Functional characterization of GABAA receptor-mediated modulation of cortical neuron network activity in microelectrode array recordings.

Traumatic brain injury (TBI) remains the leading cause of death and disability worldwide with no existing effective treatment. The early phase after TBI induction triggers numerous molecular cascades to regulate adaptive processes and cortical network activity. Kinases play a particularly prominent role in modifying peptide substrates, which include ion channels, receptors, transcription factors and inflammatory mediators. This study aimed to better understand the post-injury serine/threonine kinome; (1) Which kinases conduct phosphorylation-induced alterations of target peptides following unilateral TBI in mouse cortex? (2) How do these kinases effectuate pathological network hyperexcitability, which has detrimental long-term outcomes? We used a serine/threonine kinase assay at 4 h, 24 h and 72 h post-TBI to identify hyper-/hypo-active/phosphorylated kinases and peptides in the ipsilateral and contralateral cortical hemispheres relative to sham-operated controls. We pharmacologically mimicked the changes seen in ERK1/2 and PKC kinase activity, and using microelectrode array recordings we explored their significant electrophysiological implications on spontaneous and evoked cortical activity. We then used these findings to manipulate key kinase activity changes at 24 h post-TBI to rescue the hyperexcitability that is seen in the contralateral cortical network at this timepoint back to sham level. The contribution of specific downstream peptide target channel/receptor subunits was also shown. We conclude that volatile kinase activity has potent implications on cortical network activity after the injury and that these kinases and/or their peptide substrates should be more seriously considered as therapeutic targets for the clinical treatment of TBI.

Self-sustained recurrent activity in cortical networks is thought to be important for multiple crucial processes, including circuit development and homeostasis. Yet, the precise relationship between synaptic input patterns and the spiking output of individual neurons remains largely unresolved. Here, we developed, validated and applied a novel in vitro experimental platform and analytical procedures that provide – for individual neurons – simultaneous excitatory and inhibitory synaptic activity estimates during recurrent network activity. Our approach combines whole-network high-density microelectrode array (HD-MEA) recordings from rat neuronal cultures with patch clamping and enables a comprehensive mapping and characterization of active incoming connections to single postsynaptic neurons. We found that, during network states with excitation(E)-inhibition(I) balance, postsynaptic spiking often coincided with the maxima of fast fluctuations in the input E/I ratio. These spike-associated E/I ratio escalations were largely due to a rapid bidirectional change in synaptic inhibition that was modulated by the network-activity level. Our approach also uncovered the underlying circuit architecture and we show that individual neurons received a few key inhibitory connections – often from special hub neurons – that were instrumental in controlling postsynaptic spiking. Balanced network theory predicts dynamical regimes governed by small and rapid input fluctuation and featuring a fast neuronal responsiveness. Our findings – obtained in self-organized neuronal cultures – suggest that the emergence of these favorable regimes and associated network architectures is an inherent property of cortical networks in general.

Self-sustained recurrent activity in cortical networks is thought to be important for multiple crucial processes, including circuit development and homeostasis. Yet, the precise relationship between the synaptic input patterns and the spiking output of individual neurons remains largely unresolved. Here, we developed, validated and applied a novel in vitro experimental platform and analytical procedures that provide – for individual neurons – simultaneous excitatory and inhibitory synaptic activity estimates during recurrent network activity. Our approach combines whole-network high-density microelectrode array (HD-MEA) recordings from rat neuronal cultures with patch clamping and enables a comprehensive mapping and characterization of active incoming connections to single postsynaptic neurons. We found that, during network states with excitation(E)-inhibition(I) balance, postsynaptic spiking coincided precisely with the maxima of fast fluctuations in the input E/I ratio. These spike-associated E/I ratio escalations were largely due to a rapid bidirectional change in synaptic inhibition that was modulated by the network-activity level. Our approach also uncovered the underlying circuit architecture and we show that individual neurons received a few key inhibitory connections – often from special hub neurons – that were instrumental in controlling postsynaptic spiking. Balanced network theory predicts dynamical regimes governed by small and rapid input fluctuation and featuring a fast neuronal responsiveness. Our findings – obtained in self-organized neuronal cultures – suggest that the emergence of these favorable regimes and associated network architectures is an inherent property of cortical networks in general.

During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.

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The characterization of the subthreshold, ongoing activity in cortical neurons has been the focus of numerous studies. This activity, described as spontaneous slow waves in membrane potential, has been observed in a span of species in diverse cortical and subcortical areas. We here characterized membrane potential fluctuations in motor and the frontal association cortices cortical neurons of ketamine–xylazine anesthetized rats. We recorded from 95 neurons from a range of cortical depths to unravel the network and cellular mechanisms that shape the subthreshold ongoing spontaneous activity of these neurons. We define a unitary event that generates the subthreshold ongoing activity: giant synaptic potentials (GSPs). These events have a duration of 87 ± 50 ms and an amplitude of 19 ± 6.4 mV. They occur at a frequency of 3.7 ± 0.8 Hz and involve an increase in conductance change of 22 ± 21%. GSPs are mainly due to excitatory activity that occurs throughout all cortical layers, unaffected by the intrinsic properties of the cells. Indeed, blocking the GABAA receptors, a procedure that had a profound effect on cortical activity, did not alter these unitary events. We propose that this unitary event is composed of individual, excitatory synaptic potentials that appear at different levels of synchrony and that the level of synchrony determines the shape of the subthreshold activity.

Early-life exposure to caffeine affects the construction and activity of cortical networks in mice

BackgroundMonitoring the activity and morphology of neuron-astrocyte networks in culture is a powerful tool for studying dynamics, structure, and communication in neuron-astrocyte networks independently or as a model of the sub-brain network. These cultures are known to produce stereotypical patterns of activity, e.g., highly synchronized network bursts resembling sleep or seizure states, thus it enables the exploration of behaviors that can relate to brain function and disease. High-resolution microscopy of calcium imaging combined with simultaneous electrical recording provides a comprehensive overview on the network's dynamics. This setup makes it possible to apply global perturbations of electrical and chemical stimulation on the cultures during the recording task and to record the effects on network activity on-line. Morphological changes in the cultures can be obtained to have a complete dataset for structure-function study of neuron-astrocyte networks in vitro.FindingsThe 4 TB of data presented here was recorded and imaged as part of an accompanying study looking at in vitro structure-function of neuron-astrocyte networks. Simultaneous optical (calcium imaging) and electrical (micro-electrode array) recordings lasted 5–12 minutes and included spontaneous activity recording, electrical and chemical stimulation of neuron-astrocyte, and isolated astrocyte cultures. The data include activity recordings of 58 different cultures, with 1–2 regions of interest recorded for each culture. Production procedures, experimental protocols, and reuse options are included. The data have been suitable to reveal changes in the activity and morphology of the cultures and enabled observation and analysis of neuron-astrocyte and isolated astrocyte culture behaviors under the applied perturbations.ConclusionsOur dataset is sufficient to show significant changes in activity and morphology of neuron-astrocyte networks in culture under the applied stimulations. More than 100 recordings of 58 different cultures give insight of the observation's significance and led to conclusions about astrocyte activity and neuron-astrocyte network communication. Making it available here will allow others to test new tools for calcium imaging analysis and extracellular neuronal voltage recordings.

Irregular ongoing activity in cortical networks is often modeled as arising from recurrent connectivity. Yet it remains unclear to what extent its presence corrupts sensory signal transmission and network computational capabilities. In a recurrent cortical-like network, we have determined the activity patterns that are better transmitted and self-sustained by the network. We show that reproducible spiking and subthreshold dynamics can be triggered if the statistics of the imposed external drive are consistent with patterns previously seen in the ongoing activity. A subset of neurons in the network, constrained to replay temporal pattern segments extracted from the recorded ongoing activity of the same network, reliably drives the remaining, free-running neurons to call the rest of the pattern. Comparison with surrogate Poisson patterns indicates that the efficiency of the recall and completion process depends on the similarity between the statistical properties of the input with previous ongoing activity The reliability of evoked dynamics in recurrent networks is thus dependent on the stimulus used, and we propose that the similarity between spontaneous and evoked activity in sensory cortical areas could be a signature of efficient transmission and propagation across cortical networks.

In vivo toxicokinetic studies provide evidence for the translocation and accumulation of nanoparticles (NP) in the brain, thereby causing concern for adverse health effects, particularly for effects following chronic exposure. To date, only few studies investigated the effects of NP exposure on neuronal function in vitro, primarily focusing on short-term effects. The aim of this study was therefore to investigate the effects of two common types of NP, titanium dioxide NP (TiO2NP) and silver NP (AgNP), on neuronal function following acute (0.5 h), sub-chronic (24 h and 48 h) and chronic (14 days) exposure in vitro. Effects of NP exposure on intracellular calcium homeostasis, spontaneous neuronal (network) activity and neuronal network morphology were investigated in rat primary cortical cells using respectively, single-cell microscopy calcium imaging, micro-electrode array (MEA) recordings and immunohistochemistry. Our data demonstrate that high doses of AgNP (≥ 30 µg/mL) decrease calcium influx after 24 h exposure, although neuronal activity is not affected following acute and sub-chronic exposure. However, chronic exposure to non-cytotoxic doses of AgNP (1–10 µg/mL) potently decreases spontaneous neuronal (network) activity, without affecting network morphology and viability. Exposure to higher doses (≥ 30 µg/mL) affects network morphology and is also associated with cytotoxicity. In contrast, acute and sub-chronic exposure to TiO2NP is without effects, whereas chronic exposure only modestly reduces neuronal function without affecting morphology. Our combined findings indicate that TiO2NP exposure is of limited hazard for neuronal function whereas AgNP, in particularly during chronic exposure, has profound effects on neuronal (network) function and morphology.

GABAergic inhibitory interneurons play a pivotal role in balancing neuronal activity in the neocortex. They can be classified into different classes according to their variable morphological, electrophysiological, and neurochemical properties, including two major groups: parvalbumin-containing (PV+), fast-spiking (FS) cells and somatostatin-containing (SOM+) cells. Using transgenic mice, we identified two subgroups, distinct by all criteria, of SOM+ cells in the somatosensory (barrel) cortex of the mouse, one (called X94) in layer 4 and 5B, and the other one (X98) in deep layers (Ma et al., 2006). We found that X98 cells were calbindin-expressing (CB+), infragranular, layer 1--targeting "Martinotti" cells, and had a propensity to fire low-threshold calcium spikes, whereas X94 cells did not express CB, targeted mostly layer 4, discharged in stuttering pattern and with quasi "fast-spiking" properties. In the barrel cortex, it was previously shown that SOM+ cells mediate disynaptic inhibition in supragranular and infragranular layers. However, the roles of layer 4 SOM+ cells remain largely unknown. We used dual whole-cell recording to elucidate the synaptic circuits in layer 4 and the function of layer 4 SOM+ cells during cortical network activities. We found that layer 4 "X94" SOM+ cells received strongly facilitating excitatory input and generated relatively slow rising inhibitory postsynaptic currents (IPSCs) compared to those evoked by FS cells. Strikingly, our data showed that SOM+ cells mediated strong synaptic inhibition of FS cells with connection probability greater than 90% in layer 4, but received very little reciprocal inhibition from FS cells, and no reciprocal inhibition from other SOM+ cells. Moreover, 100% of recorded SOM+-SOM+ cell pairs were electrically coupled with higher coupling ratio compared to that of electrically coupled FS cell pairs. In order to examine the functions of SOM+ cells, we applied 0 Mg2+ artificial cerebrospinal fluid (ACSF) to induce episodes of cortical network activity and observed that, during episodes of network activity, SOM+ cells fired robustly and synchronously, and produced strong inhibition of regular-spiking (RS) excitatory cells and inhibitory FS cells, especially the latter. Taken together, our data reveal that SOM+ cells in the barrel cortex can be sub-divided into different subtypes, and that layer 4 SOM+ cells exert a powerful inhibitory effect during high frequency network activity.

We present long-term (approximately hours) measurements of the spontaneous activity of two-dimensional cortical cell neural networks placed on multielectrode arrays. We compare histograms of single neuron interspike intervals and the network intersynchronized bursting events intervals. In addition, the effect of Ca concentration on the network activity is being studied. At 1 mM Ca concentration, the network exhibits periodic synchronized bursting that fades away after about 20 min. We present a feedback-regulated integrate and fire model to account for the observations. In the model we include two additional features: dynamical threshold and synapse fatigue.

Accurate identification of bursting activity is an essential element in the characterization of neuronal network activity. Despite this, no one technique for identifying bursts in spike trains has been widely adopted. Instead, many methods have been developed for the analysis of bursting activity, often on an ad hoc basis. Here we provide an unbiased assessment of the effectiveness of eight of these methods at detecting bursts in a range of spike trains. We suggest a list of features that an ideal burst detection technique should possess and use synthetic data to assess each method in regard to these properties. We further employ each of the methods to reanalyze microelectrode array (MEA) recordings from mouse retinal ganglion cells and examine their coherence with bursts detected by a human observer. We show that several common burst detection techniques perform poorly at analyzing spike trains with a variety of properties. We identify four promising burst detection techniques, which are then applied to MEA recordings of networks of human induced pluripotent stem cell-derived neurons and used to describe the ontogeny of bursting activity in these networks over several months of development. We conclude that no current method can provide "perfect" burst detection results across a range of spike trains; however, two burst detection techniques, the MaxInterval and logISI methods, outperform compared with others. We provide recommendations for the robust analysis of bursting activity in experimental recordings using current techniques.

Local coherence oscillations in the EEG during development in the fetal baboon

The cortex processes information through intricate circuitry and outputs to multiple brain areas by different sets of pyramidal cells (PCs). PCs form intra- and inter-laminar subnetworks, depending on PC projection subtypes. However, it remains unknown how individual PC subtypes are involved in cortical network activity and, thereby, in distinct brain functions. Here, we examined the effects of optogenetic manipulations of specific PC subtypes on network activity in the motor cortex. In layer V, the beta/gamma frequency band of oscillation was evoked by photostimulation, depending on PC subtypes. Our experimental and simulation results suggest that oscillatory activity is generated in reciprocal connections between pyramidal tract (PT) and fast-spiking cells. A similar frequency band was also observed in local field potentials during a pattern learning task. Manipulation of PT cell activity affected beta/gamma band power and learning. Our results suggest that PT cell-dependent oscillations play important roles in motor learning.