complexes were defined and quantitated. Synaptology was studied on -motoneuron somata, -motoneuron primary dendrites, peripheral dendrites and interneuron somata. In addition, organelles were quantified for the pre- and postsynaptic members of the synaptic complex. All counts were made on coded material and these data were analyzed statistically.Motoneuron somata had significantly more (P < 0.01) F (58%) than S (33%) boutons. This was also the case for the motoneuron primary dendrite (P < 0.01; F, 61%; S, 37%). The small dendrites had more (P < 0.05) S (56%) than F (44%) boutons. More Cf bulbs (P < 0.01) were found on motoneuron somata (9%) than on motoneuron primary dendrites (2%) or interneuron somata (3%). The C complex presynaptic bouton contained spherical (Cs) or flattened (Cf) synaptic vesicles which were attributed to the fixation employed. Cf bulbs were not observed on small dendrites. G bulbs were observed (< 1%) only on small dendrites. M bulbs were not observed on any postsynaptic structure.The boutons of the motoneuron primary dendrites (15% of total afferents) and peripheral dendrites (14% of total afferents) were frequently branched whereas there was significantly (P < 0.01) less branching of boutons on motoneuron and interneuron somata. Small postsynaptic subsurface cisterns were associated with boutons of both the S and F type on all structures. In addition, these cisterns were observed in motoneuron somata (4%) and interneuron somata (2%) without an accompanying bouton. C postsynaptic organelles were observed in motoneuron somata (3%) and primary dendrites (1%) with an overlying neuroglial cell process and no presynaptic bouton.The synaptology of the rat ventral horn is comparable to that in the cat and monkey. However, M (R) and P bulbs were not observed in the rat. This could be due to the sampling method which indicated that synapses with less than 1% occurrence fall at the level of statistical resolution in quantitative electron microscopy. The presence of postsynaptic specialization usually associated with presynaptic boutons with no presynaptic component may be a reflection of the dynamics of normal bouton renewal in the rat ventral horn.     

Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools

Summary The effect of electrical field stimulation on the release of acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) from superfused strips of myenteric plexus-longitudinal muscle (MPLM) of guinea-pig ileum and on the transmitter content of the tissue was investigated at different frequencies and in the presence and absence of choline hemicholinium-3 and colchicine. Low frequency electrical field stimulation released ACh by more than 4 times the basal release; the simultaneously detected VIP secretion was increased only slightly above the resting level. During high frequency stimulation (50 Hz) the release of VIP was greatly increased (to 5 times the resting release) whereas the release of ACh increased to only 150% of the basal output. When choline was present, the ACh content of the tissue itself was not altered by electrical stimulation indicating a rate of synthesis sufficient to maintain release. It was reduced in a frequency-dependent manner in the absence of exogenous choline or in the presence of 10 M hemicholinium-3 (an inhibitor of choline uptake) by up to 54% of the original content. A similar but even larger reduction took place in the amount of ACh released. Neither the secretion of VIP nor the tissue VIP content was altered by these treatments. Long-lasting (>60 min) high-frequency (50 Hz) stimulation resulted in the depletion of the VIP pool (by 25%) while the ACh content remained unaltered. The stimulus-induced depletion of the VIP pool was increased when colchicine reduced the amount of VIP released and the VIP content of the stimulated tissue up to 50%; it caused a small hemicholinium-like effect on the release of ACh and on the ACh content of the tissue when the tissue was stimulated for an extended length of time (120 to 180 min) at low frequency (5 Hz) but otherwise had little effect. Depolarization by elevated potassium concentration or by veratridine increased the ACh release by 150–280% and the VIP release by 125–200% of the resting release and was thus equivalent to electrical stimulation at 8–10 Hz. At the end of prolonged chemical depolarization (60 min) the ACh content was reduced to 60–80% and the VIP content to 80–90% of the non-stimulated values respectively. These results are interpreted in terms of the different intracellular dynamics of ACh and VIP and their respective storage vesicles.     

Angiotensin II and acetylcholine differentially activate mobilization of inositol phosphates in Xenopus laevis ovarian follicles

Angiotensin II (AII) evokes a Ca²⁺-dependent Cl^– current in Xenopus laevis ovarian follicles that appears to involve a pertussis-toxin-sensitive G protein mediating phosphoinositide hydrolysis and Ca²⁺ mobilization from intracellular stores. Follicle responses to AII closely resemble the two-component response stimulated by acetylcholine (ACh) in this tissue. Intraoocyte injections of phytic acid, heparin, and inositol 1,4, 5-trisphosphate [Ins(1,4,5)P ₃], acting as inhibitors of Ins(1,4,5)P ₃-induced Ca²⁺-release, resulted in loss of responsiveness to AII and ACh. As previously reported for ACh [Moriarty et al. (1988) Proc Natl Acad Sci USA 85: 8865–8869], pertussis toxin and microinjected GTP[S] were found to inhibit follicle responses to AII, implying the involvement of a G protein. However, ACh and AII responses differ strikingly in the way they mobilize inositol phosphates and in densitization characteristics. We have previously been unable to find significant increases in inositol phosphates after 60 min stimulation (with Li⁺) by AII, although ACh potently activated increases in these [McIntosh and McIntosh (1990) Arch Biochem Biophys 283:135–140]. In the present paper, AII was found to activate rapid increases in inositol bisand trisphosphates after 1 min stimulation without Li⁺. ACh and AII also exerted different actions on follicle adenylate-cyclase-dependent responses. We conclude that at least two separate inositol-phosphate-linked receptor mechanisms may exist in ovarian follicles, resulting from involvement of one or more pertussis-toxin-sensitive G protein(s).     

Non-sympathetic synaptic innervation of the pinealocyte of the Mongolian gerbil (Meriones unguiculatus): an electron microscopic study

Summary Direct synaptic innervation of pinealocytes was observed in the superficial pineal gland of the Mongolian gerbil (Meriones unguiculatus) by electron microscopy. This innervation consisted of nerve fibres terminating in boutons with clear transmitter vesicles with a diameter of 40–60 nm. The boutons made synaptic junctions with the cell membrane of the pinealocyte displaying thickenings of both the pre- and postsynaptic membranes. Such boutons persisted in the gland 1 week after removal of both superior cervical ganglia. In contrast, all the sympathetic boutons containing transmitter vesicles with a small dense core disappeared after ganglionectomy. This direct synaptic innervation reveals a neuronal character of the pinealocyte and might underlie reports of action potentials in electrophysiological recordings from the gland.     

Storage and release of acetylcholine in a sympathetic ganglion

1. The hypotheses of preferential release of newly synthesized acetylcholine (ACh) and two compartment storage of transmitter in the cat superior cervical ganglion have been re-examined by testing, first, the assumption that ganglionic ACh stores do not alter during a 20 min rest following 60 min preganglionic nerve stimulation at 20/s, and secondly, the implication that the rate of ACh release should be high near the onset of activity and decline to a lower rate with time irrespective of the frequency of stimulation.2. The ganglionic ACh stores were found to increase by 38 +/- 8% within 20 min following 60 min preganglionic nerve stimulation at 20/s, and this extra ACh was releasable.3. The rate of ACh release from ganglia perfused with cat plasma and stimulated at 4/s increased over the first 5 min of stimulation to reach a 27% higher rate that was maintained.4. Correction of the original data to allow for the post-activation increase in ACh stores suggests that newly synthesized ACh equilibrates with most of the preformed stores. The time course of ACh release at 4/s does not support the two compartment model as currently formulated.5. These findings resolve in part a conflict between the physiological data and a recent hypothesis for ACh storage based on ganglion morphology.     

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

Summary Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd²⁺- 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 Ca²⁺-free medium for a period of 3 h, (2) 25 or 40mM 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 fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated -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 Ca²⁺-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 Ca²⁺-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 Ca²⁺-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.     

Non-electrolyte transport across renal proximal tubule cell membranes measured by tracer efflux and light scattering

Non-electrolyte transport in brush border membrane vesicles (BBMV), basolateral membrane vesicles (BLMV) and in viable cells isolated from the proximal convoluted tubule (PCT) of the rabbit kidney were measured by rapid filtration and stopped-flow light scattering techniques. Efflux of tracer solute was measured by loading packed vesicles or cells with¹⁴C solute, diluting into non-radioactive buffer and filtering rapidly at varying incubation times. In BBMV at 23°C, [¹⁴C-urea] decreased exponentially with time constant 3.2±0.3 s (S.D.,n=5) corresponding to a permeability coefficient (P _urea) of 1.6×10^–6 cm/s, assuming a BBMV surface-to-volume ratio of 2×10⁵ cm^–1.P _urea decreased to 7×10^–7 cm/s in the presence of 20 mM phenylurea. Tracer efflux determinations of BBMVP _urea (1.6×10^–6 cm/s) andP _glycerol (0.6×10^–6 cm/s), and BLMVP _urea (1.8×10^–6 cm/s) andP _thiourea (2.5×10^–6 cm/s) were in excellent agreement withP _s values determined by stopped-flow light scattering, where the time course of vesicle volume (linearly related to scattered light intensity) was measured in response to 100 mM outwardly directed solute gradients. These results establish accurateP _s value in brush border and basolateral membranes and support the application of light scattering to measureP _s in vesicles. In PCT cells however, there were systematic differences in urea and thiourea transport measured by tracer efflux and light scattering, indicating the potential difficulties in applying light scattering toP _s measurements in complex cell systems.     

Dense core vesicles resemble active-zone transport vesicles and are diminished following synaptogenesis in mature hippocampal slices

Large dense core vesicles (approximately 100 nm) contain neuroactive peptides and other co-transmitters. Smaller dense core vesicles (approximately 80 nm) are known to contain components of the presynaptic active zone and thought to transport and deliver these components during developmental synaptogenesis. It is not known whether excitatory axons in area CA1 contain such dense core vesicles, and whether they contribute to synaptic plasticity of mature hippocampus. Serial section electron microscopy was used to identify dense core vesicles in presynaptic axons in s. radiatum of area CA1 in adult rat hippocampus. Comparisons were made among perfusion-fixed hippocampus and hippocampal slices that undergo synaptogenesis during recovery in vitro. Dense core vesicles occurred in 26.1+/-3.6% of axonal boutons in perfusion fixed hippocampus, and in only 17.6+/-4.5% of axonal boutons in hippocampal slices (P<0.01). Most of the dense core vesicle positive boutons contained only one dense core vesicle, and no reconstructed axonal bouton had more than a total of 10 dense core vesicles in either condition. Overall the dense core vesicles had average diameters of 79+/-11 nm. These small dense core vesicles were usually located near nonsynaptic membranes and rarely occurred near the edge of a presynaptic active zone. Their size, low frequency, locations, and decrease following recuperative synaptogenesis in slices are novel findings that merit further study with respect to small dense core vesicle content and possible contributions to synapse assembly and plasticity in the mature hippocampus.