Activity-dependent elimination of neuromuscular synapses

At developing neuromuscular synapses in vertebrates, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. At the same time, a single input is strengthened and maintained throughout adult life, consistently recruiting muscle fibers to contract even at rapid firing rates. Work over the last decade has led to an understanding of some of the cell biological mechanisms that underlie competition and how these culminate in synapse elimination. We discuss current ideas about how activity modulates neuromuscular synaptic competition, how competition leads to synapse loss, and how these processes are modulated by cell-cell signaling. A common feature of competition at neuromuscular as well as CNS synapses is that temporally correlated activity seems to slow or prevent competition, while uncorrelated activity seems to trigger or enhance competition. Important questions that remain to be addressed include how patterns of motor neuron activity affect synaptic strength, what is the temporal relationship between changes in synaptic strength and structure, and what cellular signals mediate synapse loss. Answers to these questions will expand our understanding of the mechanisms by which activity edits synaptic structure and function, writing permanent changes in neural circuitry.     

A model of activity-dependent formation of cerebellar microzones

According to modern views of the cerebellum in motor control, each cerebellar functional unit, or microzone, learns how to execute predictive and coordinative control, based on long-term depression of the granule cell-Purkinje cell synapses. In the present paper, in light of recent experimental and theoretical studies on synaptic elimination and cerebellar motor learning, a model of the formation of cerebellar microzones by climbing fiber synaptic elimination is proposed. It is shown that competition for an activity-dependent supply of neurotrophic factor can reproduce the spatio-temporal characteristics of climbing fiber synaptic elimination. It is further shown that when this elimination is accurate, motor coordination can be acquired in an arm reaching task. In view of the results of the present study, several predictions are proposed. Received: 19 January 1998 / Accepted in revised form: 22 April 1998     

Unequal competition between axons for neuronal targets.

Precise wiring of the nervous system depends not only on a matching between neurons and their synaptic targets, but also upon competition between neurons for particular targets. Neurons in adult leeches regenerate synaptic connections with their usual neuronal targets in the central nervous system, selecting only those targets with which they connect during embryogenesis. Thus during development axons of nociceptive (N) sensory cells make contacts on the cell bodies of certain neurons in adjacent ganglia but not upon those same types of cells in their own ganglion. After injury the N cell axons accurately regenerate contacts on the appropriate target cells. An abnormal feature observed after injury is that N cell axons sprout and grow to make contacts upon cell bodies within their own ganglion. This is a consequence of the normal innervation of those cells having been removed, thereby eliminating the source of competition. Similar competition during embryogenesis may guide the formation of selective connections.     

Target-dependent morphological segregation of Aplysia sensory outgrowth in vitro.

The adult nervous system is characterized by partial or complete morphological segregation of terminals from different afferent neurons innervating the same postsynaptic target. This segregation is thought to result, in part, from competition between the afferent terminals. To explore the role of the target cell in the spatial distribution of presynaptic inputs, the sensory neurons of Aplysia were cultured either with or without a common target motor neuron. In the presence of a common target, the outgrowth from two different sensory neurons tends to occupy separate postsynaptic regions. When cultured without a target motor neuron, processes from different sensory neurons do not segregate, but rather grow freely along one another. Thus, morphological segregation of sensory outgrowth requires interaction with a target neuron and may reflect competition between presynaptic terminals for a limited number of synaptic sites on the motor neuron, or for a postsynaptic trophic factor.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.     

Spike-based synaptic plasticity and the emergence of direction selective simple cells: mathematical analysis

In the companion paper we presented extended simulations showing that the recently observed spike-timing dependent synaptic plasticity can explain the development of simple cell direction selectivity (DS) when simultaneously modifying the synaptic strength and the degree of synaptic depression. Here we estimate the spatial shift of the simple cell receptive field (RF) induced by the long-term synaptic plasticity, and the temporal phase advance caused by the short-term synaptic depression in response to drifting grating stimuli. The analytical expressions for this spatial shift and temporal phase advance lead to a qualitative reproduction of the frequency tuning curves of non-directional and directional simple cells. In agreement with in vivo recordings, the acquired DS is strongest for test gratings with a temporal frequency around 1–4 Hz. In our model this best frequency is determined by the width of the learning function and the time course of depression, but not by the temporal frequency of the training stimuli. The analysis further reveals the instability of the initially symmetric RF, and formally explains why direction selectivity develops from a non-directional cell in a natural, directionally unbiased stimulation scenario.     

Reversing the outcome of synapse elimination at developing neuromuscular junctions in vivo: evidence for synaptic competition and its mechanism

During mammalian development, neuromuscular junctions and some other postsynaptic cells transition from multiple- to single-innervation as synaptic sites are exchanged between different axons. It is unclear whether one axon invades synaptic sites to drive off other inputs or alternatively axons expand their territory in response to sites vacated by other axons. Here we show that soon-to-be-eliminated axons rapidly reverse fate and grow to occupy vacant sites at a neuromuscular junction after laser removal of a stronger input. This reversal supports the idea that axons take over sites that were previously vacated. Indeed, during normal development we observed withdrawal followed by takeover. The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated. These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.     

Ectopic sensory neurons in mutant cockroaches compete with normal cells for central targets.

The cercus of the first instar cockroach, Periplaneta americana, bears two filiform hairs, lateral (L) and medial (M), each of which is innervated by a single sensory neuron. These project into the terminal ganglion of the CNS where they make synaptic connections with a number of ascending interneurons. We have discovered mutant animals that have more hairs on the cercus; the most typical phenotype, called "Space Invader" (SI), has an extra filiform hair in a proximo-lateral position on one of the cerci. The afferent neuron of this supernumerary hair (SIN) "invades the space" occupied by L in the CNS and makes similar synaptic connections to giant interneurons (GIs). SIN and L compete for these synaptic targets: the size of the L EPSP in a target interneuron GI3 is significantly reduced in the presence of SIN. Morphometric analysis of the L afferent in the presence or absence of SIN shows no anatomical concomitant of competition. Ablation of L afferent allows SIN to increase the size of its synaptic input to GI3. Less frequently in the mutant population, we find animals with a supernumerary medical (SuM) sensillum. Its afferent projects to the same neuropilar region as the M afferent, makes the same set of synaptic connections to GIs, and competes with M for these synaptic targets. The study of these competitive interactions between identified afferents and identified target interneurons reveals some of the dynamic processes that go on in normal development to shape the nervous system.     

Coherent Motions in Confluent Cell Monolayer Sheets

Cell migration plays a pivotal role in many physiologically important processes such as embryogenesis, wound-healing, immune defense, and cancer metastasis. Although much effort has been directed toward motility of individual cells, the mechanisms underpinning collective cell migration remain poorly understood. Here we develop a collective motility model that incorporates cell mechanics and persistent random motions of individual cells to study coherent migratory motions in epithelial-like monolayers. This model, in absence of any external chemical signals, is able to explain coordinate rotational motion seen in systems ranging from two adherent cells to multicellular assemblies. We show that the competition between the active persistent force and random polarization fluctuation is responsible for the robust rotation. Passive mechanical coupling between cells is necessary but active chemical signaling between cells is not. The predicted angular motions also depend on the geometrical shape of the underlying substrate: cells exhibit collective rotation on circular substrates, but display linear back-and-forth motion on long and narrow substrates.     

Coherent Motions in Confluent Cell Monolayer Sheets

Cell migration plays a pivotal role in many physiologically important processes such as embryogenesis, wound-healing, immune defense, and cancer metastasis. Although much effort has been directed toward motility of individual cells, the mechanisms underpinning collective cell migration remain poorly understood. Here we develop a collective motility model that incorporates cell mechanics and persistent random motions of individual cells to study coherent migratory motions in epithelial-like monolayers. This model, in absence of any external chemical signals, is able to explain coordinate rotational motion seen in systems ranging from two adherent cells to multicellular assemblies. We show that the competition between the active persistent force and random polarization fluctuation is responsible for the robust rotation. Passive mechanical coupling between cells is necessary but active chemical signaling between cells is not. The predicted angular motions also depend on the geometrical shape of the underlying substrate: cells exhibit collective rotation on circular substrates, but display linear back-and-forth motion on long and narrow substrates.     

海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.     

Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that alpha and omega termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Development of feedforward receptive field structure of a simple cell and its contribution to the orientation selectivity: a modeling study

Recent experimental studies of hetero-synaptic interactions in various systems have shown the role of signaling in the plasticity, challenging the conventional understanding of Hebb's rule. It has also been found that activity plays a major role in plasticity, with neurotrophins acting as molecular signals translating activity into structural changes. Furthermore, role of synaptic efficacy in biasing the outcome of competition has also been revealed recently. Motivated by these experimental findings we present a model for the development of simple cell receptive field structure based on the competitive hetero-synaptic interactions for neurotrophins combined with cooperative hetero-synaptic interactions in the spatial domain. We find that with proper balance in competition and cooperation, the inputs from two populations (ON/OFF) of LGN cells segregate starting from the homogeneous state. We obtain segregated ON and OFF regions in simple cell receptive field. Our modeling study supports the experimental findings, suggesting the role of synaptic efficacy and the role of spatial signaling. We find that using this model we obtain simple cell RF, even for positively correlated activity of ON/OFF cells. We also compare different mechanism of finding the response of cortical cell and study their possible role in the sharpening of orientation selectivity. We find that degree of selectivity improvement in individual cells varies from case to case depending upon the structure of RF field and type of sharpening mechanism.     

A mathematical model of activity–dependent,anatomical segregation induced by competition for neurotrophic support

Mathematical or computational models of activity–dependent neural competition typically impose competition in anatomically fixed networks by the use of synaptic normalisation, for which there is very little experimental support. Recent experimental evidence, however, strongly implicates neurotrophic factors in neural plasticity and competition, in addition to their well–known potent effects on neurite outgrowth and synaptogenesis. We therefore present a simple, mathematical model of anatomical segregation induced by activity–dependent competition for a limited supply of a neurotrophic factor provided by target cells to afferents. We extract the behaviour of the model in various regimes, in which the neurotrophic factor is either in critical supply or in abundant supply, by a combination of analytical and numerical methods, and study the effects of correlations in afferent inputs on competition. We apply the model to three different systems: ocular dominance column formation; elimination of polyneuronal innervation at the vertebrate neuromuscular junction; trigeminal brain stem whisker–related structure formation. Several classes of related predictions emerge, including the prediction that kittens reared with strabismus should require a higher concentration of neurotrophic factor infusion into their primary visual cortex than normally reared cats in order to induce the anatomical desegregation of ocular dominance columns. We also speculate on the mechanisms of support of inhibitory rather than excitatory neurons, and suggest the existence of a separate, Cl–mediated activity–dependent pathway for their neurotrophic support. Received: 17 May 1996 / Accepted in revised form: 27 July 1996     

Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that α and ω termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.     

Loss of correlated motor neuron activity during synaptic competition at developing neuromuscular synapses

During late stages of neural development, synaptic circuitry is edited by neural activity. At neuromuscular synapses, the transition from multiple to single innervation is modulated by the relative pattern of activity among inputs competing for innervation of the same muscle fiber. While experimental perturbations of activity result in marked changes in the timing of neuromuscular synaptic competition, little is known about the patterns of activity present during normal development. Here, we report the temporal patterning of motor unit activity in the soleus muscle of awake, behaving neonatal mice, and that patterning is modulated by gap-junctional coupling. Our work suggests that neuromuscular synaptic competition is modulated by surprisingly low levels of activity and may be triggered by the disappearance of temporally correlated activity among inputs competing for innervation of the same muscle fiber.     

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine‐rich repeat transmembrane proteins in hippocampal neurons

Leucine‐rich repeat transmembrane proteins (LRRTMs) are single‐spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post‐synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55–56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1–4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1–2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of ‐16 to ‐13 from the C‐terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering‐deficient LRRTM2 mutant lost the ability to promote the accumulation of post‐synaptic density protein‐95 (PSD‐95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post‐synaptic molecular assembly and maturation.