Network architecture of the cerebral nuclei (basal ganglia) association and commissural connectome.

The cerebral nuclei form the ventral division of the cerebral hemisphere and are thought to play an important role in neural systems controlling somatic movement and motivation. Network analysis was used to define global architectural features of intrinsic cerebral nuclei circuitry in one hemisphere (association connections) and between hemispheres (commissural connections). The analysis was based on more than 4,000 reports of histologically defined axonal connections involving all 45 gray matter regions of the rat cerebral nuclei and revealed the existence of four asymmetrically interconnected modules. The modules form four topographically distinct longitudinal columns that only partly correspond to previous interpretations of cerebral nuclei structure-function organization. The network of connections within and between modules in one hemisphere or the other is quite dense (about 40% of all possible connections), whereas the network of connections between hemispheres is weak and sparse (only about 5% of all possible connections). Particularly highly interconnected regions (rich club and hubs within it) form a topologically continuous band extending through two of the modules. Connection path lengths among numerous pairs of regions, and among some of the network's modules, are relatively long, thus accounting for low global efficiency in network communication. These results provide a starting point for reexamining the connectional organization of the cerebral hemispheres as a whole (right and left cerebral cortex and cerebral nuclei together) and their relation to the rest of the nervous system.

New data on the nervous system of Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae): revisiting old hypotheses.

Data on the interposition of the immunoreactive nerve cords in Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae) and its chaetotaxy were obtained. The nervous system of C. parvicaudata was described using immunostaining of 5-hydroxytryptamine and FMRFamide immunoreactive nerve elements. The morphology and distribution of sensory receptors were analysed using scanning electron microscopy and the silver nitrate impregnation technique. Our integrated approach to the study of the nervous system revealed a clear colocalization of surface papillae with nerve cords and commissures in C. parvicaudata. The structure of the nervous system in C. parvicaudata differs partly from the classical model that defines the entire nomenclature of chaetotaxy.

Compartmentalized Regulation of Parkin-Mediated Mitochondrial Quality Control in the Drosophila Nervous System In Vivo.

UNLABELLED: In neurons, the normal distribution and selective removal of mitochondria are considered essential for maintaining the functions of the large asymmetric cell and its diverse compartments. Parkin, a E3 ubiquitin ligase associated with familial Parkinson's disease, has been implicated in mitochondrial dynamics and removal in cells including neurons. However, it is not clear how Parkin functions in mitochondrial turnover in vivo, or whether Parkin-dependent events of the mitochondrial life cycle occur in all neuronal compartments. Here, using the live Drosophila nervous system, we investigated the involvement of Parkin in mitochondrial dynamics, distribution, morphology, and removal. Contrary to our expectations, we found that Parkin-deficient animals do not accumulate senescent mitochondria in their motor axons or neuromuscular junctions; instead, they contain far fewer axonal mitochondria, and these displayed normal motility behavior, morphology, and metabolic state. However, the loss of Parkin did produce abnormal tubular and reticular mitochondria restricted to the motor cell bodies. In addition, in contrast to drug-treated, immortalized cells in vitro, mature motor neurons rarely displayed Parkin-dependent mitophagy. These data indicate that the cell body is the focus of Parkin-dependent mitochondrial quality control in neurons, and argue that a selection process allows only healthy mitochondria to pass from cell bodies to axons, perhaps to limit the impact of mitochondrial dysfunction. SIGNIFICANCE STATEMENT: Parkin has been proposed to police mitochondrial fidelity by binding to dysfunctional mitochondria via PTEN (phosphatase and tensin homolog)-induced putative kinase 1 (PINK1) and targeting them for autophagic degradation. However, it is unknown whether and how the PINK1/Parkin pathway regulates the mitochondrial life cycle in neurons in vivo Using Drosophila motor neurons, we show that parkin disruption generates an abnormal mitochondrial network in cell bodies in vivo and reduces the number of axonal mitochondria without producing any defects in their axonal transport, morphology, or metabolic state. Furthermore, while cultured neurons display Parkin-dependent axonal mitophagy, we find this is vanishingly rare in vivo under normal physiological conditions. Thus, both the spatial distribution and mechanism of mitochondrial quality control in vivo differ substantially from those observed in vitro.

The first data on the innervation of the lophophore in the rhynchonelliform brachiopod Hemithiris psittacea: what is the ground pattern of the lophophore in lophophorates?

BACKGROUND: The nervous system in brachiopods has seldom been studied with modern methods. An understanding of lophophore innervation in adult brachiopods is useful for comparing the innervation of the same lophophore type among different brachiopods and can also help answer questions about the monophyly of the lophophorates. Although some brachiopods are studied with modern methods, rhynchonelliform brachiopods still require investigation. The current study used transmission electron microscopy, immunocytochemistry, and confocal laser scanning microscopy to investigate the nerve system of the lophophore and tentacles in the rhynchonelliform Hemithiris psittacea. RESULTS: Four longitudinal nerves pass along each brachium of the lophophore: the main, accessory, second accessory, and lower. The main brachial nerve extends at the base of the dorsal side of the brachial fold and gives rise to the cross nerves, passing through the extracellular matrix to the tentacles. Cross nerves skirt the accessory brachial nerve, branch, and penetrate into adjacent outer and inner tentacles, where they are referred to as the frontal tentacular nerves. The second accessory nerve passes along the base of the inner tentacles. This nerve consists of Ʊ-like parts, which repetitively skirt the frontal and lateral sides of the inner tentacle and the frontal sides of the outer tentacles. The second accessory nerve gives rise to the latero-frontal nerves of the inner and outer tentacles. The abfrontal nerves of the inner tentacles also originate from the second accessory nerve, whereas the abfrontal nerves of the outer tentacles originate from the lower brachial nerve. The lower brachial nerve extends along the outer side of the lophophore brachia and gives rise to the intertentacular nerves, which form a T-like branch and penetrate the adjacent outer tentacles where they are referred to as abfrontal nerves. The paired outer radial nerves start from the lower brachial nerve, extend into the second accessory nerve, and give rise to the lateroabfrontal tentacular nerves of the outer tentacles. CONCLUSIONS: The innervation of the lophophore in the rhynchonelliform Hemithiris psittacea differs from that in the inarticulate Lingula anatina in several ways. The accessory brachial nerve does not participate in the innervation of the tentacles in H. psittacea as it does in L. anatina. The second accessory nerve is present in H. psittacea but not in L. anatina. There are six tentacular nerves in the outer tentacles of H. psittacea but only four in all other brachiopods studied to date. The reduced contribution of the accessory brachial nerve to tentacle innervation may reflect the general pattern of reduction of the inner lophophoral nerve in both phoronids and brachiopods. Bryozoan lophophores, in contrast, have a weakened outer nerve and a strengthened inner nerve. Our results suggest that the ancestral lophophore of all lophophorates had a simple shape but many nerve elements.

Ground plan of the larval nervous system in phoronids: Evidence from larvae of viviparous phoronid.

Nervous system organization differs greatly in larvae and adults of many species, but has nevertheless been traditionally used for phylogenetic studies. In phoronids, the organization of the larval nervous system depends on the type of development. With the goal of understanding the ground plan of the nervous system in phoronid larvae, the development and organization of the larval nervous system were studied in a viviparous phoronid species. The ground plan of the phoronid larval nervous system includes an apical organ, a continuous nerve tract under the preoral and postoral ciliated bands, and two lateral nerves extending between the apical organ and the nerve tract. A bilobed larva with such an organization of the nervous system is suggested to be the primary larva of the taxonomic group Brachiozoa, which includes the phyla Brachiopoda and Phoronida. The ground plan of the nervous system of phoronid larvae is similar to that of the early larvae of annelids and of some deuterostomians. The protostome- and deuterostome-like features, which are characteristic of many organ systems in phoronids, were probably inherited by phoronids from the last common bilaterian ancestor. The information provided here on the ground plan of the larval nervous system should be useful for future analyses of phoronid phylogeny and evolution.

Musculoskeletal and nervous systems of the attachment organ in three species of Diplectanum (Monogenea: Dactylogyroidea).

The taxonomy of Diplectanum Diesing, 1858, a genus of monopisthocotylean monogeneans, remains unsettled and needs to be revised based on new morphological criteria. Recent studies in monopisthocotyleans have shown that the muscle arrangement in the posterior attachment organ (haptor) differs between congeneric species and can be used as an additional criterion in genus-level taxonomy. To explore the possibility of using the haptoral musculature and nervous system in the taxonomy of Diplectanum, we conducted a detailed confocal-microscopy study of three species of Diplectanum (D. aculeatum Parona et Perugia, 1889, D. sciaenae van Beneden et Hesse, 1863 and D. similis Bychowsky, 1957) with phalloidin staining for muscle and indirect immunostaining for 5HT and FMRFamide. A further goal was to clarify the functional mechanics of the haptor and the role of its essential components (squamodiscs and anchors) in attachment to the host. The system of connecting bars and gaffing anchors was found to have a complex musculature consisting of 23 muscles in D. aculeatum and D. sciaenae, and 21 muscles in D. similis. The squamodiscs were shown to be operated by several groups of muscles attached primarily to the area termed the squamodisc fulcrum. Most of the haptoral musculature is identical in D. aculeatum and D. sciaenae and these species differ only in the presence of a muscle sheath around the tissue strand between the squamodiscs in D. sciaenae and in the different patterns of superficial squamodisc muscles. Diplectanum similis shows more significant differences from the other two species: besides lacking two of the haptoral muscles, it also differs in the shapes and arrangement of several other muscles. The nervous system of all three species conforms to the general pattern typical for the Dactylogyroidea and shows little variation between species.

The nervous system of the lophophore in the ctenostome Amathia gracilis provides insight into the morphology of ancestral ectoprocts and the monophyly of the lophophorates.

BACKGROUND: The Bryozoa (=Ectoprocta) is a large group of bilaterians that exhibit great variability in the innervation of tentacles and in the organization of the cerebral ganglion. Investigations of bryozoans from different groups may contribute to the reconstruction of the bryozoan nervous system bauplan. A detailed investigation of the polypide nervous system of the ctenostome bryozoan Amathia gracilis is reported here. RESULTS: The cerebral ganglion displays prominent zonality and has at least three zones: proximal, central, and distal. The proximal zone is the most developed and contains two large perikarya giving rise to the tentacle sheath nerves. The neuroepithelial organization of the cerebral ganglion is revealed. The tiny lumen of the cerebral ganglion is represented by narrow spaces between the apical projections of the perikarya of the central zone. The cerebral ganglion gives rise to five groups of main neurite bundles of the lophophore and the tentacle sheath: the circum-oral nerve ring, the lophophoral dorso-lateral nerves, the pharyngeal and visceral neurite bundles, the outer nerve ring, and the tentacle sheath nerves. Serotonin-like immunoreactive nerve system of polypide includes eight large perikarya located between tentacles bases. There are two analmost and six oralmost perikarya with prominent serotonergic "gap" between them. Based on the characteristics of their innervations, the tentacles can be subdivided into two groups: four that are near the anus and six that are near the mouth. Two longitudinal neurite bundles - medio-frontal and abfrontal - extend along each tentacle. CONCLUSION: The zonality of the cerebral ganglion, the presence of three commissures, and location of the main nerves emanating from each zone might have caused by directive innervation of the various parts of the body: the tentacles sheath, the lophohpore, and the digestive tract. Two alternative scenarios of bryozoan lophophore evolution are discussed. The arrangement of large serotonin-like immunoreactive perikarya differs from the pattern previously described in ctenostome bryozoans. In accordance with its position relative to the same organs (tentacles, anus, and mouth), the lophophore outer nerve ring corresponds to the brachiopod lower brachial nerve and to the phoronid tentacular nerve ring. The presence of the outer nerve ring makes the lophophore innervation within the group (clade) of lophophorates similar and provides additional morphological evidence of the lophophore homology and monophyly of the lophophorates.

Functions of Nogo proteins and their receptors in the nervous system.

The membrane protein Nogo-A was initially characterized as a CNS-specific inhibitor of axonal regeneration. Recent studies have uncovered regulatory roles of Nogo proteins and their receptors--in precursor migration, neurite growth and branching in the developing nervous system--as well as a growth-restricting function during CNS maturation. The function of Nogo in the adult CNS is now understood to be that of a negative regulator of neuronal growth, leading to stabilization of the CNS wiring at the expense of extensive plastic rearrangements and regeneration after injury. In addition, Nogo proteins interact with various intracellular components and may have roles in the regulation of endoplasmic reticulum (ER) structure, processing of amyloid precursor protein and cell survival.

Peptide-gated ion channels and the simple nervous system of Hydra.

Neurons either use electrical or chemical synapses to communicate with each other. Transmitters at chemical synapses are either small molecules or neuropeptides. After binding to their receptors, transmitters elicit postsynaptic potentials, which can either be fast and transient or slow and longer lasting, depending on the type of receptor. Fast transient potentials are mediated by ionotropic receptors and slow long-lasting potentials by metabotropic receptors. Transmitters and receptors are well studied for animals with a complex nervous system such as vertebrates and insects, but much less is known for animals with a simple nervous system like Cnidaria. As cnidarians arose early in animal evolution, nervous systems might have first evolved within this group and the study of neurotransmission in cnidarians might reveal an ancient mechanism of neuronal communication. The simple nervous system of the cnidarian Hydra extensively uses neuropeptides and, recently, we cloned and functionally characterized an ion channel that is directly activated by neuropeptides of the Hydra nervous system. These results demonstrate the existence of peptide-gated ion channels in Hydra, suggesting they mediate fast transmission in its nervous system. As related channels are also present in the genomes of the cnidarian Nematostella, of placozoans and of ctenophores, it should be considered that the early nervous systems of cnidarians and ctenophores have co-opted neuropeptides for fast transmission at chemical synapses.

Variation in the schedules of somite and neural development in frogs.

The timing of notochord, somite, and neural development was analyzed in the embryos of six different frog species, which have been divided into two groups, according to their developmental speed. Rapid developing species investigated were Xenopus laevis (Pipidae), Engystomops coloradorum, and Engystomops randi (Leiuperidae). The slow developers were Epipedobates machalilla and Epipedobates tricolor (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Blastopore closure, notochord formation, somite development, neural tube closure, and the formation of cranial neural crest cell-streams were detected by light and scanning electron microscopy and by immuno-histochemical detection of somite and neural crest marker proteins. The data were analyzed using event pairing to determine common developmental aspects and their relationship to life-history traits. In embryos of rapidly developing frogs, elongation of the notochord occurred earlier relative to the time point of blastopore closure in comparison with slowly developing species. The development of cranial neural crest cell-streams relative to somite formation is accelerated in rapidly developing frogs, and it is delayed in slowly developing frogs. The timing of neural tube closure seemed to be temporally uncoupled with somite formation. We propose that these changes are achieved through differential timing of developmental modules that begin with the elongation of the notochord during gastrulation in the rapidly developing species. The differences might be related to the necessity of developing a free-living tadpole quickly in rapid developers.

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells.

Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function.

A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome.

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

Ventral nerve cord in Phoronopsis harmeri larvae.

The nervous system organization is considered a phylogenetically important character among metazoans. The phylum Phoronida is included in a supraphyletic taxon known as Lophotrochozoa. Many lophotrochozoans possess a metameric ventral nerve cord as adults or larvae. Phoronids do not exhibit external metamery either as larvae or as adults. The current study describes the ventral nerve cord in the young larva of Phoronopsis harmeri. This structure is apparent both in the serotonergic and FMRF-amidergic nervous system in young larvae. The ventral nerve cord extends from the mouth to the tentacular ridge. Both serotonergic and FMRF-amidergic components consist of two ventrolateral nerves, each with several unipolar neurons. The ventrolateral nerves connect to each other by means of thin repetitive transversal nerves ("commissures"). The abundance of neurons and nerves in the epidermis of the oral field of actinotrocha larva likely reflects the importance of this area in collection of food particles. The ventral nerve cords of the actinotrocha and the metatrochophore differ in their positions with respect to ciliated bands: the cord is located between the preoral and postoral ciliated bands in the actinotrocha but between the postoral ciliated band and telotroch in the metatrochophore. The presence of the ventral nerve cord, which contains repetitive elements (neurons and "commissures"), in the early development of P. harmeri may recapitulate some stages of nervous system development during phoronid phylogeny. The larval nervous system does not contain nervous centers under the tentacular ridge that can correlate with the catastrophic metamorphosis and unique body plan of phoronids.

Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy.

Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 microm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.

The neuro-glandular brain of the Pyramicocephalus phocarum plerocercoid (Cestoda, Diphyllobothriidea): Immunocytochemical and ultrastructural study.

А novel type of a complex neuro-glandular brain structure including both nervous and glandular elements and associated with sensory ones is detected in Pyramicocephalus phocarum plerocercoid (Cestoda: Diphyllobothriidea), parasite of Gadus morua from the White Sea. The brain has two lateral lobes connected by a long cellular median commissure. The brain is tightly surrounded by glandular cells, which receive numerous synapses from the brain neurons. A complex of sensory organs associated with ducts and terminal pores of the frontal glands lies in the scolex tegument. Serotonin, FMRFamide- and GABA-like immunoreactive (IR) neurons are found in the brain, the main nerve cords, and the plexus of the plerocercoid. The innervation of the frontal gland ducts by FMRFamide-IR neurites is detected for the first time proving that they function under control of the nervous system and thus evidencing the eccrine nature of the secretion mechanism. Ultrastructural data show that light, dark and neurosecretory neurons are present in the brain lobes. The median commissure consists of loosely arranged thin parallel axons and several giant and small neurons. The commissure is stratified and penetrated by frontal glandular cells and their processes. Such neuro-glandular morpho-functional brain complex is suggested as a model for Diphyllobothriidae family. Five structural types of sensory organs are described in the scolex of P. phocarum; their colocalization with eccrine gland terminals is supposedly specific for Diphyllobothriidae family. Within the order Diphyllobothriidea, there are significant differences in the architecture of the plerocercoid brain at the family level. We suppose homology of giant commissural neurons among Diphyllobothriidea. Differences between diphyllobothriidean nervous system and that of other cestodes are discussed.

PKA activation bypasses the requirement for UNC-31 in the docking of dense core vesicles from C. elegans neurons.

The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. We address this technical limitation by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, we have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. We also demonstrate that UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis.

C. elegans G protein regulator RGS-3 controls sensitivity to sensory stimuli.

Signal transduction through heterotrimeric G proteins is critical for sensory response across species. Regulator of G protein signaling (RGS) proteins are negative regulators of signal transduction. Herein we describe a role for C. elegans RGS-3 in the regulation of sensory behaviors. rgs-3 mutant animals fail to respond to intense sensory stimuli but respond normally to low concentrations of specific odorants. We find that loss of RGS-3 leads to aberrantly increased G protein-coupled calcium signaling but decreased synaptic output, ultimately leading to behavioral defects. Thus, rgs-3 responses are restored by decreasing G protein-coupled signal transduction, either genetically or by exogenous dopamine, by expressing a calcium-binding protein to buffer calcium levels in sensory neurons or by enhancing glutamatergic synaptic transmission from sensory neurons. Therefore, while RGS proteins generally act to downregulate signaling, loss of a specific RGS protein in sensory neurons can lead to defective responses to external stimuli.

A principal skeleton algorithm for standardizing confocal images of fruit fly nervous systems.

MOTIVATION: The fruit fly (Drosophila melanogaster) is a commonly used model organism in biology. We are currently building a 3D digital atlas of the fruit fly larval nervous system (LNS) based on a large collection of fly larva GAL4 lines, each of which targets a subset of neurons. To achieve such a goal, we need to automatically align a number of high-resolution confocal image stacks of these GAL4 lines. One commonly employed strategy in image pattern registration is to first globally align images using an affine transform, followed by local non-linear warping. Unfortunately, the spatially articulated and often twisted LNS makes it difficult to globally align the images directly using the affine method. In a parallel project to build a 3D digital map of the adult fly ventral nerve cord (VNC), we are confronted with a similar problem. RESULTS: We proposed to standardize a larval image by best aligning its principal skeleton (PS), and thus used this method as an alternative of the usually considered affine alignment. The PS of a shape was defined as a series of connected polylines that spans the entire shape as broadly as possible, but with the shortest overall length. We developed an automatic PS detection algorithm to robustly detect the PS from an image. Then for a pair of larval images, we designed an automatic image registration method to align their PSs and the entire images simultaneously. Our experimental results on both simulated images and real datasets showed that our method does not only produce satisfactory results for real confocal larval images, but also perform robustly and consistently when there is a lot of noise in the data. We also applied this method successfully to confocal images of some other patterns such as the adult fruit fly VNC and center brain, which have more complicated PS. This demonstrates the flexibility and extensibility of our method. AVAILABILITY: The supplementary movies, full size figures, test data, software, and tutorial on the software can be downloaded freely from our website http://penglab.janelia.org/proj/principal_skeleton.

SV2 modulates the size of the readily releasable pool of secretory vesicles.

The exocytosis of neurotransmitters is regulated by calcium and is plastic - features that suggest specialized regulation of the basic membrane trafficking process. Here we show that Synaptic Vesicle Protein 2 (SV2), a protein specific to neurons and endocrine cells, is required to maintain a pool of vesicles available for calcium-stimulated exocytosis. Direct measures of exocytosis in adrenal chromaffin cells showed that the calcium-induced exocytotic burst, which operationally defines the readily releasable pool of vesicles, was significantly reduced in mice lacking SV2A. Burst kinetics were normal in cells from SV2A knockout animals, however, indicating that SV2 functions before the final events of fusion. Analyses of SDS-resistant SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion) attachment protein receptor) complexes in brain tissue showed that loss of SV2A was associated with fewer SDS-resistant complexes. Our observations indicate that SV2 may modulate the formation of protein complexes required for fusion and therefore the progression of vesicles to a fusion-competent state.