Spatiotemporal memory is an intrinsic property of networks of dissociated cortical neurons.

The ability to process complex spatiotemporal information is a fundamental process underlying the behavior of all higher organisms. However, how the brain processes information in the temporal domain remains incompletely understood. We have explored the spatiotemporal information-processing capability of networks formed from dissociated rat E18 cortical neurons growing in culture. By combining optogenetics with microelectrode array recording, we show that these randomly organized cortical microcircuits are able to process complex spatiotemporal information, allowing the identification of a large number of temporal sequences and classification of musical styles. These experiments uncovered spatiotemporal memory processes lasting several seconds. Neural network simulations indicated that both short-term synaptic plasticity and recurrent connections are required for the emergence of this capability. Interestingly, NMDA receptor function is not a requisite for these short-term spatiotemporal memory processes. Indeed, blocking the NMDA receptor with the antagonist APV significantly improved the temporal processing ability of the networks, by reducing spontaneously occurring network bursts. These highly synchronized events have disastrous effects on spatiotemporal information processing, by transiently erasing short-term memory. These results show that the ability to process and integrate complex spatiotemporal information is an intrinsic property of generic cortical networks that does not require specifically designed circuits.

miR-27b shapes the presynaptic transcriptome and influences neurotransmission by silencing the polycomb group protein Bmi1.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood. RESULTS: Here, we identify miR-27b-an abundant neuronal miRNA implicated in neurological disorders-as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1. CONCLUSIONS: We conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation.

Short-term memory in networks of dissociated cortical neurons.

Short-term memory refers to the ability to store small amounts of stimulus-specific information for a short period of time. It is supported by both fading and hidden memory processes. Fading memory relies on recurrent activity patterns in a neuronal network, whereas hidden memory is encoded using synaptic mechanisms, such as facilitation, which persist even when neurons fall silent. We have used a novel computational and optogenetic approach to investigate whether these same memory processes hypothesized to support pattern recognition and short-term memory in vivo, exist in vitro. Electrophysiological activity was recorded from primary cultures of dissociated rat cortical neurons plated on multielectrode arrays. Cultures were transfected with ChannelRhodopsin-2 and optically stimulated using random dot stimuli. The pattern of neuronal activity resulting from this stimulation was analyzed using classification algorithms that enabled the identification of stimulus-specific memories. Fading memories for different stimuli, encoded in ongoing neural activity, persisted and could be distinguished from each other for as long as 1 s after stimulation was terminated. Hidden memories were detected by altered responses of neurons to additional stimulation, and this effect persisted longer than 1 s. Interestingly, network bursts seem to eliminate hidden memories. These results are similar to those that have been reported from similar experiments in vivo and demonstrate that mechanisms of information processing and short-term memory can be studied using cultured neuronal networks, thereby setting the stage for therapeutic applications using this platform.

A Proteome-Wide Effect of PHF8 Knockdown on Cortical Neurons Shows Downregulation of Parkinson's Disease-Associated Protein Alpha-Synuclein and Its Interactors.

Synaptic dysfunction may underlie the pathophysiology of Parkinson's disease (PD), a presently incurable condition characterized by motor and cognitive symptoms. Here, we used quantitative proteomics to study the role of PHD Finger Protein 8 (PHF8), a histone demethylating enzyme found to be mutated in X-linked intellectual disability and identified as a genetic marker of PD, in regulating the expression of PD-related synaptic plasticity proteins. Amongst the list of proteins found to be affected by PHF8 knockdown were Parkinson's-disease-associated SNCA (alpha synuclein) and PD-linked genes DNAJC6 (auxilin), SYNJ1 (synaptojanin 1), and the PD risk gene SH3GL2 (endophilin A1). Findings in this study show that depletion of PHF8 in cortical neurons affects the activity-induced expression of proteins involved in synaptic plasticity, synaptic structure, vesicular release and membrane trafficking, spanning the spectrum of pre-synaptic and post-synaptic transmission. Given that the depletion of even a single chromatin-modifying enzyme can affect synaptic protein expression in such a concerted manner, more in-depth studies will be needed to show whether such a mechanism can be exploited as a potential disease-modifying therapeutic drug target in PD.

Enhanced Neuronal Activity and Asynchronous Calcium Transients Revealed in a 3D Organoid Model of Alzheimer's Disease.

Advances in the development of three-dimensional (3D) brain organoids maintained in vitro have provided excellent opportunities to study brain development and neurodegenerative disorders, including Alzheimer's disease (AD). However, there remains a need to generate AD organoids bearing patient-specific genomic backgrounds that can functionally recapitulate the key features observed in the AD patient's brain. To address this need, we described a strategy to generate self-organizing 3D cerebral organoids which develop a functional neuronal network connectivity. This was achieved by neuroectoderm induction of human pluripotent stem cell (hPSCs) aggregates and subsequent differentiation into desired neuroepithelia and mature neurons in a 3D Matrigel matrix. Using this approach, we successfully generated AD cerebral organoids from human pluripotent stem cells (hPSCs) derived from a familial AD patient with a common mutation in presenilin 2 (PSEN2N141I). An isogenic control with an identical genetic background but wild-type PSEN2 was generated using CRISPR/Cas9 technology. Both control and AD organoids were characterized by analyzing their morphology, the Aß42/Aß40 ratio, functional neuronal network activity, drug sensitivity, and the extent of neural apoptosis. The spontaneous activity of the network and its synchronization was measured in the organoids via calcium imaging. We found that compared with the mutation-corrected control organoids, AD organoids had a higher Aß42/Aß40 ratio, asynchronous calcium transients, and enhanced neuronal hyperactivity, successfully recapitulating an AD-like pathology at the molecular, cellular, and network level in a human genetic context. Moreover, two drugs which increase neuronal activity, 4-aminopyridine (4-AP) and bicuculline methochloride, induced high-frequency synchronized network bursting to a similar extent in both organoids. Therefore, our study presents a promising organoid-based biosystem for the study of the pathophysiology of AD and a platform for AD drug development.

Selective increase of correlated activity in Arc-positive neurons after chemically induced long-term potentiation in cultured hippocampal neurons.

The activity-dependent expression of immediate-early genes (IEGs) has been utilised to label memory traces. However, their roles in engram specification are incompletely understood. Outstanding questions remain as to whether expression of IEGs can interplay with network properties such as functional connectivity and also if neurons expressing different IEGs are functionally distinct. In order to connect IEG expression at the cellular level with changes in functional-connectivity, we investigated the expression of 2 IEGs, Arc and c-Fos, in cultured hippocampal neurons. Primary neuronal cultures were treated with a chemical cocktail (4-aminopyridine, bicuculline, and forskolin) to increase neuronal activity, IEG expression, and induce chemical long-term potentiation. Neuronal firing is assayed by intracellular calcium imaging using GCaMP6m and expression of IEGs is assessed by immunofluorescence staining. We noted an emergent network property of refinement in network activity, characterized by a global downregulation of correlated activity, together with an increase in correlated activity between subsets of specific neurons. Subsequently, we show that Arc expression correlates with the effects of refinement, as the increase in correlated activity occurs specifically between Arc-positive neurons. The expression patterns of the IEGs c-Fos and Arc strongly overlap, but Arc was more selectively expressed than c-Fos. A subpopulation of neurons positive for both Arc and c-Fos shows increased correlated activity, while correlated firing between Arc+/cFos- neurons is reduced. Our results relate neuronal activity-dependent expression of the IEGs Arc and c-Fos on the individual cellular level to changes in correlated activity of the neuronal network.SIGNIFICANCEEstablishing a stable long-lasting memory requires neuronal network-level changes in connection strengths in a subset of neurons, which together constitute a memory trace or engram. Two genes, c-Fos and Arc, have been implicated to play critical roles in the formation of the engram. They have been studied extensively at the cellular/molecular level, and have been used as markers of memory traces in mice. We have correlated Arc and c-Fos cellular expression with refinement of correlated neuronal activity following pharmacological activation of networks formed by cultured hippocampal neurons. Whereas there is a global loss of correlated activity, Arc-positive neurons show selectively increased correlated activity. Arc is more selectively expressed than c-Fos, but the two genes act together in encoding information about changes in correlated firing.

Familiarity Detection and Memory Consolidation in Cortical Assemblies.

Humans have a large capacity of recognition memory (Dudai, 1997), a fundamental property of higher-order brain functions such as abstraction and generalization (Vogt and Magnussen, 2007). Familiarity is the first step towards recognition memory. We have previously demonstrated using unsupervised neural network simulations that familiarity detection of complex patterns emerges in generic cortical microcircuits with bidirectional synaptic plasticity. It is therefore meaningful to conduct similar experiments on biological neuronal networks to validate these results. Studies of learning and memory in dissociated rodent neuronal cultures remain inconclusive to date. Synchronized network bursts (SNBs) that occur spontaneously and periodically have been speculated to be an intervening factor. By optogenetically stimulating cultured cortical networks with random dot movies (RDMs), we were able to reduce the occurrence of SNBs, after which an ability for familiarity detection emerged: previously seen patterns elicited higher firing rates than novel ones. Differences in firing rate were distributed over the entire network, suggesting that familiarity detection is a system level property. We also studied the change in SNB patterns following familiarity encoding. Support vector machine (SVM) classification results indicate that SNBs may be facilitating memory consolidation of the learned pattern. In addition, using a novel network connectivity probing method, we were able to trace the change in synaptic efficacy induced by familiarity encoding, providing insights on the long-term impact of having SNBs in the cultures.

The NR1 M3 domain mediates allosteric coupling in the N-methyl-D-aspartate receptor.

N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits.

Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arc's molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the betaIV spectrin splice variant betaSpIVSigma5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to beta-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and betaSpIVSigma5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

Familiarity Detection is an Intrinsic Property of Cortical Microcircuits with Bidirectional Synaptic Plasticity.

Humans instantly recognize a previously seen face as "familiar." To deepen our understanding of familiarity-novelty detection, we simulated biologically plausible neural network models of generic cortical microcircuits consisting of spiking neurons with random recurrent synaptic connections. NMDA receptor (NMDAR)-dependent synaptic plasticity was implemented to allow for unsupervised learning and bidirectional modifications. Network spiking activity evoked by sensory inputs consisting of face images altered synaptic efficacy, which resulted in the network responding more strongly to a previously seen face than a novel face. Network size determined how many faces could be accurately recognized as familiar. When the simulated model became sufficiently complex in structure, multiple familiarity traces could be retained in the same network by forming partially-overlapping subnetworks that differ slightly from each other, thereby resulting in a high storage capacity. Fisher's discriminant analysis was applied to identify critical neurons whose spiking activity predicted familiar input patterns. Intriguingly, as sensory exposure was prolonged, the selected critical neurons tended to appear at deeper layers of the network model, suggesting recruitment of additional circuits in the network for incremental information storage. We conclude that generic cortical microcircuits with bidirectional synaptic plasticity have an intrinsic ability to detect familiar inputs. This ability does not require a specialized wiring diagram or supervision and can therefore be expected to emerge naturally in developing cortical circuits.

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells.

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.

Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.

Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential.

The nonkinase phorbol ester receptor alpha 1-chimerin binds the NMDA receptor NR2A subunit and regulates dendritic spine density.

Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie synaptic plasticity. The key regulators of cytoskeletal reorganization in dendrites and spines are the Rho GTPases, which modify actin polymerization in response to synaptic signaling. Rho GTPase activity is modulated by multiple regulatory proteins, some of which have been found to associate with proteins localized to spines. Here, we show that the nonkinase phorbol ester receptor alpha1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. Alpha1-chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of alpha1-chimerin in cultured hippocampal neurons inhibits formation of new spines and removes existing spines. This reduction in spine density is mediated by Rac1 inhibition, because it depends critically on the presence of a functional GAP domain. Conversely, depletion of alpha1-chimerin leads to an increase in spine density, indicating that a basal inhibition of Rac1 maintains the number of spines at a submaximal level. The ability of alpha1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an alpha1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that alpha1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.

Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.

K+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.

K channel subconductance levels result from heteromeric pore conformations.

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes.

Cellular functions of the Golgi are determined by the unique distribution of its resident proteins. Currently, electron microscopy is required for the localization of a Golgi protein at the sub-Golgi level. We developed a quantitative sub-Golgi localization method based on centers of fluorescence masses of nocodazole-induced Golgi ministacks under conventional optical microscopy. Our method is rapid, convenient, and quantitative, and it yields a practical localization resolution of ∼ 30 nm. The method was validated by the previous electron microscopy data. We quantitatively studied the intra-Golgi trafficking of synchronized secretory membrane cargoes and directly demonstrated the cisternal progression of cargoes from the cis- to the trans-Golgi. Our data suggest that the constitutive efflux of secretory cargoes could be restricted at the Golgi stack, and the entry of the trans-Golgi network in secretory pathway could be signal dependent.

Molecular Features Underlying Neurodegeneration Identified through In Vitro Modeling of Genetically Diverse Parkinson's Disease Patients.

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis, we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro, we generated neurons harboring disease-causing mutations from patient-specific, induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons, reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro, including oxidative stress vulnerability and altered neuronal activity. Notably, PD neurons show that elevated RBFOX1, a gene previously linked to neurodevelopmental diseases, underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes.

The NMDA receptor M3 segment is a conserved transduction element coupling ligand binding to channel opening.

Ion channels alternate stochastically between two functional states, open and closed. This gating behavior is controlled by membrane potential or by the binding of neurotransmitters in voltage- and ligand-gated channels, respectively. Although much progress has been made in defining the structure and function of the ligand-binding cores and the voltage sensors, how these domains couple to channel opening remains poorly understood. Here we show that the M3 transmembrane segments of the NMDA receptor allosterically interact with both the ligand-binding cores and the channel gate. It is proposed that M3 functions as a transduction element whose conformational change couples ligand binding with channel opening. Furthermore, amino acid homology between glutamate receptor M3 segments and the equivalent S6 or TM2 segments in K(+) channels suggests that ion channel activation and gating are both structurally and functionally conserved.

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts.

In both humans and animals brief synchronizing bursts of epileptiform activity known as interictal epileptiform discharges (IEDs) can, even in the absence of overt seizures, cause transient cognitive impairments (TCI) that include problems with perception or short-term memory. While no evidence from single units is available, it has been assumed that IEDs destroy information represented in neuronal networks. Cultured neuronal networks are a model for generic cortical microcircuits, and their spontaneous activity is characterized by the presence of synchronized network bursts (SNBs), which share a number of properties with IEDs, including the high degree of synchronization and their spontaneous occurrence in the absence of an external stimulus. As a model approach to understanding the processes underlying IEDs, optogenetic stimulation and multielectrode array (MEA) recordings of cultured neuronal networks were used to study whether stimulus information represented in these networks survives SNBs. When such networks are optically stimulated they encode and maintain stimulus information for as long as one second. Experiments involved recording the network response to a single stimulus and trials where two different stimuli were presented sequentially, akin to a paired pulse trial. We broke the sequential stimulus trials into encoding, delay and readout phases and found that regardless of which phase the SNB occurs, stimulus-specific information was impaired. SNBs were observed to increase the mean network firing rate, but this did not translate monotonically into increases in network entropy. It was found that the more excitable a network, the more stereotyped its response was during a network burst. These measurements speak to whether SNBs are capable of transmitting information in addition to blocking it. These results are consistent with previous reports and provide baseline predictions concerning the neural mechanisms by which IEDs might cause TCI.