Large Synaptic Vesicles Research Articles

Presynaptic quantal size enhancement counteracts post-tetanic release depression.

Repetitive synaptic stimulation can induce different forms of synaptic plasticity but may also limit the robustness of synaptic transmission by exhausting key resources. Little is known about how synaptic transmission is stabilized after high-frequency stimulation.In the present study, we observed that tetanic stimulation of the Drosophila neuromuscular junction (NMJ) decreases quantal content, release-ready vesicle pool size and synaptic vesicle density for minutes after stimulation. This was accompanied by a pronounced increase in quantal size. Interestingly, action potential-evoked synaptic transmission remained largely unchanged. EPSC amplitude fluctuation analysis confirmed the post-tetanic increase in quantal size and the decrease in quantal content, suggesting that the quantal size increase counteracts release depression to maintain evoked transmission. The magnitude of the post-tetanic quantal size increase and release depression correlated with stimulation frequency and duration, indicating activity-dependent stabilization of synaptic transmission. The post-tetanic quantal size increase persisted after genetic ablation of the glutamate receptor subunits GluRIIA or GluRIIB, and glutamate receptor calcium permeability, as well as blockade of postsynaptic calcium channels. By contrast, it was strongly attenuated by pharmacological or presynaptic genetic perturbation of the GTPase dynamin. Similar observations were made after inhibition of the H+-ATPase, suggesting that the quantal size increase is presynaptically driven. Additionally, dynamin and H+-ATPase perturbation resulted in a post-tetanic decrease in evoked amplitudes. Finally, we observed an increase in synaptic vesicle diameter after tetanic stimulation. Thus, a presynaptically-driven quantal size increase, likely mediated by larger synaptic vesicles, counterbalances post-tetanic release depression, thereby conferring robustness to synaptic transmission on the minute time scale. KEY POINTS: Many synapses transmit robustly aftersustained activity despite the limitation of key resources, such as release-ready synaptic vesicles. We report robust synaptic transmission after sustained high-frequency stimulation of the Drosophila neuromuscular junction despite a reduction in release-ready vesicle number. An increased postsynaptic response to individual vesicles, likely driven by an increase in vesicle size due to endocytosis defects, stabilizes synaptic efficacy for minutes after sustained activity. Our study provides novel insights into the mechanisms governing synaptic stability after sustained neural activity.

Increased Cholinergic Tone Causes Pre-synaptic Neuromuscular Degeneration and is Associated with Impaired Diaphragm Function

In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.

Neuromuscular transmission and muscle fatigue changes by nanostructured oxygen.

Oxygen (O2 ) nanobubbles offer a new method for tissue oxygenation. The effects of O2 nanobubbles on transmission at neuromuscular junctions (NMJs) and muscle function were explored in murine diaphragm. Electrophysiological parameters, NMJ ultrastructure, muscle force, and muscle fatigue were studied during superfusion with solutions with different oxygen levels or oxygen nanobubbles. High frequency nerve stimulation of muscles superfused with O2 nanobubble solution slowed neurotransmission decline over those with either control or hyperoxic solution. O2 nanobubble solution increased the amplitude of evoked end plate potentials and quantal content but did not affect spontaneous activity. Electron microscopy of stimulated O2 nanobubble treated NMJs showed accumulation of large synaptic vesicles and endosome-like structures. O2 nanobubble solution had no effects on isometric muscle force, but it significantly decreased fatigability and maximum force recovery time in nerve stimulated muscles. O2 nanobubbles increase neurotransmission and reduce the probability of neurotransmission failure in muscle fatigue. Muscle Nerve 55: 555-563, 2017.

Drosophila S6 Kinase like inhibits neuromuscular junction growth by downregulating the BMP receptor thickveins.

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the α7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

Involvement of neuropeptide mechanisms in the process of integration of heterotopic dental fascia transplants with recipient brains

Embryonic dentate fascia was transplanted into the somatosensory area of the neocortex of adult rats. Ultrastructural and morphometric analyses of giant synapses formed by the granule neurons of transplants with inappropriate neuronal targets in the recipient brains were performed after nine months. As compared with intact synaptic terminals in the control hippocampus, there were differences in the quantity and distribution of large synaptic vesicles with electron-dense centers storing neuropeptide cotransmitters. The proportion of peptidergic vesicles (of the total number of vesicles) in ectopic giant synapses was 5.8 +/- 0.6%, compared with 3.3 +/- 0.6% in controls. Accumulations of large, dense vesicles close to the active zones of aberrant connections were seen almost 7.9 times more often than in controls. These results show that neuropeptide transmitters are critical for maintaining synaptic connections between heterotopic dentate fascia transplants and recipient brains.

Neuromuscular junction in abdominal muscles of Drosophila melanogaster during adulthood and aging

The neuromuscular junction (NMJ) of Drosophila melanogaster has been established as a productive model for the study of synaptogenesis, synaptic plasticity, vesicle recycling, and other synaptic functions in embryos and larvae. It also has potential for the study of long-term plasticity during adult life and degenerative processes associated with aging. Here we provide a detailed description of the morphology and ultrastructure of the NMJ on abdominal dorsal longitudinal muscles throughout adult life from eclosion to senescence. In contrast to the case in the larva, the predominant type of terminals in these muscles in the adult fly consists of only two or three branches with tightly packed synaptic boutons. We observed qualitative and quantitative changes as mean bouton size increased gradually during adulthood, and the largest boutons were present in the old fly. The length of nerve branches first increased and thereafter decreased gradually during most of adult life. Branch diameter also decreased progressively, but branch number did not change. The subsynaptic reticulum became progressively thinner, and "naked" boutons were found in old flies. Ultrastructural traits gave indications of an age-associated increment in autophagy, larger synaptic vesicles, and impaired endocytosis. We propose that NMJ aging in the fly correlates with impaired endocytosis and membrane dynamics. This view finds a functional correlate in flies carrying a temperature-sensitive mutation in shibire that reversible blocks endocytosis; age significantly reduces the time required for complete paralysis and increases the time of recovery, thus confirming the age-dependent alteration in vesicle dynamics.

The vesicular monoamine transporter 2 contains trafficking signals in both its N‐glycosylation and C‐terminal domains

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.

Fast Flies Take a Quantum Leap

Presynaptic regulation of quantal size is an appealing mechanism for changing synapse strength. In this issue of Neuron, Steinert et al. describe an activity-dependent increase in synapse strength mediated by the formation and release of large synaptic vesicles at the Drosophila neuromuscular junction.

Synaptic remodeling in the dorsal motor nucleus of the vagus nerve following vagal-hypoglossal nerve anastomosis in the cat.

Horseradish peroxidase (HRP) retrograde tracing techniques and morphometric analyses were performed to investigate synaptic remodeling associated with neuronal and glial changes in the dorsal motor nucleus of the vagus (DMV) of cats after vagal-hypoglossal nerve anastomosis (VHA). At 25 days postoperation (dpo), in the early target-reinnervation stage, there were 50% fewer presynaptic boutons containing round vesicles (R) or round and large dense-cored synaptic vesicles (R+D) contacting HRP-labeled DMV motoneurons. The loss of R boutons was maintained throughout the remaining postoperative intervals up to 500 dpo, whereas R+D boutons were further reduced at 123 dpo but were restored at 315 dpo, so that, by 500 dpo, 71.4% of them had gained access to the DMV motoneurons. Boutons containing pleomorphic synaptic vesicles (P) were completely disconnected from the DMV motoneurons at 25 dpo and did not reappear even in the long-term reinnervation stage. Loss and recovery of presynaptic boutons occurred in parallel with changes in astroglial ensheathment of the DMV motoneurons. It is suggested that synaptic remodeling associated with astroglial ensheathment in the DMV may be influenced by some retrogradely transported factors/signals derived from the newly acquired target organ, viz. tongue skeletal musculature. Our results further suggest that the observed changes in boutonal configurations may be attributable to modified functions of the DMV motoneurons induced by VHA.

Colocalization of neurotransmitters in presynaptic boutons of inhibitory synapses in the lamprey spinal cord.

Electron-microscopic immunocytochemical studies were performed to detect GABA and glycine immunoreactivity in presynaptic axon terminals in the central gray matter of the spinal cord of the lamprey Lampetra fluviatilis. The immunopositive presynaptic terminals contacting identified dendrites of motoneurons and unidentified postsynaptic profiles included terminals immunopositive for GABA only (44%) and glycine only (26%), as well as terminals containing GABA and glycine (30%). Glycine-immunopositive presynaptic terminals contained flattened synaptic vesicles. Large synaptic vesicles with dense cores were present along with classical synaptic vesicles in 74% of GABA-immunopositive boutons.

Resolution of Patch Capacitance Recordings and of Fusion Pore Conductances in Small Vesicles

We investigated the noise levels in cell-attached patch capacitance recordings with a lock-in amplifier. The capacitance noise level decreases with increasing sine wave frequency up to 20–40 kHz. With a 20-mV rms sine wave the rms noise level above 8 kHz is <50 aF. With increasing sine wave amplitudes a further reduction down to 14 aF could be achieved. Capacitance measurements with a lock-in amplifier may also be used to measure the conductance of fusion pores connecting the vesicular lumen to the extracellular space. It is estimated that at noise levels of 14 aF fusion pore conductances between 20 pS and 700 pS may be resolved in vesicles with 380-aF capacitance by using a 50-kHz sine wave. This corresponds to vesicles with a ∼110-nm diameter. It is suggested that with low-noise techniques fusion pores may be detectable in vesicles approaching the size of large synaptic vesicles.

Relationship of substance P- and CGRP-immunoreactive central endings of the primary afferent neurons to GABAergic interneurons in the guinea pig substantia gelatinosa.

The synaptic relationships between primary afferent central endings containing substance P (SP) and calcitonin gene-related peptide (CGRP) and GABAergic interneurons in the guinea pig substantia gelatinosa of the lumbar spinal dorsal horn were studied. The pre-embedding PAP method was used for detection of GABA and the post-embedding double immunogold labeling method for SP and CGRP detection. Immunogold particles specific for SP and CGRP were mainly localized separately or together in large synaptic vesicles devoid of dense cores. SP and CGRP immunoreactivity was separate or co-localized in small roundish, slender, sinuous or large scalloped (fan-like) terminals with closely packed round agranular synaptic vesicles of various sizes and few large dense core vesicles and mitochondria, which are thought to be capsaicin-sensitive primary afferent terminals. These SP- and CGRP-immunoreactive boutons make presynaptic or symmetric contacts with GABAergic dendrites and soma. These findings suggest that the central endings of nociceptive primary afferents transmit pain stimuli to intrinsic inhibitory interneurons, thereby modulating nociceptive information via a postsynaptic circuit.

The synaptic structure of PACAP immunoreactive axons in the intermediolateral nucleus of the rat

Immuno-electronmicroscopic studies were performed to detect the presence and features of synaptic contacts between pituitary adenylate cyclase activating polypeptide immunoreactive (PACAP-ir) axons and cholera toxin B-horseradish peroxidase labeled preganglionic sympathetic neurons (PSNs) in the intermediolateral nucleus of the rat thoracic spinal cord. PACAP-ir axon varicosities, which contained small clear and large core synaptic vesicles, were found to form asymmetric type of synaptic contacts with dendrites and infrequently with somata of labeled preganglionic neurons. The present study provides ultrastructural evidence of PACAP-ir synaptic contacts with PSNs, raising the possibility that the peptide may function as a transmitter/modulator to these neurons.

Ultrastructure of synapses in the first-evolved nervous systems.

The phylum Cnidaria represents the first group of animals to evolve a recognizable nervous system. A comparison of the ultrastructural features of synaptic loci in animals representing all four classes of the cnidaria has provided an overview of the first-evolved synapses that can be compared morphologically to synapses in higher forms. Synapses in these watery jellylike animals with unmyelinated axons are sparse and difficult to fix well. However, we now have sufficient evidence to define an early synapse as one with paired electron dense plasma membranes separated by a 13-25 nm gap containing intracleft filaments and with vesicles on one or both sides of the synaptic cleft. The vesicles are of three types: dense-cored, clear, and opaque. Neuromuscular synapses resemble neuronal synapses and lack the postsynaptic specializations of higher animals. However, some coelenterates, such as the jellyfish Chrysaora, have a postsynaptic cisterna in the muscle. Neuromuscular and neuronematocyte synapses can have either clear or dense-cored vesicles. Opaque vesicles at two-way interneuronal synapses and at neuromuscular synapses in the oral sphincter muscle of sea anemones can be labelled with antisera to the neuropeptides Antho-RFamide (Antho-Arg-Phe-NH2) and Antho-RWamides (Antho-Arg-Trp-NH2) I and II, respectively. That suggests that neuropeptides evolved as neurotransmitters early in the animal kingdom. The basic differences between first evolved synapses and synapses of higher animals are the lack of postjunctional folds at neuromuscular synapses and the presence of fewer and somewhat larger synaptic vesicles, generally containing granular cores, in the more primitive animals.

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig.

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electron-dense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.

Chromogranin A in Cerebrospinal Fluid: A Biochemical Marker for Synaptic Degeneration in Alzheimer’s Disease?

Biochemical markers for AD would be of great value both to improve the clinical diagnostic accuracy in scientific studies and to increase the knowledge of the pathogenesis of the disorder. One of the main features of AD is a degeneration of synapses. Therefore, we examined if chromogranin A (CrA), the major protein of large dense-core synaptic vesicles, in cerebrospinal fluid (CSF) may be of value as a biochemical marker for the synaptic function in AD. The mean concentration of CrA in CSF was about 7.5 times higher than its concentration in serum, and there was no significant correlation between CSF-CrA and the blood-brain barrier function (measured as the CSF/serum albumin ratio), nor between CSF-CrA and serum-CrA. These findings suggest that the major portion of CSF-CrA is locally produced within the CNS. There were no significant differences in CSF-CrA between the AD (n = 29), vascular dementia (n = 13), and age-matched control (n = 9) groups (99.9 +/- 58.9 ng/ml, 108.0 +/- 69.4 ng/ml, and 115.1 +/- 44.4 ng/ml, respectively). However, when the AD group was subdivided into AD type I (n = 12) and AD type II (n = 17), a lower concentration of CSF-CrA was found in AD type I (72.8 +/- 28.9 ng/ml) compared with controls (115.1 +/- 44.4 ng/ml), p < 0.02, and compared with AD type II (119.1 +/- 67.5 ng/ml), p < 0.05, while CSF-CrA did not significantly differ between AD type II and controls. These findings suggest that CSF-CrA has a potential as a biochemical marker for the synaptic degeneration in AD type I, and gives further support for the relevance of identifying the AD type I (pure AD) subgroup in scientific studies.

Localization of substance P and leucine enkephalin in the nerve terminals of the guinea pig paracervical ganglion.

Immunoreactivities (IR) of substance P and leucine enkephalin have been demonstrated in the guinea-pig paracervical ganglion by an immunogold electron microscope method. Both substance P-IR and leucine enkephalin-IR were detected in large synaptic vesicles with electron-dense cores. The former neuropeptide was detected in nerve terminals and varicosities comprised mainly of large vesicles with electron-dense cores; the latter was detected in nerve terminals and varicosities that also included small, clear synaptic vesicles. In a minority of nerve terminals and varicosities coexistence of both immunoreactivities could be demonstrated within vesicles with an electron-dense core. Also present in these nerve terminals and varicosities were small, clear synaptic vesicles, though these were unreactive.

Subcellular distribution of serotonin in the lamprey spinal cord

The subcellular distribution of serotonin (5-hydroxytryptamine; 5-HT) in the lamprey ( Ichtyomyzon unicuspis, Lampetra fluviatilis) spinal cord was investigated by using ultracentrifugation on continuous density gradients combined with an electron microscopic analysis of the gradients and of immunostained tissue. Endogenous 5-HT was analyzed by high-performance liquid chromatography with electrochemical detection. After differential centrifugation, the highest levels of 5-HT were found in the particulate fractions. After ultracentrifugation of lysed synaptosomal fractions on continouus sucrose gradients and the subsequent sedimentation of the individual fractions, 5-HT showed a biphasic distribution in the gradient. The two peaks corresponded to 0.30–0.40 M and 0.85–1.05 M sucrose. Electron microoscopy of intact tissue showed that some of the boutons were strongly immunoreactive to 5-HT with dense precipitates over large granular vesicles. The area around these large vesicles, however, also showed reaction product. Large granular vesicles could be clearly distinguished in the immunostained axonal varicosities. In tissue not processed for 5-HT immunoreactivity it was seen that the varicosities contained not only large dense-cored vesicles, but also small agranular vesicles. An electron microscopical analysis of the subcellular fractions revealed that the fraction corresponding to the “light” 5-HT peak contained numerous vesicular structures, which in most cases were electron lucent. In the “heavy” fractions, nerve ending particles containing vesicles of various sizes were observed. The results suggest that 5-HT in the lamprey spinal cord may be distributed in more than one subcellular compartment wwhich, apart from the cytosol, possibly corresponds to small and large synaptic vesicles.

Ultrastructure and Neuronal Control of Luminous Cells in the Copepod Gaussia princeps.

The physiology of light production in copepods is largely unknown. The mesopelagic copepod Gaussia princeps possesses luminous glands, each consisting of a single large cell discharging through a cuticular pore. Slow flashes external to the cuticle are triggered from excised abdomens by electrical stimulation of the ventral nerve cord. Each luminous cell contains UV fluorescent secretory vesicles distally, which are secreted through a valved cuticular pore. Each luminous cell, except for the most proximal portion, is surrounded by a cellular sheath, which appears to form the distal valve. Luminous cells have a stem containing small, electron-lucent precursors to secretory vesicles proximal to the fluorescent vesicles. Nerve terminals, filled with large synaptic vesicles, are associated with the unsheathed proximal cell membrane. Gap junctions interconnect the nerve terminals, and possibly serve to accelerate conduction to the luminous cell to achieve a synchronous effector output.