Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

RIC-7 promotes neuropeptide secretion

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV-mediated secretion.     

大鼠孤束核内脑啡肽样免疫反应阳性结构及其轴突终末的突触联系

本文应用免疫细胞化学方法在光镜与电镜下观察了大鼠孤束核内脑啡肽样免疫反应（ＥＮＫ－ＬＩ）阳性结构的分布特征和ＥＮＫ－ＬＩ轴突终末的突触联系以及非突触性关系。结果表明：（１）经秋水仙素处理的大鼠,其孤束核内有许多ＥＮＫ－ＬＩ胞体的分布;而未经秋水仙素处理的大鼠,其孤束核内可见密集的ＥＮＫ－ＬＩ纤维与终末;ＥＮＫ－ＬＩ胞体、纤维和终末主要分布于锥体交叉平面至闩平面的孤束核内侧亚核与胶状质亚核。（２）ＥＮＫ－ＬＩ阳性产物主要定位于小圆形清亮囊泡外表面、大颗粒囊泡内和线粒体外表面等处。（３）ＥＮＫ－ＬＩ轴突终末主要与阴性树突形成轴－树突触。（４）阴性轴突终末终止于ＥＮＫ－ＬＩ轴突终末上,形成轴－轴突触。（５）ＥＮＫ－ＬＩ轴突终末与阴性轴突终末形成非突触性的轴－轴并靠。以上结果提示孤束核内的ＥＮＫ－ＬＩ神经成分主要通过突触后机制、也不排除突触前作用,参与孤束核中内脏信息的整合过程,而且这一作用又受到非ＥＮＫ－ＬＩ神经成分的调控。     

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

Summary 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.     

Mechanisms of facilitation of synaptic glutamate release by nicotinic agonists in the nucleus of the solitary tract

The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory information. Functional nicotinic acetylcholine receptors (nAChRs) have been found on presynaptic glutamatergic terminals in subsets of caudal NTS neurons. Activation of these receptors has been shown to enhance synaptic release of glutamate and thus may modulate autonomic sensory-motor integration and visceral reflexes. However, the mechanisms of nAChR-mediated facilitation of synaptic glutamate release in the caudal NTS remain elusive. This study uses rat horizontal brainstem slices, patch-clamp electrophysiology, and fluorescent Ca(2+) imaging to test the hypothesis that a direct Ca(2+) entrance into glutamatergic terminals through active presynaptic non-α7- or α7-nAChR-mediated ion channels is sufficient to trigger synaptic glutamate release in subsets of caudal NTS neurons. The results of this study demonstrate that, in the continuous presence of 0.3 μM tetrodotoxin, a selective blocker of voltage-activated Na(+) ion channels, facilitation of synaptic glutamate release by activation of presynaptic nAChRs (detected as an increase in the frequency of miniature excitatory postsynaptic currents) requires external Ca(2+) but does not require activation of presynaptic Ca(2+) stores and presynaptic high- and low-threshold voltage-activated Ca(2+) ion channels. Expanding the knowledge of mechanisms and pharmacology of nAChRs in the caudal NTS should benefit therapeutic approaches aimed at restoring impaired autonomic homeostasis.     

Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats

Summary Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.     

Ultrastructure and morphometric parameters of glutamatergic synapses on nigrothalamic and unidentified neurons of the reticular zone of theSubstantia nigra in cats

Nigrothalamic neurons were identified into thesubstantia nigra by their retrograde labelling with horseradish peroxidase. Axon terminals that contain glutamate (the excitatory transmitter) were revealed immunocytochemically with an immunogold electron microscopic technique. Ultrastructural parameters (the large and small diameters of axon terminals, area of their profiles, coefficient of form of profiles, large and small diameters of synaptic vesicles) were analyzed in all 240 synapses under study. Synaptic contacts localized on both nigrothalamic and unidentified neurons belonged to three morphologically specific groups. Synapses of the groups I and III, according to classification by Rinvik and Grofova, were characterized by a symmetric type of synaptic contact and contained polymorphic synaptic vesicles. Contacts in group-II synapses were asymmetric, and respective terminals contained round vesicles. Among the studied synapses, 65.8% were classified as group-I contacts, 25.0% belonged to group II, and 9.2% belonged to group III. Glutamate-positive axon terminals formed predominantly group-II synapses; such connections constituted 70% of this group's synapses. Sixty percent of glutamate-positive synapses were localized on the distal dendrites and 23% on the proximal dendrites, while 17% of such synapses were distributed on the somata of nigral neurons. Such a pattern of distribution of glutamate-positive synapses was observed on both nigrothalamic and unidentified nigral neurons. About 7% of glutamate-positive synapses were formed by very large axon terminals containing round synaptic vesicles; yet, the contacts of these terminals were of a symmetric type. Twenty percent of group-I synapses, i.e., synapses considered inhibitory connections, were found to manifest a weak immune reaction to glutamate.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 285–295, November–December, 1996.     

Signaling for Vesicle Mobilization and Synaptic Plasticity

The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity.     

Neuronal and synaptic organization of the lateral geniculate nucleus of the tree shrew,Tupaia glis

Summary The ultrastructural study of the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia glis) revealed two types of neurons: (1) a large thalamocortical relay cell (TCR), which may bear cilia, and (2) a small Golgi type-II interneuron (IN) with an invaginated nucleus. The narrow rim of pale cytoplasm of the IN contains fewer lysosomes and fewer Nissl bodies than the cytoplasm of the TCR. The IN perikarya, which in some cases establish somatosomatic contacts, frequently contain flattened or pleomorphic synaptic vesicles. The ratio of TCR to IN is 31.Three types of axon terminals were observed in the LGN. Two of them contain round synaptic vesicles but differ in size. The large RL boutons undergo dark degeneration after enucleation; they are the terminals of retino-geniculate fibers. The smaller RS boutons show dark degeneration after ablation of the visual cortex; they are the terminals of the cortico-geniculate fibers. The third type of bouton (F₁ does not degenerate after either intervention. The boutons of this type are filled with flattened vesicles and are believed to be intrageniculate terminals. F₂-profiles were interpreted as presynaptic dendrites of the IN. The characteristic synaptic glomeruli found in the LGN contain in their center an optic terminal. These optic terminals establish synaptic contacts with dendrites or spine-like dendritic protrusions of TCRs as well as with presynaptic dendrites. Synaptic triads were also seen. The distribution of the individual types of synaptic contacts in layers 3 and 4 was determined. Layer 4 contains only one third of the retino-geniculate synapses and of the synaptic contacts of F₁-terminals.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus. Dual electron microscopic immunolabeling

Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diaminobenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

Visualization of neuropeptide expression,transport, and exocytosis in Drosophila melanogaster

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP‐tagged atrial natriuretic factor fusion (preproANF‐EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF‐EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF‐EMD was concentrated in nerve terminals. We compared the release of proANF‐EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate‐containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46‐fold more neuropeptide and released much more proANF‐EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 159–172, 2001     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Neuropeptide messenger plasticity in the CNS neurons following axotomy

Neuronal peptides exert neurohormonal and neurotransmitter (neuromodulator) functions in the central nervous system (CNS). Besides these functions, a group of neuropeptides may have a capacity to create cell proliferation, growth, and survival. Axotomy induces transient (1–21 d) upregulation of synthesis and gene expression of neuropeptides, such as galanin, corticotropin releasing factor, dynorphin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, cholecystokinin, angiotensin II, and neuropeptide Y. These neuropeptides are colocalized with “classic” neurotransmitters (acetylcholine, aspartate, glutamate) or neurohormones (vasopressin, oxytocin) that are downregulated by axotomy in the same neuronal cells. It is more likely that neuronal cells, in response to axotomy, increase expression of neuropeptides that promote their survival and regeneration, and may downregulate substances related to their transmitter or secretory activities.     

Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea-Pig Small Intestine

The acetylcholine-rich myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine has been subjected to subcellular fractionation using modifications of both classical methods and that originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by means of density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 × S.E.M. 1.8 (5) nmol of acetylcholine × mg of protein^?1. Electron-microscopical examination showed that it consisted of a homogeneous preparation of vesicles of mean spherical diameter 61 ×sd 4 (108) nm, with little or no contamination with other lipoprotein membrane structures, mixed how-ever with considerable amounts of actomyosin fibrils, presumably derived from the longitudinal muscle. Slab-gel electrophoresis in sodium dodecyl sulphate showed that, in addition to prominent peaks attributable to actin and myosin, there was a relatively simple pattern of (presumably) vesicle protein among which all the proteins thought to be characteristic of Torpedo synaptic vesicles were present. Dowe G. H. C. et al. Isolation of cholinergic synaptic vesicles from the myenteric plexus of guinea-pig small intestine. J. Neurochem. 35, 993–1003 (1980).     

Dense cored vesicles in presynaptic profiles of the rabbit dorsal lateral geniculate nucleus

We are carrying out a study about the synaptic relations between identified synaptic profiles in the dorsal lateral geniculate nucleus (dLGN) of the rabbit. Here, the types of synaptic vesicle containing profiles of the dLGN are described. There are presynaptic large profiles containing round vesicles and pale mitochondria (RLP terminals) and small profiles that contain round vesicles and dark mitochondria (RSD terminals) which respectively arise from the retina and the visual cortex. Another type of presynaptic profile contains elliptical vesicles (F-boutons) which can be subdivided according to their cytoplasmic content. These F-boutons arise from dLGN interneurons. We have found different sized vesicles that have a dense core within RLP, and F terminals and a possible RSD terminal. The significance of the coexistance of pale and dense cored vesicles in the presynaptic profiles of the rabbit dLGN is discussed.     

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD^HOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD^HOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD^HOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD^HOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.