Morphological characteristics of various segments of local connections in the rat somatosensory cortex

Pyramidal, aspinous, sparsely-spinous bipolar and multipolar neurons of the rat sensomotor cerebral cortex, impregnated after Golgi method, have been studied at an electron microscopical level. The ultrastructural characteristics of the pyramidal neurons differs from that of the nonpyramidal cells. Distribution of various synaptic contacts on the cellular surface and cortical postsynaptic targets of the axonal arborizations of the neurons are revealed. On the body of the pyramidal cells only symmetrical synapses exist, on large dendritic trunks symmetrical synapses prevail, on the spines and the terminal dendritic branches assymetrical synapses mainly predominate. Axonal collateralies of the pyramidal cells form asymmetrical synapses on the spines, small and middle dendrites. There are more axo-somatic synapses on the bodies of the nonpyramidal neurons than on the pyramidal cells, among them both symmetrical and asymmetrical types of the synapses occur. On the trunks and small dendrites of the nonpyramidal cells both types of synaptic contacts are revealed. In the distal direction of the dendrites the number of the asymmetrical synapses becomes predominating. Axons of the bipolar cells form asymmetrical synapses on the spines, small and middle dendrites. Axons of the multipolar cells form symmetrical synapses on the dendrites and the dendritic trunks of the nondifferentiated cells. Differences in the distribution character of the synaptic inlets and various postsynaptic targets of the axonal systems in the cells assume various functional role of the identified neurons.     

In vivo time-lapse imaging and serial section electron microscopy reveal developmental synaptic rearrangements

Dendrites, axons, and synapses are dynamic during circuit development; however, changes in microcircuit connections as branches stabilize have not been directly demonstrated. By combining in?vivo time-lapse imaging of Xenopus tectal neurons with electron microscope reconstructions of imaged neurons, we report the distribution and ultrastructure of synapses on individual vertebrate neurons and relate these synaptic properties to dynamics in dendritic and axonal arbor structure over hours or?days of imaging. Dynamic dendrites have a high density of immature synapses, whereas stable dendrites have sparser, mature synapses. Axons initiate contacts from multisynapse boutons on stable branches. Connections are refined by decreasing convergence from multiple inputs to postsynaptic dendrites and by decreasing divergence from multisynapse boutons to postsynaptic sites. Visual deprivation or NMDAR antagonists decreased synapse maturation and elimination, suggesting that coactive input activity promotes microcircuit development by concurrently regulating synapse elimination and maturation of remaining contacts.     

Convergence among Non-Sister Dendritic Branches: An Activity-Controlled Mean to Strengthen Network Connectivity

The manner by which axons distribute synaptic connections along dendrites remains a fundamental unresolved issue in neuronal development and physiology. We found in vitro and in vivo indications that dendrites determine the density, location and strength of their synaptic inputs by controlling the distance of their branches from those of their neighbors. Such control occurs through collective branch convergence, a behavior promoted by AMPA and NMDA glutamate receptor activity. At hubs of convergence sites, the incidence of axo-dendritic contacts as well as clustering levels, pre- and post-synaptic protein content and secretion capacity of synaptic connections are higher than found elsewhere. This coupling between synaptic distribution and the pattern of dendritic overlapping results in ‘Economical Small World Network’, a network configuration that enables single axons to innervate multiple and remote dendrites using short wiring lengths. Thus, activity-mediated regulation of the proximity among dendritic branches serves to pattern and strengthen neuronal connectivity.     

A phenomenological theory of spatially structured local synaptic connectivity

The structure of local synaptic circuits is the key to understanding cortical function and how neuronal functional modules such as cortical columns are formed. The central problem in deciphering cortical microcircuits is the quantification of synaptic connectivity between neuron pairs. I present a theoretical model that accounts for the axon and dendrite morphologies of pre- and postsynaptic cells and provides the average number of synaptic contacts formed between them as a function of their relative locations in three-dimensional space. An important aspect of the current approach is the representation of a complex structure of an axonal/dendritic arbor as a superposition of basic structures—synaptic clouds. Each cloud has three structural parameters that can be directly estimated from two-dimensional drawings of the underlying arbor. Using empirical data available in literature, I applied this theory to three morphologically different types of cell pairs. I found that, within a wide range of cell separations, the theory is in very good agreement with empirical data on (i) axonal–dendritic contacts of pyramidal cells and (ii) somatic synapses formed by the axons of inhibitory interneurons. Since for many types of neurons plane arborization drawings are available from literature, this theory can provide a practical means for quantitatively deriving local synaptic circuits based on the actual observed densities of specific types of neurons and their morphologies. It can also have significant implications for computational models of cortical networks by making it possible to wire up simulated neural networks in a realistic fashion.     

Differential effects of NgCAM and N-cadherin on the development of axons and dendrites by cultured hippocampal neurons

A fundamental step in neuronal development is the acquisition of a polarized form, with distinct axons and dendrites. Although the ability to develop a polarized form appears to be largely an intrinsic property of neurons, it can be influenced by environmental cues. For example, in cell cultures substrate and diffusible factors can enhance and orient axonal development. In this study we examine the effects of growth on each of two cell adhesion molecules (CAMs), NgCAM and N-cadherin, on the development of polarity by cultured hippocampal neurons. We find that although the same pattern of development occurs on control substrates and the CAMs, the CAMs greatly accelerate the rate and extent of development of axons—axons form sooner and grow longer on the CAMs than on the control substrate. In contrast, the CAMs have opposite effects on dendritic development—N-cadherin enhances, but NgCAM reduces dendritic growth compared to control. These results provide further evidence that the development of polarity is largely determined by a cell-autonomous program, but that environmental cues can independently regulate axonal and dendritic growth.     

Dendritic Branch Intersections Are Structurally Regulated Targets for Efficient Axonal Wiring and Synaptic Clustering

Synaptic clustering on dendritic branches enhances plasticity, input integration and neuronal firing. However, the mechanisms guiding axons to cluster synapses at appropriate sites along dendritic branches are poorly understood. We searched for such a mechanism by investigating the structural overlap between dendritic branches and axons in a simplified model of neuronal networks - the hippocampal cell culture. Using newly developed software, we converted images of meshes of overlapping axonal and dendrites into topological maps of intersections, enabling quantitative study of overlapping neuritic geometry at the resolution of single dendritic branch-to-branch and axon-to-branch crossings. Among dendro-dendritic crossing configurations, it was revealed that the orientations through which dendritic branches cross is a regulated attribute. While crossing angle distribution among branches thinner than 1 µm appeared to be random, dendritic branches 1 µm or wider showed a preference for crossing each other at angle ranges of either 50°–70° or 80°–90°. It was then found that the dendro-dendritic crossings themselves, as well as their selective angles, both affected the path of axonal growth. Axons displayed 4 fold stronger tendency to traverse within 2 µm of dendro-dendritic intersections than at farther distances, probably to minimize wiring length. Moreover, almost 70% of the 50°–70° dendro-denritic crossings were traversed by axons from the obtuse angle’s zone, whereas only 15% traversed through the acute angle’s zone. By contrast, axons showed no orientation restriction when traversing 80°–90° crossings. When such traverse behavior was repeated by many axons, they converged in the vicinity of dendro-dendritic intersections, thereby clustering their synaptic connections. Thus, the vicinity of dendritic branch-to-branch crossings appears to be a regulated structure used by axons as a target for efficient wiring and as a preferred site for synaptic clustering. This synaptic clustering mechanism may enhance synaptic co-activity and plasticity.     

Class-specific features of neuronal wiring

Brain function relies on specificity of synaptic connectivity patterns among different classes of neurons. Yet, the substrates of specificity in complex neuropil remain largely unknown. We search for imprints of specificity in the layout of axonal and dendritic arbors from the rat neocortex. An analysis of 3D reconstructions of pairs consisting of pyramidal cells (PCs) and GABAergic interneurons (GIs) revealed that the layout of GI axons is specific. This specificity is manifested in a relatively high tortuosity, small branch length of these axons, and correlations of their trajectories with the positions of postsynaptic neuron dendrites. Axons of PCs show no such specificity, usually taking a relatively straight course through neuropil. However, wiring patterns among PCs hold a large potential for circuit remodeling and specificity through growth and retraction of dendritic spines. Our results define distinct class-specific rules in establishing synaptic connectivity, which could be crucial in formulating a canonical cortical circuit.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Developmental mechanisms that regulate retinal ganglion cell dendritic morphology

One of the fundamental features of retinal ganglion cells (RGCs) is that dendrites of individual RGCs are confined to one or a few narrow strata within the inner plexiform layer (IPL), and each RGC synapses only with a small group of presynaptic bipolar and amacrine cells with axons/dendrites ramified in the same strata to process distinct visual features. The underlying mechanisms which control the development of this laminar-restricted distribution pattern of RGC dendrites have been extensively studied, and it is still an open question whether the dendritic pattern of RGCs is determined by molecular cues or by activity-dependent refinement. Accumulating evidence suggests that both molecular cues and activity-dependent refinement might regulate RGC dendrites in a cell subtype-specific manner. However, identification of morphological subtypes of RGCs before they have achieved their mature dendritic pattern is a major challenge in the study of RGC dendritic development. This problem is now being circumvented through the use of molecular markers in genetically engineered mouse lines to identify RGC subsets early during development. Another unanswered fundamental question in the study of activity-dependent refinement of RGC dendrites is how changes in synaptic activity lead to the changes in dendritic morphology. Recent studies have started to shed light on the molecular basis of activity-dependent dendritic refinement of RGCs by showing that some molecular cascades control the cytoskeleton reorganization of RGCs.     

Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites

Secreted semaphorins act as guidance cues in the developing nervous system and may have additional functions in mature neurons. How semaphorins are transported and secreted by neurons is poorly understood. We find that endogenous semaphorin 3A (Sema3A) displays a punctate distribution in axons and dendrites of cultured cortical neurons. GFP-Sema3A shows a similar distribution and co-localizes with secretory vesicle cargo proteins. Live-cell imaging reveals highly dynamic trafficking of GFP-Sema3A vesicles with distinct properties in axons and dendrites regarding directionality, velocity, mobility and pausing time. In axons, most GFP-Sema3A vesicles move fast without interruption, almost exclusively in the anterograde direction, while in dendrites many GFP-Sema3A vesicles are stationary and move equally frequent in both directions. Disruption of microtubules, but not of actin filaments, significantly impairs GFP-Sema3A transport. Interestingly, depolarization induces a reversible arrest of axonal transport of GFP-Sema3A vesicles but has little effect on dendritic transport. Conversely, action potential blockade using tetrodotoxin (TTX) accelerates axonal transport, but not dendritic transport. These data indicate that axons and dendrites regulate trafficking of Sema3A and probably other secretory vesicles in distinct ways, with axons specializing in fast, uninterrupted, anterograde transport. Furthermore, neuronal activity regulates secretory vesicle trafficking in axons by a depolarization-evoked trafficking arrest.     

Synapse formation proceeds independently of dendritic elongation in cultured hippocampal neurons

In central neurons, dendritic differentiation begins well after axonal elongation and is accompanied by the compartmentation of the microtubule-associated protein 2 (MAP2) in the somatodendritic domain. Whether MAP2 plays a role in the morphological and functional maturation of dendrites remains an open question and is the focus of this study. Cultured hippocampal neurons depleted of MAP2 by means of antisense oligonucleotides failed to elongate their dendrites. On the other hand, MAP2-depleted neurons were capable of receiving synapses within the same time course as their control counterparts. However, both the number of synapses per cell and the synaptic density were markedly reduced in neurons in which dendritic elongation has been impaired. Taken collectively, these results suggest that the expression of MAP2 is required for the morphological differentiation of dendrites. Dendritic elongation, however, is not a prerequisite for synapse formation in cultured hippocampal neurons.     

Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.     

A Morpho-Density Approach to Estimating Neural Connectivity

Neuronal signal integration and information processing in cortical neuronal networks critically depend on the organization of synaptic connectivity. Because of the challenges involved in measuring a large number of neurons, synaptic connectivity is difficult to determine experimentally. Current computational methods for estimating connectivity typically rely on the juxtaposition of experimentally available neurons and applying mathematical techniques to compute estimates of neural connectivity. However, since the number of available neurons is very limited, these connectivity estimates may be subject to large uncertainties. We use a morpho-density field approach applied to a vast ensemble of model-generated neurons. A morpho-density field (MDF) describes the distribution of neural mass in the space around the neural soma. The estimated axonal and dendritic MDFs are derived from 100,000 model neurons that are generated by a stochastic phenomenological model of neurite outgrowth. These MDFs are then used to estimate the connectivity between pairs of neurons as a function of their inter-soma displacement. Compared with other density-field methods, our approach to estimating synaptic connectivity uses fewer restricting assumptions and produces connectivity estimates with a lower standard deviation. An important requirement is that the model-generated neurons reflect accurately the morphology and variation in morphology of the experimental neurons used for optimizing the model parameters. As such, the method remains subject to the uncertainties caused by the limited number of neurons in the experimental data set and by the quality of the model and the assumptions used in creating the MDFs and in calculating estimating connectivity. In summary, MDFs are a powerful tool for visualizing the spatial distribution of axonal and dendritic densities, for estimating the number of potential synapses between neurons with low standard deviation, and for obtaining a greater understanding of the relationship between neural morphology and network connectivity.     

Fast recruitment of recurrent inhibition in the cat visual cortex

Neurons of the same column in L4 of the cat visual cortex are likely to share the same sensory input from the same region of the visual field. Using visually-guided patch clamp recordings we investigated the biophysical properties of the synapses of neighboring layer 4 neurons. We recorded synaptic connections between all types of excitatory and inhibitory neurons in L4. The E-E, E-I, and I-E connections had moderate CVs and failure rates. However, E-I connections had larger amplitudes, faster rise-times, and shorter latencies. Identification of the sites of putative synaptic contacts together with compartmental simulations on 3D reconstructed cells, suggested that E-I synapses tended to be located on proximal dendritic branches, which would explain their larger EPSP amplitudes and faster kinetics. Excitatory and inhibitory synapses were located at the same distance on distal dendrites of excitatory neurons. We hypothesize that this co-localization and the fast recruitment of local inhibition provides an efficient means of modulating excitation in a precisely timed way.     

Cerebellar Golgi cell models predict dendritic processing and mechanisms of synaptic plasticity

The Golgi cells are the main inhibitory interneurons of the cerebellar granular layer. Although recent works have highlighted the complexity of their dendritic organization and synaptic inputs, the mechanisms through which these neurons integrate complex input patterns remained unknown. Here we have used 8 detailed morphological reconstructions to develop multicompartmental models of Golgi cells, in which Na, Ca, and K channels were distributed along dendrites, soma, axonal initial segment and axon. The models faithfully reproduced a rich pattern of electrophysiological and pharmacological properties and predicted the operating mechanisms of these neurons. Basal dendrites turned out to be more tightly electrically coupled to the axon initial segment than apical dendrites. During synaptic transmission, parallel fibers caused slow Ca-dependent depolarizations in apical dendrites that boosted the axon initial segment encoder and Na-spike backpropagation into basal dendrites, while inhibitory synapses effectively shunted backpropagating currents. This oriented dendritic processing set up a coincidence detector controlling voltage-dependent NMDA receptor unblock in basal dendrites, which, by regulating local calcium influx, may provide the basis for spike-timing dependent plasticity anticipated by theory.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

A cooperative switch determines the sign of synaptic plasticity in distal dendrites of neocortical pyramidal neurons

Pyramidal neurons in the cerebral cortex span multiple cortical layers. How the excitable properties of pyramidal neuron dendrites allow these neurons to both integrate activity and store associations between different layers is not well understood, but is thought to rely in part on dendritic backpropagation of action potentials. Here we demonstrate that the sign of synaptic plasticity in neocortical pyramidal neurons is regulated by the spread of the backpropagating action potential to the synapse. This creates a progressive gradient between LTP and LTD as the distance of the synaptic contacts from the soma increases. At distal synapses, cooperative synaptic input or dendritic depolarization can switch plasticity between LTD and LTP by boosting backpropagation of action potentials. This activity-dependent switch provides a mechanism for associative learning across different neocortical layers that process distinct types of information.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.