Independently Outgrowing Neurons and Geometry-Based Synapse Formation Produce Networks with Realistic Synaptic Connectivity

Neuronal signal integration and information processing in cortical networks critically depend on the organization of synaptic connectivity. During development, neurons can form synaptic connections when their axonal and dendritic arborizations come within close proximity of each other. Although many signaling cues are thought to be involved in guiding neuronal extensions, the extent to which accidental appositions between axons and dendrites can already account for synaptic connectivity remains unclear. To investigate this, we generated a local network of cortical L2/3 neurons that grew out independently of each other and that were not guided by any extracellular cues. Synapses were formed when axonal and dendritic branches came by chance within a threshold distance of each other. Despite the absence of guidance cues, we found that the emerging synaptic connectivity showed a good agreement with available experimental data on spatial locations of synapses on dendrites and axons, number of synapses by which neurons are connected, connection probability between neurons, distance between connected neurons, and pattern of synaptic connectivity. The connectivity pattern had a small-world topology but was not scale free. Together, our results suggest that baseline synaptic connectivity in local cortical circuits may largely result from accidentally overlapping axonal and dendritic branches of independently outgrowing neurons.     

A quantitative analysis of the local connectivity between pyramidal neurons in layers 2/3 of the rat visual cortex

This study provides a detailed quantitative estimate for local synaptic connectivity between neocortical pyramidal neurons. A new way of obtaining such an estimate is presented. In acute slices of the rat visual cortex, four layer 2 and four layer 3 pyramidal neurons were intracellularly injected with biocytin. Axonal and dendritic arborizations were three-dimensionally reconstructed with the aid of a computer-based camera lucida system. In a computer experiment, pairs of pre- and postsynaptic neurons were formed and potential synaptic contacts were calculated. For each pair, the calculations were carried out for a whole range of distances (0 to 500 μm) between the presynaptic and the postsynaptic neuron, in order to estimate cortical connectivity as a function of the spatial separation of neurons. It was also differentiated whether neurons were situated in the same or in different cortical layers. The data thus obtained was used to compute connection probabilities, the average number of contacts between neurons, the frequency of specific numbers of contacts and the total number of contacts a dendritic tree receives from the surrounding cortical volume. Connection probabilities ranged from 50% to 80% for directly adjacent neurons and from 0% to 15% for neurons 500 μm apart. In many cases, connections were mediated by one contact only. However, close neighbors made on average up to 3 contacts with each other. The question as to whether the method employed in this study yields a realistic estimate of synaptic connectivity is discussed. It is argued that the results can be used as a detailed blueprint for building artificial neural networks with a cortex-like architecture. Received: 30 March 1999 / Accepted in revised form: 5 August 1999     

Synaptic relationships between GABA-immunoreactive neurons and an identified uniglomerular projection neuron in the antennal lobe of Periplaneta americana: a double-labeling electron microscopic study

Summary Two types of central neurons in the antennal lobe of the American cockroach Periplaneta americana were labeled with a combination of two specific markers. Their synaptic contacts were characterized and their distribution on the neurons examined. A uniglomerular pheromone-sensitive projection neuron with dendritic arbor in the male-specific macroglomerulus (attractant neuron) was characterized physiologically by intracellular recording and then filled with biocytin, which was converted to a marker for this individual neuron by a preembedding procedure. In a postembedding procedure local, multiglomerular interneurons were marked by immunogold labeling of GABA. Two kinds of synaptic contacts were found on the attractant neuron. (i) Input synapses from GABA-immunoreactive profiles. There were many of these, which (together with results of previous studies) suggests that local interneurons mediate polysynaptic transmission from antennal receptor fibers to the projection neuron. (ii) Output synapses onto GABA-immunoreactive profiles and onto non-identified neurons. These contacts indicate that signals generated by the projection neurons in a given glomerulus are passed back to multiglomerular interneurons and hence are also transmitted to other glomeruli.     

Synaptic relationships between GABA-immunoreactive neurons and an identified uniglomerular projection neuron in the antennal lobe of Periplaneta americana: a double-labeling electron microscopic study

Two types of central neurons in the antennal lobe of the American cockroach Periplaneta americana were labeled with a combination of two specific markers. Their synaptic contacts were characterized and their distribution on the neurons examined. A uniglomerular pheromone-sensitive projection neuron with dendritic arbor in the male-specific macroglomerulus (attractant neuron) was characterized physiologically by intracellular recording and then filled with biocytin, which was converted to a marker for this individual neuron by a preembedding procedure. In a postembedding procedure local, multiglomerular interneurons were marked by immunogold labeling of GABA. Two kinds of synaptic contacts were found on the attractant neuron. (i) Input synapses from GABA-immunoreactive profiles. There were many of these, which (together with results of previous studies) suggests that local interneurons mediate polysynaptic transmission from antennal receptor fibers to the projection neuron. (ii) Output synapses onto GABA-immunoreactive profiles and onto non-identified neurons. These contacts indicate that signals generated by the projection neurons in a given glomerulus are passed back to multiglomerular interneurons and hence are also transmitted to other glomeruli.     

Location-dependent effects of inhibition on local spiking in pyramidal neuron dendrites

Cortical computations are critically dependent on interactions between pyramidal neurons (PNs) and a menagerie of inhibitory interneuron types. A key feature distinguishing interneuron types is the spatial distribution of their synaptic contacts onto PNs, but the location-dependent effects of inhibition are mostly unknown, especially under conditions involving active dendritic responses. We studied the effect of somatic vs. dendritic inhibition on local spike generation in basal dendrites of layer 5 PNs both in neocortical slices and in simple and detailed compartmental models, with equivalent results: somatic inhibition divisively suppressed the amplitude of dendritic spikes recorded at the soma while minimally affecting dendritic spike thresholds. In contrast, distal dendritic inhibition raised dendritic spike thresholds while minimally affecting their amplitudes. On-the-path dendritic inhibition modulated both the gain and threshold of dendritic spikes depending on its distance from the spike initiation zone. Our findings suggest that cortical circuits could assign different mixtures of gain vs. threshold inhibition to different neural pathways, and thus tailor their local computations, by managing their relative activation of soma- vs. dendrite-targeting interneurons.     

Structural homeostasis: compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input

As the nervous system develops, there is an inherent variability in the connections formed between differentiating neurons. Despite this variability, neural circuits form that are functional and remarkably robust. One way in which neurons deal with variability in their inputs is through compensatory, homeostatic changes in their electrical properties. Here, we show that neurons also make compensatory adjustments to their structure. We analysed the development of dendrites on an identified central neuron (aCC) in the late Drosophila embryo at the stage when it receives its first connections and first becomes electrically active. At the same time, we charted the distribution of presynaptic sites on the developing postsynaptic arbor. Genetic manipulations of the presynaptic partners demonstrate that the postsynaptic dendritic arbor adjusts its growth to compensate for changes in the activity and density of synaptic sites. Blocking the synthesis or evoked release of presynaptic neurotransmitter results in greater dendritic extension. Conversely, an increase in the density of presynaptic release sites induces a reduction in the extent of the dendritic arbor. These growth adjustments occur locally in the arbor and are the result of the promotion or inhibition of growth of neurites in the proximity of presynaptic sites. We provide evidence that suggest a role for the postsynaptic activity state of protein kinase A in mediating this structural adjustment, which modifies dendritic growth in response to synaptic activity. These findings suggest that the dendritic arbor, at least during early stages of connectivity, behaves as a homeostatic device that adjusts its size and geometry to the level and the distribution of input received. The growing arbor thus counterbalances naturally occurring variations in synaptic density and activity so as to ensure that an appropriate level of input is achieved.     

Preferential control of basal dendritic protrusions by EphB2

The flow of information between neurons in many neural circuits is controlled by a highly specialized site of cell-cell contact known as a synapse. A number of molecules have been identified that are involved in central nervous system synapse development, but knowledge is limited regarding whether these cues direct organization of specific synapse types or on particular regions of individual neurons. Glutamate is the primary excitatory neurotransmitter in the brain, and the majority of glutamatergic synapses occur on mushroom-shaped protrusions called dendritic spines. Changes in the morphology of these structures are associated with long-lasting modulation of synaptic strength thought to underlie learning and memory, and can be abnormal in neuropsychiatric disease. Here, we use rat cortical slice cultures to examine how a previously-described synaptogenic molecule, the EphB2 receptor tyrosine kinase, regulates dendritic protrusion morphology in specific regions of the dendritic arbor in cortical pyramidal neurons. We find that alterations in EphB2 signaling can bidirectionally control protrusion length, and knockdown of EphB2 expression levels reduces the number of dendritic spines and filopodia. Expression of wild-type or dominant negative EphB2 reveals that EphB2 preferentially regulates dendritic protrusion structure in basal dendrites. Our findings suggest that EphB2 may act to specify synapse formation in a particular subcellular region of cortical pyramidal neurons.     

Reconstructing the three-dimensional GABAergic microcircuit of the striatum

A system's wiring constrains its dynamics, yet modelling of neural structures often overlooks the specific networks formed by their neurons. We developed an approach for constructing anatomically realistic networks and reconstructed the GABAergic microcircuit formed by the medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs) of the adult rat striatum. We grew dendrite and axon models for these neurons and extracted probabilities for the presence of these neurites as a function of distance from the soma. From these, we found the probabilities of intersection between the neurites of two neurons given their inter-somatic distance, and used these to construct three-dimensional striatal networks. The MSN dendrite models predicted that half of all dendritic spines are within 100μm of the soma. The constructed networks predict distributions of gap junctions between FSI dendrites, synaptic contacts between MSNs, and synaptic inputs from FSIs to MSNs that are consistent with current estimates. The models predict that to achieve this, FSIs should be at most 1% of the striatal population. They also show that the striatum is sparsely connected: FSI-MSN and MSN-MSN contacts respectively form 7% and 1.7% of all possible connections. The models predict two striking network properties: the dominant GABAergic input to a MSN arises from neurons with somas at the edge of its dendritic field; and FSIs are inter-connected on two different spatial scales: locally by gap junctions and distally by synapses. We show that both properties influence striatal dynamics: the most potent inhibition of a MSN arises from a region of striatum at the edge of its dendritic field; and the combination of local gap junction and distal synaptic networks between FSIs sets a robust input-output regime for the MSN population. Our models thus intimately link striatal micro-anatomy to its dynamics, providing a biologically grounded platform for further study.     

Effects of Neural Morphology and Input Distribution on Synaptic Processing by Global and Focal NMDA-Spikes

Cortical neurons can respond to glutamatergic stimulation with regenerative N-Methyl-D-aspartic acid (NMDA)-spikes. NMDA-spikes were initially thought to depend on clustered synaptic activation. Recent work had shown however a new variety of a global NMDA-spike, which can be generated by randomly distributed inputs. Very little is known about the factors that influence the generation of these global NMDA-spikes, as well the potentially distinct rules of synaptic integration and the computational significance conferred by the two types of NMDA-spikes. Here I show that the input resistance (R_IN) plays a major role in influencing spike initiation; while the classical, focal NMDA-spike depended upon the local (dendritic) R_IN, the threshold of global NMDA-spike generation was set by the somatic R_IN. As cellular morphology can exert a large influence on R_IN, morphologically distinct neuron types can have dissimilar rules for NMDA-spikes generation. For example, cortical neurons in superficial layers were found to be generally prone to global NMDA-spike generation. In contrast, electric properties of cortical layer 5b cells clearly favor focal NMDA-spikes. These differences can translate into diverse synaptic integration rules for the different classes of cortical cells; simulated superficial layers neurons were found to exhibit strong synaptic interactions between different dendritic branches, giving rise to a single integrative compartment mediated by the global NMDA-spike. In these cells, efficiency of postsynaptic activation was relatively little dependent on synaptic distribution. By contrast, layer 5b neurons were capable of true multi-unit computation involving independent integrative compartments formed by clustered synaptic input which could trigger focal NMDA-spikes. In a sharp contrast to superficial layers neurons, randomly distributed synaptic inputs were not very effective in driving firing the layer 5b cells, indicating a possibility for different computation performed by these important cortical neurons.     

Dendritic spines and distributed circuits

Dendritic spines receive most excitatory connections in pyramidal cells and many other principal neurons. But why do neurons use spines, when they could accommodate excitatory contacts directly on their dendritic shafts? One suggestion is that spines serve to connect with passing axons, thus increasing the connectivity of the dendrites. Another hypothesis is that spines are biochemical compartments that enable input-specific synaptic plasticity. A third possibility is that spines have an electrical role, filtering synaptic potentials and electrically isolating inputs from each other. In this review, I argue that, when viewed from the perspective of the circuit function, these three functions dovetail with one another to achieve a single overarching goal: to implement a distributed circuit with widespread connectivity. Spines would endow these circuits with nonsaturating, linear integration and input-specific learning rules, which would enable them to function as neural networks, with emergent encoding and processing of information.     

Protein synthesis and processing in cytoplasmic microdomains beneath postsynaptic sites on CNS neurons

Recent studies have shown that protein synthetic machinery consisting of polyribosomes and associated membranous cisterns is selectively localized beneath synaptic sites on neurons. In the present paper, the role of this machinery in neuronal function will be considered. We will: 1. Summarize the studies that characterize the polyribosomes and define their associations with membranous cisterns. Taken together, these observations suggest the existence of a system for the synthesis and posttranslational processing of proteins at individual synaptic sites; 2. Review the evidence that the protein synthetic machinery is particularly prominent during the initial formation of synaptic contacts (during early development), and during lesion-induced synaptogenesis in mature animals. These observations have led to the hypothesis that the polyribosomes produce proteins that play a role in the formation of the synaptic junction; 3. Review evidence that supports the hypothesis that there is a local synthesis of protein within dendrites, as well as local glycosylation; 4. Describe the evidence suggesting that at least some of the protein constituents of the synaptic junction itself are synthesized locally; and 5. Describe our studies that reveal a mechanism for selective dendritic transport of RNA; this transport mechanism permits the delivery of RNA to postsynaptic sites throughout the dendritic arbor. We will advance the hypothesis that neurons position protein synthetic machinery together with the mRNA's that are appropriate for particular synapses beneath synaptic contact regions. At the synaptic site, this machinery could then direct the synthesis of particular proteins that are critical for synapse formation or maintenance. The positioning of protein synthetic machinery at postsynaptic sites permits a rapid local regulation of the production of key proteins by events at individual synapses.     

Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex

A key feature of the mammalian brain is its capacity to adapt in response to experience, in part by remodeling of synaptic connections between neurons. Excitatory synapse rearrangements have been monitored in vivo by observation of dendritic spine dynamics, but lack of a vital marker for inhibitory synapses has precluded their observation. Here, we simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbor of pyramidal neurons in the adult mammalian cortex using large-volume, high-resolution dual-color two-photon microscopy. We find that inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience. Further, we find inhibitory synapse and dendritic spine remodeling to be spatially clustered and that clustering is influenced by sensory input. Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.     

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.     

Homer proteins shape Xenopus optic tectal cell dendritic arbor development in vivo

Considerable evidence suggests that the Homer family of scaffolding proteins contributes to synaptic organization and function. We investigated the role of both Homer 1b, the constitutively expressed, and developmentally regulated form of Homer, and Homer 1a, the activity-induced immediate early gene, in dendritic arbor elaboration and synaptic function of developing Xenopus optic tectal neurons. We expressed exogenous Homer 1a or Homer 1b in developing Xenopus tectal neurons. By collecting in vivo time lapse images of individual, EGFP-labeled and Homer-expressing neurons over 3 days, we found that Homer 1b leads to a significant decrease in dendritic arbor growth rate and arbor size. Synaptic transmission was also altered in developing neurons transfected with Homer 1b. Cells expressing exogenous Homer 1b over 3 days had a significantly greater AMPA to NMDA ratios, and increased AMPA mEPSC frequency. These data suggest that increasing Homer 1b expression increases excitatory synaptic inputs, increases synaptic maturation, and slows dendritic arbor growth rate. Exogenous Homer 1a expression increases AMPA mEPSC frequency, but did not significantly affect tectal cell dendritic arbor development. Changes in the ratio of Homer 1a to Homer 1b may signal the neuron that overall activity levels in the cell have changed, and this in turn could affect protein interactions at the synapse, synaptic transmission, and structural development of the dendritic arbor.     

Dynamics underlying synaptic gain between pairs of cortical pyramidal neurons

Changes in connectivity between pairs of neurons can serve as a substrate for information storage and for experience-dependent changes in neuronal circuitry. Early in development, synaptic contacts form and break, but how these dynamics influence the connectivity between pairs of neurons is not known. Here we used time-lapse imaging to examine the synaptic interactions between pairs of cultured cortical pyramidal neurons, and found that the axon-dendrite contacts between each neuronal pair were composed of both a relatively stable and a more labile population. Under basal conditions, loss and gain of contacts within this labile population was well balanced and there was little net change in connectivity. Selectively increasing the levels of activated CaMKII in the postsynaptic neuron increased connectivity between pairs of neurons by increasing the rate of gain of new contacts without affecting the probability of contact loss, or the proportion of stable and labile contacts, and this increase required Calcium/calmodulin binding to CaMKII. Our data suggest that activating CaMKII can increase synaptic connectivity through a CaM-dependent increase in contact formation, followed by stabilization of a constant fraction of new contacts.     

A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia

Synapse elimination is an important process underlying the establishment of functional neuronal networks during development. Here, we tested the idea that neurons select among potential synaptic partners already during initial contact formation between dendritic filopodia and axons-well before mature synapses are established. We show that filopodia frequently make contact with axons, and while some contacts are selectively stabilized, many are short-lived. More specifically, we demonstrate that contacts with a certain population of GABAergic axons never get stabilized, indicating that filopodia already early on select between different types of axons. Local dendritic calcium transients that are independent of glutamate occur within seconds after contact formation, and their frequency is high where contacts become stabilized and low at short-lived contacts. Thus, filopodia are capable of choosing between potential synaptic partners well before a mature synapse is established.     

Spike-Timing Dependence of Structural Plasticity Explains Cooperative Synapse Formation in the Neocortex

Structural plasticity governs the long-term development of synaptic connections in the neocortex. While the underlying processes at the synapses are not fully understood, there is strong evidence that a process of random, independent formation and pruning of excitatory synapses can be ruled out. Instead, there must be some cooperation between the synaptic contacts connecting a single pre- and postsynaptic neuron pair. So far, the mechanism of cooperation is not known. Here we demonstrate that local correlation detection at the postsynaptic dendritic spine suffices to explain the synaptic cooperation effect, without assuming any hypothetical direct interaction pathway between the synaptic contacts. Candidate biomolecular mechanisms for dendritic correlation detection have been identified previously, as well as for structural plasticity based thereon. By analyzing and fitting of a simple model, we show that spike-timing correlation dependent structural plasticity, without additional mechanisms of cross-synapse interaction, can reproduce the experimentally observed distributions of numbers of synaptic contacts between pairs of neurons in the neocortex. Furthermore, the model yields a first explanation for the existence of both transient and persistent dendritic spines and allows to make predictions for future experiments.     

Multiple clusters of release sites formed by individual thalamic afferents onto cortical interneurons ensure reliable transmission

Thalamic afferents supply the cortex with sensory information by contacting both excitatory neurons and inhibitory interneurons. Interestingly, thalamic contacts with interneurons constitute such a powerful synapse that even one afferent can fire interneurons, thereby driving feedforward inhibition. However, the spatial representation of this potent synapse on interneuron dendrites is poorly understood. Using Ca imaging and electron microscopy we show that an individual thalamic afferent forms multiple contacts with the interneuronal proximal dendritic arbor, preferentially near branch points. More contacts are correlated with larger amplitude synaptic responses. Each contact, consisting of a single bouton, can release up to seven vesicles simultaneously, resulting in graded and reliable Ca transients. Computational modeling indicates that the release of multiple vesicles at each contact minimally reduces the efficiency of the thalamic afferent in exciting the interneuron. This strategy preserves the spatial representation of thalamocortical inputs across the dendritic arbor over a wide range of release conditions.     

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.     

Impact of active dendrites and structural plasticity on the memory capacity of neural tissue

We consider the combined effects of active dendrites and structural plasticity on the storage capacity of neural tissue. We compare capacity for two different modes of dendritic integration: (1) linear, where synaptic inputs are summed across the entire dendritic arbor, and (2) nonlinear, where each dendritic compartment functions as a separately thresholded neuron-like summing unit. We calculate much larger storage capacities for cells with nonlinear subunits and show that this capacity is accessible to a structural learning rule that combines random synapse formation with activity-dependent stabilization/elimination. In a departure from the common view that memories are encoded in the overall connection strengths between neurons, our results suggest that long-term information storage in neural tissue could reside primarily in the selective addressing of synaptic contacts onto dendritic subunits.