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The spatial distribution of neurons and activity-dependent neurite outgrowth shape long-range interaction, recurrent local connectivity and the modularity in neuronal networks. We investigated how this mesoscale architecture develops by interaction of neurite outgrowth, cell migration and activity in cultured networks of rat cortical neurons and sh...
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... migration promotes neuronal clustering 175 To quantify the structural development, we seeded networks at lower densities of about 300 176 neurons per mm 2 that were more suitable for morphometric analyses ( (Table 1). At 22 DIV, clustering was moderate in PKC N networks 183 (CI=0.75±0.03) ...
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... size was quantified as the ratio between the total length of detected dendrite 194 stretches and the number of neurons within regions of interest (Table 1). The measure 195 estimates the average contribution of each neuron to the dendritic mesh. ...
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... all conditions, dendrite size did not change 201 significantly between 22 and 29 DIV, indicating stabilization of the dendritic network after the 202 third week in vitro. As in the model (Figure 2-figure supplement 1), dendrite size in mature 203 networks was negatively correlated with the degree of cell body clustering and, thus, the 204 distance between neurons ( Figure 3D Table 1) and the dendritic occupancy as the 211 number of synapses per unit dendrite length ( Figure 3F, Table 1). Manipulating PKC activity 212 had no significant influence on early synaptogenesis up to 8 DIV, consistent with the 213 comparable dendrite density in different PKC conditions at this stage. ...
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... all conditions, dendrite size did not change 201 significantly between 22 and 29 DIV, indicating stabilization of the dendritic network after the 202 third week in vitro. As in the model (Figure 2-figure supplement 1), dendrite size in mature 203 networks was negatively correlated with the degree of cell body clustering and, thus, the 204 distance between neurons ( Figure 3D Table 1) and the dendritic occupancy as the 211 number of synapses per unit dendrite length ( Figure 3F, Table 1). Manipulating PKC activity 212 had no significant influence on early synaptogenesis up to 8 DIV, consistent with the 213 comparable dendrite density in different PKC conditions at this stage. ...
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... degree of connectivity realized, however, could be lower 232 because of multiple structural synapses between neuron pairs. Although the density of 233 neurons decreased during early development ( Figure 3H, Table 1 Mesoscale architecture and the development of spontaneous activity 240 We recently showed that the specific spatiotemporal patterns of spontaneous bursting 241 depended considerably on the mesoscale architecture of the network ( Okujeni et al., 2017;242 Okujeni and Egert, 2019) (Figure 4-figure supplement 1). In all networks types, spikes were 243 typically organized in bursts that were synchronized across micro-electrode arrays (MEA; 244 networks. ...
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... This resulted in increasing node densities around the initially placed nodes (Fig. 1). Cluster sizes were expected to have a mean size 5 (200), 10 (100), 20 (50) or 50 (20) nodes (initially placed nodes), corresponding to different regimes of clustering found in in vitro studies of neural networks (Okujeni and Egert, 2019b). Due to the distance from the initially placed node being determined by a power-law, most nodes were placed close to the initial node with a non-zero probability of a node being found at a large distance (Fig. 1c,e,g,i). ...
Biological neural networks are characterized by short average path lengths, high clustering, and modular and hierarchical architectures. These complex network topologies strike a balance between local specialization and global synchronization via long-range connections, resulting in highly efficient communication. Here, we use a geometric network model with either an intermediate or a long-range connection probability to investigate the effects of wiring cost principles on network complexity for different spatial conformations. We find that both long-range and intermediate wiring probabilities only conform to small-world architectures for neurons in dense spatial clusters due to a decrease in wiring cost within clusters. Furthermore, both small-worldness and modularity were reduced in systems with long-range connections caused by a reduction in network clustering, allowing for novel insight into mechanisms underlying adaptive or maladaptive network alterations. Our findings corroborate previous work showing that both wiring probability and spatial distributions play a key role in neural network development.
... These models cannot encode certainty in bump [32,33] and modeling [34,35] studies of metastable neural circuits, stronger and/or longer stimuli successively and discretely recruit more active microclusters locally in a neural circuit. Such microclusters may emerge spontaneously in development due to interactions of self-sustained activity with neuronal migration and outgrowth [36,37]. Macroscale connectivity has ring topology akin to that inferred and observed in recordings of neural circuits encoding periodic continuum variables [14,[38][39][40]. ...
Localized persistent neural activity has been shown to serve delayed estimation of continuous variables. Common experiments require that subjects store and report the feature value (e.g., orientation) of a particular cue (e.g., oriented bar on a screen) after a delay. Visualizing recorded activity of neurons according to their feature tuning reveals activity bumps whose centers wander stochastically, degrading the estimate over time. Bump position therefore represents the remembered estimate. Recent work suggests that bump amplitude may represent estimate certainty reflecting a probabilistic population code for a Bayesian posterior. Idealized models of this type are fragile due to the fine tuning common to constructed continuum attractors in dynamical systems. Here we propose an alternative metastable model for robustly supporting multiple bump amplitudes by extending neural circuit models to include quantized nonlinearities. Asymptotic projections of circuit activity produce low-dimensional evolution equations for the amplitude and position of bump solutions in response to external stimuli and noise perturbations. Analysis of reduced equations accurately characterizes phase variance and the dynamics of amplitude transitions between stable discrete values. More salient cues generate bumps of higher amplitude which wander less, consistent with the experimental finding that greater certainty correlates with more accurate memories.
... A similar increase in NBs similarity was previously proposed, effectively reducing the number of burst initiation zones in postnatal cortical cultures by restraining protein kinase C (PKC) (26). ...
Background: Cellular signals orchestrating synapse formation and neuronal network function remain poorly understood. To explore the critical signaling pathways in neurons and their influence on network development, pharmacological assays were employed to inhibit multiple signaling pathways in cultured neurons.
Methods: Immunofluorescence and western blotting are applied to identify the expression of synapse-related proteins within neurons. micro-electrode arrays (MEAs) are employed to study the developmental characteristics of neuronal networks. RNA sequencing is utilized to determine the gene expression profiles pertaining to multiple signaling pathways.
Results: Canonical c-jun N-terminal kinases (JNK) pathway is necessary for pre- and post-synaptic specializations, while phosphatidylinositide3-kinases (PI3K) is a key to postsynaptic specialization and affects the puncta sizes of presynaptic marker. Unexpectedly, pharmacological inhibition of JNK pathway significantly suppressed the mean firing rate (MFR), network burst frequency (NBF) and regularity of network firing after 4 weeks, but did not alter the synchrony of the network. During network development, PI3K pathway regulates the longer burst duration and lower network synchrony. Gene sets associated with neurodevelopmental processes and myelination was disturbed during restraining these signal pathways. Furthermore, inhibition of the PI3K signaling pathway obviously transformed voltage-gated ion channel activity, synaptic transmission and synaptic plasticity of neurons.
Conclusion: This study reveals that JNK and PI3K signaling pathways play different roles during synapse formation, and these signaling pathways have a lasting impact on the development of neuronal networks. Thus, this study provides further insights into the intracellular signaling pathways associated with synapse formation in the development of neuronal networks.
... These findings suggest that the morphological changes are secondary to the electrophysiological phenotype. We speculate that this could be a homeostatic adaptation to the exceptionally high spontaneous miniature release rates and/or to increased AP-induced total release per synapse in these neurons [49], since no morphological defects were reported for Syt1 knockout (KO) neurons [34]. ...
Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1PL-rescued neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous miniature release events per synapse were increased more than 500% in Syt1PL-rescued neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential-induced asynchronous release was increased more than 100%, while synchronous release was unaffected. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1PL was overexpressed in wild type neurons. Our findings show that Syt1PL desynchronizes neurotransmission by increasing the readily releasable pool for asynchronous release and reducing the suppression of spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increased synaptic input. Syt1PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1’s primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other SYT1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.
... How synapse formation between rods and bipolar cells contributes to rod translocation is unclear; however, reverse signalling at the synapse may be of importance. Indeed, in early postnatal development, cortical neuronal migration is regulated by activity-dependent changes in intracellular Ca 2+ concentration (50). Similarly, activity dependent Ca 2+ spiking at the synapse can act as a signal to communicate with the nucleus, resulting in transcriptional regulation via nuclear factor of activated T cells (NFAT) (51). ...
Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.
... The difference could originate not only from the culture age or the duration of stimulation but also from extracellular calcium condition (68), opening future applications of in vitro cortical networks to the understanding of the cellular mechanisms underlying learning and memory. Besides the precision micropatterning approach used in the present work, the overall extent of network modularity can also be controlled through the modulation of cell affinity of the scaffold (54,55) or through pharmacological manipulation of neurite outgrowth (69,70), which could be beneficial in tuning network structures at a larger scale. ...
High-level information processing in the mammalian cortex requires both segregated processing in specialized circuits and integration across multiple circuits. One possible way to implement these seemingly opposing demands is by flexibly switching between states with different levels of synchrony. However, the mechanisms behind the control of complex synchronization patterns in neuronal networks remain elusive. Here, we use precision neuroengineering to manipulate and stimulate networks of cortical neurons in vitro, in combination with an in silico model of spiking neurons and a mesoscopic model of stochastically coupled modules to show that (i) a modular architecture enhances the sensitivity of the network to noise delivered as external asynchronous stimulation and that (ii) the persistent depletion of synaptic resources in stimulated neurons is the underlying mechanism for this effect. Together, our results demonstrate that the inherent dynamical state in structured networks of excitable units is determined by both its modular architecture and the properties of the external inputs.
... This bursting appears by the end of the first week in vitro [42], may remain for the entire lifetime of the culture [111], and it is in general resilient to biochemical perturbations [6] or damage [112]. Although connectivity evolves in a non-random manner and rich micro-architectures emerge during development [113][114][115], such a dominant network-wide bursting may be problematic in those studies that explore development or alterations in a neuronal culture from a functional point of view, since the ever present bursting portrays a highly correlated network in which details of interest at the micro-scale are masked or difficult to resolve. This may explain the challenge in assessing strong functional differences between healthy and diseased neuronal networks in models of neurological disorders in vitro, e.g., as in our work in Ref. [26]. ...
Neuronal cultures are one of the most important experimental models in modern interdisciplinary neuroscience, allowing to investigate in a control environment the emergence of complex behavior from an ensemble of interconnected neurons. Here, I review the research that we have conducted at the neurophysics laboratory at the University of Barcelona over the last 15 years, describing first the neuronal cultures that we prepare and the associated tools to acquire and analyze data, to next delve into the different research projects in which we actively participated to progress in the understanding of open questions, extend neuroscience research on new paradigms, and advance the treatment of neurological disorders. I finish the review by discussing the drawbacks and limitations of neuronal cultures, particularly in the context of brain-like models and biomedicine.
... Indeed, important network aspects such as self-organization (Pasquale et al., 2008), activity patterns (Chiappalone et al., 2006), synchronization (Eytan and Marom, 2006;Schroeter et al., 2015), mesoscale architecture (Okujeni and Egert, 2019;Tibau et al., 2020), the emergence of spontaneous activity (Orlandi et al., 2013), or functional network formation (Poli et al., 2015;Schroeter et al., 2015) have been widely studied in rodent primary cultures, both in two-dimensional (2D) and three-dimensional (3D) preparations (Dingle et al., 2020), providing invaluable insight on the functional organization of neuronal circuits. However, it is not clear whether such investigations apply to hiPSCs network formation and functionality. ...
... This strong synchronization, in turn, led to functional networks that were excessively integrated, a condition that is considered pathological when compared with healthy brain-like behavior (Deco et al., 2015). A richer repertoire of activity and functionality in rat primary cultures could be favored by imprinting modular characteristics in the network, for instance through strong aggregation (Teller et al., 2014;Okujeni and Egert, 2019;Tibau et al., 2020) or fine-tuned neuro-engineering (Yamamoto et al., 2018;Montalà-Flaquer et al., 2022). By contrast, hiPSC-derived cultures exhibited a broad repertoire of activity patterns (functional segregation), and the synchronous-like behavior present at late stages did not dominate network dynamics. ...
Models for human brain-oriented research are often established on primary cultures from rodents, which fails to recapitulate cellular specificity and molecular cues of the human brain. Here we investigated whether neuronal cultures derived from human induced pluripotent stem cells (hiPSCs) feature key advantages compared with rodent primary cultures. Using calcium fluorescence imaging, we tracked spontaneous neuronal activity in hiPSC-derived, human, and rat primary cultures and compared their dynamic and functional behavior as they matured. We observed that hiPSC-derived cultures progressively changed upon development, exhibiting gradually richer activity patterns and functional traits. By contrast, rat primary cultures were locked in the same dynamic state since activity onset. Human primary cultures exhibited features in between hiPSC-derived and rat primary cultures, although traits from the former predominated. Our study demonstrates that hiPSC-derived cultures are excellent models to investigate development in neuronal assemblies, a hallmark for applications that monitor alterations caused by damage or neurodegeneration.
... Interestingly, dendrites of single Syt1 PL -expressing neurons embedded in a mass culture of wild type cells developed normally, presumably receiving normal synaptic input from neighboring neurons, suggesting that the morphological changes are secondary to the electrophysiological phenotype. We speculate that this could be a homeostatic adaptation speci cally to the exceptionally high spontaneous release rates beyond KO levels and/or increased AP-induced total release per synapse in these neurons 42 , since no morphological defects were reported for Syt1 KO neurons 43 . ...
Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1 PL -expressing neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous neurotransmitter release per synapse was increased more than 500% in Syt1 PL -expressing neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential induced asynchronous release was increased more than 100%, while synchronous release was not changed. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1 PL was expressed in wild type neurons. Our findings show that Syt1 PL desynchronizes neurotransmission by reducing the suppression spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increase in release. Syt1 PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1’s primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other Syt1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1 -associated neurodevelopmental disorder.
... [23][24][25] Despite the importance of these neuroengineering efforts, it has been shown that an initially homogeneous distribution of neurons undergo significant reorganization that shape complex network features such as small-worldness and rich-club topology, 26,27 whereas cultures exhibiting mild fluctuations in the spatial distribution of neurons in combination with activity-depend mechanisms evolve to exhibit modularity traits and balanced local-to-global connectivity. 28,29 Although the above studies show that self-organization in neuronal circuits suffice to imprint rich functional traits, an aspect that remains unexplored is whether these traits can be accelerated or strengthened by incorporating coarse spatial constraints that break the isotropy of the substrate in which neurons grow. To advance in this quest, here we used mesoscopic neuronal cultures 6 mm in diameter grown on PDMS topographical substrates that contained elevations shaped as either parallel tracks or squares. ...
... Primary neuronal cultures are one of the most celebrated techniques in several multidisciplinary research fields, including physics of complex systems, neuroengineering and medicine. Their versatility, accessibility and ease of manipulation have made them ideal to investigate in a controlled manner phenomena as diverse as self-organization, [26][27][28] repertoire of activity patterns, 22,31 structure-to-function relationship, 12,14,29 resilience to perturbations 32,33 and alteration upon disease. 34,35 However, primary cultures grown on flat surfaces typically exhibit a strong bursting behavior in which all neurons activate together in a short time iScience Article window and remain practically silent in between bursts. ...
... The effort of imprinting 'mesoscale architecture' while allowing self-organization is conceptually similar to the studies on neuronal cultures with spatial aggregation. 28,29,46 Aggregation helped neurons to connect within their neighborhood but without hindering long-range connectivity, shaping networks with a richer dynamic behavior and more varied activity initiation, as in our case. ...
Neuronal cultures are a prominent experimental tool to understand complex functional organization in neuronal assemblies. However, neurons grown on flat surfaces exhibit a strongly coherent bursting behavior with limited functionality. To approach the functional richness of naturally formed neuronal circuits, here we studied neuronal networks grown on polydimethylsiloxane (PDMS) topographical patterns shaped as either parallel tracks or square valleys. We followed the evolution of spontaneous activity in these cultures along 20 days in vitro using fluorescence calcium imaging. The networks were characterized by rich spatiotemporal activity patterns that comprised from small regions of the culture to its whole extent. Effective connectivity analysis revealed the emergence of spatially compact functional modules that were associated to both the underpinned topographical features and predominant spatiotemporal activity fronts. Our results show the capacity of spatial constraints to mold activity and functional organization, bringing new opportunities to comprehend the structure-function relationship in living neuronal circuits.
