Growth and derived life-history characteristics of the Brazilian electric ray Narcine brasiliensis.

The majority of batoids are listed as Threatened (20.4%) or Data Deficient (41%) by the IUCN Red List. A key challenge to assessing Data-Deficient species is obtaining estimates of key life-history characteristics. Here, a Bayesian approach was used to estimate derived life-history characteristics from a growth model applied to the Data-Deficient Brazilian electric ray Narcine brasiliensis. The age of 170 specimens (107 females, 63 males) was estimated from vertebral centra, and total length, disc width, total weight and birth size were used in a joint estimation of sex-specific length-weight models and two-dimensional von Bertalanffy growth models. Estimates of age at length zero, age at maturity, longevity and mortality at age were derived simultaneously. The Bayesian joint modelling approach was robust to small sample sizes by adding a likelihood to constrain L0 and sharing parameters, such as Brody growth coefficient between length measurements. The median growth parameter estimates were a shared L0 = 38.8 mm, female L∞ = 515 mm, 𝑘 = 0.125 and male L∞ = 387 mm, 𝑘 = 0.194. Age at maturity was estimated to be 7.40-7.49 years for females and 4.45-4.47 years for males, whereas longevity was 22.5-22.6 years for females and 14.2 years for males depending on length measurement. Age-1 natural mortality was estimated to be 0.199-0.207 for females and 0.211-0.213 for males. The derived life-history characteristics indicate N. brasiliensis is earlier maturing, but slower growing relative to other Torpediniformes. These characteristics along with the species' endemism to southern Brazil and high by-catch rates indicate that one of the IUCN Red List threatened categories may be more appropriate for the currently Data-Deficient status. The Bayesian approach used for N. brasiliensis can prove useful for utilizing limited age-growth data in other Data-Deficient batoid species to inform necessary life characteristics for conservation and management.

Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

BACKGROUND: The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. RESULTS: Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). CONCLUSIONS: In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.

Notes on the reproductive biology of the Brazilian electric ray Narcine brasiliensis (Elasmobranchii: Narcinidae).

This study provides information on the reproductive biology of Narcine brasiliensis based on 105 individuals (72 females and 33 males) sampled in São Paulo State, Brazil. The total length at maturity for females was 318·9 mm and for males was 279·8 mm; pregnant females were observed only during summer and autumn. The peak of the gonado-somatic index for females and condition factor for males in the spring suggest a preparation for pregnancy and a mating period during this season. The capture of immature individuals indicates a need for management of the species in this region.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor.

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4ß2 and α2ß2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αßεδ) and Torpedo (αßγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 µM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 µM) than in the resting state ([(3)H]tetracaine; IC50 = 60 µM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-ß subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.

Comparative study on trace metal accumulation in the liver of two fish species (Torpedinidae): concentration-size relationship.

Metal concentrations (Hg, Cd, Pb, As, Cr, Cu, Zn and Ni) were measured in the liver of two fish, Torpedo nobiliana (electric ray) and Torpedo marmorata (marbled electric ray), from the Mediterranean Sea in order to comparatively investigate their current pollution status. Maximum mean levels of Hg were detected in electric ray (mean: 2.16µgg(-1) ww), while marbled electric ray accumulated especially Cd (mean: 0.06µgg(-1) ww), Cu (mean: 3.83µgg(-1) ww) and As (mean: 32.64µgg(-1) ww). The metal concentrations are similar to those reported in literature, except for Cd, As and Ni. Hg concentrations increased with increasing fish body length in both species, whilst no significant concentration-size relationship was found for other metals. To the best of our knowledge this is the first report providing information on trace metal levels and relationship between concentration and size of these cartilaginous fishes. Future studies on the concentrations and effects of environmental contaminants in various torpedinid species are surely needed.

Comparative cranio-mandibular myology of three species of Batoidea from the Southern Gulf of California, Mexico.

The mandibular apparatus of batoids (skates, electric rays, guitarfishes, stingrays, and sawfishes) is composed of a few skeletal elements to which the muscular bundles, responsible for all movements involved in the feeding mechanism, are inserted. The description of the different mandibular morphologies can help to understand the different feeding guilds in this group. In this study, we examined the cranio-mandibular myology of adult Rostroraja velezi, Narcine entemedor, and Zapteryx exasperata, three species of rays that coexist in the Southern Gulf of California, Mexico. This study described the muscles on the ventral and the dorsal surfaces for each species, identified the origins and insertions of these muscles, as well as the general characteristics of muscle morphology. There were 17 and 18 muscle bundles attached to the feeding apparatus, including five on the dorsal surface. Only the levator rostri, which elevates the rostrum during feeding, showed considerable differences in shape and size among species. The muscles of the adductor complex showed the greatest differences in size among the three species. N. entemedor presented the exclusive muscle X in the lower mandibular area and the extreme reduction of the coracohyoideus in the pharyngeal area derived from the absence of the basihyal cartilage. The information generated in our study supports the morphological specialization of electric rays (N. entemedor) for an almost exclusive suction feeding strategy.

Phylogenetic significance of clasper morphology of electric rays (Chondrichthyes: Batoidea: Torpediniformes).

Torpediniformes (electric rays) is a monophyletic group strongly supported by morphological and molecular phylogenetic studies. The claspers of electric rays, however, are poorly documented in comparation to the clasper of other batoids, especially skates, and the knowledge of their anatomical variation is restricted to the description of a few species. The present article analyzes the external and skeletal clasper anatomy of electric rays and reports newly discovered characters that can be useful for taxonomic diagnoses and higher-level systematic studies. The family Torpedinidae exclusively presents the integumental flap, a poorly calcified clasper skeleton, and a dorsal marginal cartilage with a medial flange on its distal portion. Derived or diagnostic characters were not found in the clasper of the reportedly nonmonophyletic families Narcinidae and Narkidae; however, the claspers of species and genera of narcinids and narkids present different anatomical patterns that can be useful for taxonomic and phylogenetic studies.

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiêm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.

Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

Tooth reorientation affects tooth function during prey processing and tooth ontogeny in the lesser electric ray, Narcine brasiliensis.

The dental anatomy of elasmobranch fishes (sharks, rays and relatives) creates a functional system that is more dynamic than that of mammalian dentition. Continuous dental replacement (where new teeth are moved rostrally to replace older ones) and indirect fibrous attachment of the dentition to the jaw allow teeth to reorient relative to the jaw over both long- and short-term scales, respectively. In this study, we examine the processing behavior and dental anatomy of the lesser electric ray Narcine brasiliensis (Olfers, 1831) to illustrate that the freedom of movement of elasmobranch dentition allows a functional flexibility that can be important for complex prey processing behaviors. From static manipulations of dissected jaws and observations of feeding events in live animals, we show that the teeth rotate during jaw protrusion, resulting in a secondary grasping mechanism that likely serves to hold prey while the buccal cavity is flushed free of sediment. The function of teeth is not always readily apparent from morphology; in addition to short-term reorientation, the long-term dental reorientation during replacement allows a given tooth to serve multiple functions during tooth ontogeny. Unlike teeth inside the mouth, the cusps of external teeth (on the portion of the tooth pad that extends past the occlusal plane) lay flat, such that the labial faces act as a functional battering surface, protecting the jaws during prey excavation.

ATP dependence of the non-specific ion channel in Torpedo synaptic vesicles.

Synaptic vesicles of Torpedo electromotor neurons contain a high amount of ATP. The concentration of total ATP is around 120 mM, whereas the free [ATP] is about 5-6 mM. We examined the effect of intravesicular ATP on the non-specific ion channel in Torpedo-fused synaptic vesicles. It was found that this channel is closed when the ATP concentration is above 2 mM, but it is very frequently open at lower ATP concentrations. Unmasking this ion channel at a low ATP concentration may be significant for post-fusion control of transmitter release by the 'kiss and run' mechanism in normal conditions, while during metabolic stress it may underlie dissipation of important gradients across the vesicle membrane.

Functional morphology of jaw trabeculation in the lesser electric ray Narcine brasiliensis, with comments on the evolution of structural support in the Batoidea.

The design of minimum-weight structures that retain their integrity under dynamic loading regimes has long challenged engineers. One solution to this problem found in both human and biological design is the optimization of weight and strength by hollowing a structure and replacing its inner core with supportive struts. In animals, this design is observed in sand dollar test, avian beak, and the cancellous bone of tetrapod limbs. Additionally, within the elasmobranch fishes, mineralized trabeculae (struts) have been reported in the jaws of durophagous myliobatid stingrays (Elasmobranchii: Batoidea), but were believed to be absent in basal members of the batoid clade. This study, however, presents an additional case of batoid trabeculation in the lesser electric ray, Narcine brasiliensis (Torpediniformes). The trabeculae in these species likely play different functional roles. Stingrays use their reinforced jaws to crush bivalves, yet N. brasiliensis feeds by ballistically protruding its jaws into the sediment to capture polychaetes. In N. brasiliensis, trabeculae are localized to areas likely to experience the highest load: the quadratomandibular jaw joints, hyomandibular-cranial joint, and the thinnest sections of the jaws immediately lateral to the symphyses. However, the supports perform different functions dependent on location. In regions where the jaws are loaded transversely (as in durophagous rays), "load leading" trabeculae distribute compressive forces from the cortex through the lumen of the jaws. In the parasymphyseal regions of the jaws, "truss" trabeculae form cross-braces perpendicular to the long axes of the jaws. At peak protrusion, the jaw arch is medially compressed and the jaw loaded axially such that these trabeculae are positioned to resist buckling associated with excavation forces. "Truss" trabeculae function to maintain the second moment of area in the thinnest regions of the jaws, illustrating a novel function for batoid trabeculation. Thus, this method of structural support appears to have arisen twice independently in batoids and performs strikingly different ecological functions associated with the distribution of extreme loading environments.

Narcine baliensis, a new species of electric ray from southeast Asia (Chondrichthyes: Torpediniformes).

A new species of numbfish, Narcine baliensis, sp. nov., is described from the tropical eastern Indian Ocean from Indonesia. It is superficially similar to N. brevilabiata and N. atzi in aspects of its color pattern, but is distinguished from both congeners in details of its color pattern, in tooth band morphology, and in proportions of its dorsal fins, among other features. Narcine baliensis, sp. nov., is unique in having a dorsal color pattern composed of large, circular, ovoid or elongate dark brown spots or blotches on dorsal disc along with more numerous small (about eye-sized or slightly greater) brownish, subcircular spots, with large blotches and small spots surrounded by a very slender creamy-white pattern, as well as in having broadly circular upper and lower tooth bands of about the same width and shape. The genus Narcine is now composed of 20 valid species, but uncertainty remains concerning the identification and morphological variation of some of its species in the tropical Indo-West Pacific region.

Tetronarce cowleyi, sp. nov., a new species of electric ray from southern Africa (Chondrichthyes: Torpediniformes: Torpedinidae).

A new species of torpedo ray, Tetronarce cowleyi, sp. nov., is described from specimens collected from the southeastern Atlantic Ocean. The new species is placed in the genus Tetronarce based on a uniform dorsal coloration and absence of papillae around the spiracles. The new species is distinguished from its closest congeners, the North Atlantic Tetronarce nobiliana Bonnaparte, 1835, and southwestern Atlantic Tetronarce puelcha Lahille, 1926, by a combination of morphological characteristics including a shorter spiracular length, a proportionally greater head length as measured between snout margin and fifth gill openings, a proportionally greater preoral snout length, a uniform shiny black or dark gray dorsal surface, lacking any prominent markings, and a creamy white ventral color with dark edges in juveniles but fading with growth. Teteronarce cowleyi, sp. nov., is further distinguished from T. nobiliana by its more circular anterior disc shape (vs. relatively straight in T. nobiliana), fewer tooth rows (32/28 vs. 38-53/38-52 in T. nobiliana), greater mouth width (1.5-1.7 times as great as interorbital width vs. 0.5-0.6 times interorbital width in T. nobiliana), smaller distance between second dorsal and caudal fins (3.5-4.9% vs. 6.6-6.8% in T. nobiliana), and a clasper length extending nearly to lower caudal fin origin (claspers in T. nobiliana that extend only two-thirds distance between second dorsal and caudal fins). Teteronarce cowleyi, sp. nov., is known from Walvis Bay, Namibia to Algoa Bay, Eastern Cape, South Africa, at depths of 110 to 457 m.

Presence of long and short dystrophin and/or utrophin products in Torpedo marmorata peripheral nerves.

Peripheral nerves from rabbit and Torpedo marmorata were comparatively analyzed for the presence of short dystrophin products. Western blot analyses of Torpedo marmorata peripheral nerve extracts revealed the existence of three proteins belonging to the dystrophin family: a M(r) 400 kDa protein band detected with dystrophin/utrophin, dystrophin-specific and Torpedo utrophin-specific antibodies, a molecule identified as Dp116 and, for the first time at the protein level, a new protein probably corresponding to Up116. All of these products were carefully identified according to the specificities of the monoclonal antibodies used. In immunofluorescence studies, clear staining of the thin rim surrounding each Schwann cell-axon unit was observed in both Torpedo marmorata and rabbit peripheral nerves, showing colocalization of all of these molecules. Their potential functions were discussed in comparison to similar products found in rabbit peripheral nerves.

Torpedo electromotor system development: neuronal cell death and electric organ development in the fourth branchial arch.

The fourth branchial arch of Torpedo marmorata has been examined at the light and electron microscopic level during development. Of interest was the determination of the extent of electric organ tissue reported to be present in this arch and its possible relationship to electromotoneuron cell death in the electric lobes. The main electric organ of the torpedo is derived from the hyoid and first three branchial arches and is innervated by four major electromotor nerves. Extensive electromotoneuron cell death occurs in the electric lobes and most notably in the posterior poles. This feature could be due to a tendency for these neurons to innervate the fourth branchial arch where little or no electric tissue is formed. Our findings support this conclusion but are not entirely consistent with the idea that a population mismatch has occurred. This is because cell death precedes the genesis of the target cells. The presence of innervated differentiated electric tissue in this arch is also reported, leading to the conclusion that Torpedo marmorata possesses an accessory electric organ.

A brain-derived neurotrophic factor (BDNF) related system is involved in the maintenance of the polyinnervate Torpedo electric organ.

Target-derived molecules are essential for the maintenance of neuron survival. In the present work, we introduce the electric organ of Torpedo marmorata as a tool for the study of trophic interactions in a polyinervate system. This electric organ maintains a large number of cholinergic terminals on the postsynaptic cell surface. We have observed that a soluble extract derived from the electric organ induces the maturation of Xenopus oocytes injected with presynaptic plasma membranes (PSPM), indicating that a trophic system may exist. Moreover, we have detected a p75NGFR related protein in PSPM by Western blot analysis. These results suggest the presence of a neurotrophin-related system maintaining the polyinnervate electric organ. Furthermore, molecular experiments showed that the brain-derived neurotrophic factor (BDNF) is the neurotrophin operating in our model. Using degenerate oligonucleotides which comprise a conserved fragment of all neurotrophins, we have only amplified by polymerase chain reaction a BDNF fragment. In a similar way, we have amplified and cloned a fragment of the TrkB/C high affinity BDNF receptor. The fact that degenerate oligonucleotides only amplify BDNF allows us to conclude that the polyinnervation is maintained by this neurotrophin either alone or in combination with other trophic factors.

Expression of CTL1 in myelinating structures of Torpedo marmorata.

We initially isolated CTL1 from the electric lobe of brain through functional complementation of a yeast mutant deficient in choline transport. Here, we present the first characterization of an antibody to the C-terminal of CTL1. When full length torpedo CTL1 was expressed in oocytes, a broad 60 kDa band appeared concomitant with the detection of immunoreactivity at the plasma membrane. In, the native protein was prominent throughout the CNS and along the electric nerves. CTL1 immunolabeling was particularly conspicuous in the myelin sheath surrounding the electric nerve and in central myelinated structures. The association of the presumptive choline transporter, CTL1, with myelin membranes suggests a role for this new protein in lipid production.

Targeting of acetylcholine receptor and 43 kDa rapsyn to the postsynaptic membrane in Torpedo marmorata electrocyte.

In this study we have investigated the intracellular routing of two major components of the postsynaptic membrane in Torpedo electrocytes, the nicotinic acetylcholine receptor and the extrinsic 43 kDa protein rapsyn, and of a protein from the non-innervated membrane, the Na+,K+ ATPase. We isolated subpopulations of post-Golgi vesicles (PGVs) enriched either in AChR or in Na+,K+ ATPase. Rapsyn was associated to AChR-containing PGVs suggesting that both AChR and rapsyn are targeted to intracellular organelles in the secretory pathway before delivery to the postsynaptic membrane. In vitro assays further show that rapsyn-containing PVGs do bind more efficiently to microtubules compared to Na+,K+ ATPase-enriched PVGs. These data provide evidence in favor of the contribution of the secretory pathway to the delivery of synaptic components.

A morphometric analysis of Torpedo synaptic vesicles isolated by iso-osmotic sucrose gradient separation.

The presynaptic terminal vesicle population of Torpedo electric organ is heterogeneous in size, consisting of two prominent subpopulations that comprise 80% of the total. The use of standard iso-osmotic sucrose gradients with zonal centrifugation to isolate vesicle fractions that co-localize with the acetylcholine (ACh) peak results in the recovery of: (1) 10% of the total estimated vesicle population; and (2) a single 68-nm diameter vesicle size class. The whereabouts of the major 90-nm subclass, which accounts for 60% of the total terminal population and which has long been considered to represent the resident ACh population, has been investigated. Assuming this subclass to have undergone severe osmotic stress, the effects of hypo- and hyper-osmotic salines, buffers and fixatives were examined and found to produce only negligible changes on vesicle size. Isolation of vesicles by hypo-osmotic shocking of synaptosomes purified on a Ficoll gradient, however, resulted in a reasonable approximation of the in situ distribution. As the iso-osmotic sucrose gradient procedure utilizes frozen blocks of electric tissue, this step is suspected of being involved in the loss, perhaps because of the slow freezing rates employed. These findings indicate that the 90 nm subclass is lost rather than transformed during isolation by sucrose gradient separation and that dimensionally, the cholinergic vesicle is a constant-sized and relatively stable structure.