A NWB-based dataset and processing pipeline of human single-neuron activity during a declarative memory task.

A challenge for data sharing in systems neuroscience is the multitude of different data formats used. Neurodata Without Borders: Neurophysiology 2.0 (NWB:N) has emerged as a standardized data format for the storage of cellular-level data together with meta-data, stimulus information, and behavior. A key next step to facilitate NWB:N adoption is to provide easy to use processing pipelines to import/export data from/to NWB:N. Here, we present a NWB-formatted dataset of 1863 single neurons recorded from the medial temporal lobes of 59 human subjects undergoing intracranial monitoring while they performed a recognition memory task. We provide code to analyze and export/import stimuli, behavior, and electrophysiological recordings to/from NWB in both MATLAB and Python. The data files are NWB:N compliant, which affords interoperability between programming languages and operating systems. This combined data and code release is a case study for how to utilize NWB:N for human single-neuron recordings and enables easy re-use of this hard-to-obtain data for both teaching and research on the mechanisms of human memory.

Mechanism of homophilic adhesion by the neural cell adhesion molecule: use of multiple domains and flexibility.

The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for sequentially synergistic bond formation and the ability to accommodate differences in intercellular space.

Dynamic regulation of polysialylated neural cell adhesion molecule in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) prominently expresses polysialic acid (PSA), a carbohydrate polymer that is attached to neural cell adhesion molecule (NCAM) and promotes changes in cell interactions. Previous studies have shown that expression of PSA is important for circadian rhythm stability under constant darkness, and for photic entrainment of the SCN circadian clock. In the present study, immunoblot analyses of the Syrian hamster SCN revealed marked diurnal fluctuations in PSA under a 24-h light/dark cycle. PSA levels were reduced by >90% during the mid-to-late dark phase, and were elevated to maximal daytime levels approximately 1 h after lights-on. A similar pattern of PSA fluctuation persisted under constant darkness. Exposure of animals under a 24-h light/dark cycle to a 30-min light pulse during the late dark phase dramatically increased SCN contents of PSA within 60 min, and these values returned to basal levels 1-2 h later. There was no effect of light-on expression of PSA in the hippocampus. Parallel studies revealed changes in the NCAM-180 isoform that carries PSA in the brain, suggesting that regulation of PSA may include protein as well as carbohydrate-associated mechanisms. Immunohistological analysis revealed light-induced enhancement of PSA in the SCN subregion containing calbindin D(28K) cells. PSA staining was also closely associated with the majority of SCN cells expressing light-inducible Fos protein. This rhythmic, light-inducible expression of PSA within the SCN suggests that dynamic cell interactions are important for the photic regulation of circadian clock phase.

Dual function of polysialic acid during zebrafish central nervous system development.

Polysialic acid (PSA), a carbohydrate epitope attached to the neural cell adhesion molecule, serves as a modulator of axonal interactions during vertebrate nervous system development. We have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish central nervous system development. PSA is transiently expressed on all cell bodies and, except for the posterior commissure, it is not found on axons. Floorplate cells in the spinal cord and hindbrain strongly express PSA throughout development. Enzymatic removal of PSA leads to a defasciculated growth pattern of the posterior commissure and also affects distinct subsets of commissural axons in the hindbrain, which fail to cross the midline. Whereas the disordered growth pattern of hindbrain commissures produced by PSA-removal could be mimicked by injections of soluble PSA, the growth of axons in the posterior commissure was unaffected by such treatment. These results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.

Roles, regulation, and mechanism of polysialic acid function during neural development.

The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) appeared during the evolution of vertebrates as a new mechanism for regulation of cell interactions. This large and abundant glycoprotein can exert steric effects at the cell surface that lead to the attenuation of cell-cell bonds mediated not only by NCAM but also a variety of other adhesion receptors. PSA-NCAM expression changes both as a result of developmental programs and physiological inputs. This global modulation of cell-cell attachment has been shown to facilitate cell migration, axon pathfinding and targeting, and plastic changes in the embryonic and adult nervous system.

Regulation of cell adhesion by polysialic acid. Effects on cadherin, immunoglobulin cell adhesion molecule, and integrin function and independence from neural cell adhesion molecule binding or signaling activity.

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself.

Membrane lipid rafts are necessary for the maintenance of the (alpha)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons.

Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta-cyclodextrin. This treatment caused the dispersion of lipid rafts and the redistribution of alpha7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering. However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites. To assess whether interaction between rafts and alpha7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX. The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin. Under these conditions, large patches of CTX persisted and were colocalized with alpha7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was found that the alpha7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.

Inhibitory mechanism by polysialic acid for lamina-specific branch formation of thalamocortical axons.

During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a week in vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. This in vitro system permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain.

During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment.

The neural cell adhesion molecules L1 and NCAM-180 act in different steps of neurite outgrowth.

The formation of neurocircuitry depends on the control of neurite outgrowth that, in turn, can be divided into two processes: nerve growth cone protrusion and neurite extension. It has long been known that the neural cell adhesion molecules L1 and NCAM-180 promote neurite outgrowth, but how they function in growth cones is unclear. We addressed the roles of L1 and NCAM-180 in neurite outgrowth by using microscale chromophore-assisted laser inactivation (micro-CALI) of these proteins to perturb their functions at precise times in single growth cones of embryonic chick dorsal root ganglion neurons grown in culture. Micro-CALI of L1 causes neurite retraction after a 10 min lag period but does not affect growth cone protrusion. In contrast, micro-CALI of NCAM-180 causes rapid growth cone retraction but does not affect neurite extension. The simultaneous inactivation of both these molecules resulted in both distinct effects that were segregated in time. The behavior of growth cones after these micro-CALI treatments resemble the drug-induced perturbation of microtubules for L1 and F-actin for NCAM-180. These findings suggest distinct roles in the growth cone for L1 and NCAM-180 in different steps of neurite outgrowth: L1 functions in neurite extension,whereas NCAM-180 functions in growth cone protrusion.

Polysialic acid facilitates migration of luteinizing hormone-releasing hormone neurons on vomeronasal axons.

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the olfactory placode to the forebrain in association with vomeronasal nerves (VNN) that express the polysialic acid-rich form of the neural cell adhesion molecule (PSA-NCAM). Two approaches were used to investigate the role of PSA-NCAM: injection of mouse embryos with endoneuraminidase N, followed by the analysis of LHRH cell positions, and examination of LHRH cell positions in mutant mice deficient in the expression of NCAM or the NCAM-180 isoform, which carries nearly all PSA in the brain. The enzymatic removal of PSA at embryonic day 12 significantly inhibited the migration of nearly half of the LHRH neuron population, without affecting the VNN tract itself. Surprisingly, the absence of NCAM or NCAM-180 did not produce this effect. However, a shift in the route of migration, resulting in an excess number of LHRH cells in the accessory olfactory bulb, was observed in the NCAM-180 mutant. Furthermore, it was found that PSA expressed by the proximal VNN and its distal branch leading to the accessory bulb, but not the branch leading to the forebrain, was associated with the NCAM-140 isoform and thus was retained in the NCAM-180 mutant. These results provide two types of evidence that PSA-NCAM plays a role in LHRH cell migration: promotion of cell movement along the VNN tract that is sensitive to acute (enzymatic), but not chronic (genetic), removal of PSA-NCAM, and a preference of a subset of migrating LHRH cells for a PSA-positive axon branch over a PSA-negative branch in the NCAM-180 mutant.

LHRH neurons migrate into the trigeminal nerve when the developing olfactory nerve fibers are physically interrupted in chick embryos.

Most LHRH neurons actively migrate from the olfactory epithelium to the forebrain during embryonic days (ED) 3.5-8. When a small piece of the membrane filter was placed on the central course of the olfactory nerve in ED 3.5-5 chick embryos, LHRH neurons deviated from their regular migratory course at ED 6.5-7.5 to follow a route along the PSA-NCAM-positive medial and lateral nasal branches of the ophthalmic nerve of the trigeminal nerve. The olfactory nerve fibers which were specifically immunoreactive for somatostatin also deviated into the ophthalmic nerve. Enzymatic removal of PSA using endoneuraminidase did not interfere with the migration of LHRH neurons into the ophthalmic nerve bundle of the trigeminal nerve. The presence of structural supports seems to be primarily of importance in the migration of LHRH neurons along the olfactory and trigeminal nerve bundles. PSA may be less important for the migration of the LHRH neurons along peripheral neural elements.

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans.

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.

Spatio-temporal diversity in the microenvironments for neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule expression by sensory neurons and their targets during cochleo-vestibular innervation.

Sixteen phases in the microenvironments were defined for the structural development and innervation of the cochleo-vestibular ganglion and its targets. In each phase the cell adhesion molecules, neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule, were expressed differentially by cochleo-vestibular ganglion cells, their precursors, and the target cells on which they synapse. Detected by immunocytochemistry in staged chicken embryos, in the otocyst, neural cell adhesion molecule, but not L1-cell adhesion molecule, was localized to the ganglion and hair cell precursors. Ganglionic precursors, migrating from the otocyst, only weakly expressed neural cell adhesion molecule. Epithelial hair cell precursors, remaining in the otocyst, expressed neural cell adhesion molecule, but not L1-cell adhesion molecule. Post-migratory ganglion cell processes expressed both molecules in all stages. The cell adhesion molecules were most heavily expressed by axons penetrating the otic epithelium and accumulated in large amounts in the basal lamina. In the basilar papilla (cochlea), cell adhesion molecule expression followed the innervation gradient. Neural cell adhesion molecule and L1 were heavily concentrated on axonal endings peripherally and centrally. In the rhombencephalon, primitive epithelial cells expressed neural cell adhesion molecule, but not L1-cell adhesion molecule, except in the floorplate. The neuroblasts and their axons expressed L1-cell adhesion molecule, but not neural cell adhesion molecule, when they began to migrate and form the dorsal commissure. There was a stage-dependent, differential distribution of the cell adhesion molecules in the floorplate. Commissural axons expressed both cell adhesion molecules, but their polysialic acid disappeared within the floorplate at later stages. In conclusion, the cell adhesion molecules are expressed by the same cells at different times and places during their development. They are positioned to play different roles in migration, target penetration, and synapse formation by sensory neurons. A multiphasic model provides a morphological basis for experimental analyses of the molecules critical for the changing roles of the microenvironment in neuronal specification.

Multiple roles of neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1 adhesion molecules during sensory innervation of the otic epithelium in vitro.

To explore the role of cell adhesion molecules in the innervation of the inner ear, antibody perturbation was used on histotypic co-cultures of the ganglionic and epithelial anlagen derived from the otocyst. When unperturbed, these tissues survived and differentiated in this culture system with outgrowth of fasciculated neuronal fibers which expressed neural cell adhesion molecule and L1. The fibers exhibited target choice and penetration, then branching and spreading within the otic epithelium as individual axons. Treatment of the co-cultures, or of the ganglionic anlagen alone, with anti-neural cell adhesion molecule or anti-L1 Fab fragments produced a defasciculation of fibers but did not affect neurite outgrowth. In the co-cultures this defasciculation was accompanied by a small increase in the number of fibers found in inappropriate tissues. However, the antibodies did not prevent fiber entry to the otic epithelium. In contrast, removal of polysialic acid from neural cell adhesion molecule with endoneuraminadase-N, while producing a similar fiber defasciculation, also increased the incidence of fibers entering the epithelium. Nevertheless, once within the target tissue, the individual fibers responded to either Fab or to desialylation by spreading out more rapidly, branching, and growing farther into the epithelium. The findings suggest that fasciculation is not essential for specific sensory fibers to seek out and penetrate the appropriate target, although it may improve their tracking efficiency. Polysialic acid on neural cell adhesion molecule appears to limit initial penetration of the target epithelium. Polysialic acid as well as neural cell adhesion molecule and L1 function are involved in fiber-target interactions that influence the arborization of sensory axons within the otic epithelium.

Polysialic acid at the cell surface: biophysics in service of cell interactions and tissue plasticity.

Polysialic acid (PSA) is a long polymer of negatively-charged sialic acid associated with the neural cell adhesion molecule. PSA serves as a potent negative regulator of cell interactions via its unusual biophysical properties. During development the abundant and regulated expression of this carbohydrate is closely correlated with axon pathfinding and targeting, and with certain aspects of muscle formation. Its level can also be modulated by synaptic activity. PSA expression is more restricted in the neonatal and adult brain, being primarily associated with regions capable of morphological or physiological changes. Studies on the function of PSA studies suggest that its primary role is to promote developmentally-controlled and activity-dependent plasticity in cell interactions and thereby facilitate changes in the structure and function of the nervous system. The presence of PSA on a variety of metastatic tumor lines has also attracted the attention of oncologists, and its late appearance in evolution raises interesting questions about the phylogeny of complex tissue formation.

Removal of polysialic acid-neural cell adhesion molecule induces aberrant mossy fiber innervation and ectopic synaptogenesis in the hippocampus.

The mossy fiber axons of both the developing and adult dentate gyrus express the highly polysialylated form of neural cell adhesion molecule (NCAM) as they innervate the proximal apical dendrites of pyramidal cells in the CA3 region of the hippocampus. The present study used polysialic acid (PSA)-deficient and NCAM mutant mice to evaluate the role of PSA in mossy fiber development. The results indicate that removal of PSA by either specific enzymatic degradation or mutation of the NCAM-180 isoform that carries PSA in the brain causes an aberrant and persistent innervation of the pyramidal cell layer by mossy fibers, including excessive collateral sprouting and/or defasciculation of these processes, as well as formation of ectopic mossy fiber synaptic boutons. These results are considered in terms of two possible effects of PSA removal: an increase in the number of mossy fibers that can grow into the pyramidal cell layer and an inhibition of process retraction by formation of stable junctions including synapses. As these defects on granule cells in the adult animal and PSA-positive granule cells continue to be produced in the mature brain, the present findings may be relevant to previous studies suggesting that PSA-NCAM function is required for long-term potentiation, long-term depression, and learning behaviors associated with hippocampus.

Regulation of neural cell adhesion molecule polysialylation: evidence for nontranscriptional control and sensitivity to an intracellular pool of calcium.

The up- and downregulation of polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression on motorneurons during development is associated respectively with target innervation and synaptogenesis, and is regulated at the level of PSA enzymatic biosynthesis involving specific polysialyltransferase activity. The purpose of this study has been to describe the cellular mechanisms by which that regulation might occur. It has been found that developmental regulation of PSA synthesis by ciliary ganglion motorneurons is not reflected in the levels of polysialyltransferase-1 (PST) or sialyltransferase-X (STX) mRNA. On the other hand, PSA synthesis in both the ciliary ganglion and the developing tectum appears to be coupled to the concentration of calcium in intracellular compartments. This study documents a calcium dependence of polysialyltransferase activity in a cell-free assay over the range of 0.1-1 mM, and a rapid sensitivity of new PSA synthesis, as measured in a pulse-chase analysis of tissue explants, to calcium ionophore perturbation of intracellular calcium levels. Moreover, the relevant calcium pool appears to be within a specific intracellular compartment that is sensitive to thapsigargin and does not directly reflect the level of cytosolic calcium. Perturbation of other major second messenger systems, such as cAMP and protein kinase-dependent pathways, did not affect polysialylation in the pulse chase analysis. These results suggest that the shuttling of calcium to different pools within the cell can result in the rapid regulation of PSA synthesis in developing tissues.