Entropy estimation within in vitro neural-astrocyte networks as a measure of development instability.

The brain demands a significant fraction of the energy budget in an organism; in humans, it accounts for 2% of the body mass, but utilizes 20% of the total energy metabolized. This is due to the large load required for information processing; spiking demands from neurons are high but are a key component to understanding brain functioning. Astrocytic brain cells contribute to the healthy functioning of brain circuits by mediating neuronal network energy and facilitating the formation and stabilization of synaptic connectivity. During development, spontaneous activity influences synaptic formation, shaping brain circuit construction, and adverse astrocyte mutations can lead to pathological processes impacting cognitive impairment due to inefficiencies in network spiking activity. We have developed a measure that quantifies information stability within in vitro networks consisting of mixed neural-astrocyte cells. Brain cells were harvested from mice with mutations to a gene associated with the strongest known genetic risk factor for Alzheimer's disease, APOE. We calculate energy states of the networks and using these states, we present an entropy-based measure to assess changes in information stability over time. We show that during development, stability profiles of spontaneous network activity are modified by exogenous astrocytes and that network stability, in terms of the rate of change of entropy, is allele dependent.

Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks.

Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca2+ and are capable of regulating the release of glutamate and γ-aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory-inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed-pregnant Sprague-Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath-applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array. We found that nicotine modulated network dynamics in a concentration-dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that ß4-containing nAChRs are necessary for the observed increases in spiking, bursting, and synchrony, while the activation of α7 nAChRs augments nicotine-mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca2+ /calmodulin-dependent kinase II (CaMKII) and serine 831 phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network-wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Dopamine-dependent effects on basal and glutamate stimulated network dynamics in cultured hippocampal neurons.

Oscillatory activity occurs in cortical and hippocampal networks with specific frequency ranges thought to be critical to working memory, attention, differentiation of neuronal precursors, and memory trace replay. Synchronized activity within relatively large neuronal populations is influenced by firing and bursting frequency within individual cells, and the latter is modulated by changes in intrinsic membrane excitability and synaptic transmission. Published work suggests that dopamine, a potent modulator of learning and memory, acts on dopamine receptor 1-like dopamine receptors to influence the phosphorylation and trafficking of glutamate receptor subunits, along with long-term potentiation of excitatory synaptic transmission in striatum and prefrontal cortex. Prior studies also suggest that dopamine can influence voltage gated ion channel function and membrane excitability in these regions. Fewer studies have examined dopamine's effect on related endpoints in hippocampus, or potential consequences in terms of network burst dynamics. In this study, we record action potential activity using a microelectrode array system to examine the ability of dopamine to modulate baseline and glutamate-stimulated bursting activity in an in vitro network of cultured murine hippocampal neurons. We show that dopamine stimulates a dopamine type-1 receptor-dependent increase in number of overall bursts within minutes of its application. Notably, however, at the concentration used herein, dopamine did not increase the overall synchrony of bursts between electrodes. Although the number of bursts normalizes by 40 min, bursting in response to a subsequent glutamate challenge is enhanced by dopamine pretreatment. Dopamine-dependent potentiation of glutamate-stimulated bursting was not observed when the two modulators were administered concurrently. In parallel, pretreatment of murine hippocampal cultures with dopamine stimulated lasting increases in the phosphorylation of the glutamate receptor subunit GluA1 at serine 845. This effect is consistent with the possibility that enhanced membrane insertion of GluAs may contribute to a more slowly evolving dopamine-dependent potentiation of glutamate-stimulated bursting. Together, these results are consistent with the possibility that dopamine can influence hippocampal bursting by at least two temporally distinct mechanisms, contributing to an emerging appreciation of dopamine-dependent effects on network activity in the hippocampus.

The University of Ibadan/Grass Foundation Workshop in Neuroscience Teaching.

The University of Ibadan/Grass Foundation Workshop in Neuroscience Teaching (March 31st to April 2nd, 2017) in Ibadan, Nigeria was sponsored by the Grass Foundation as a "proof of principle" outreach program for young neuroscience faculty at Nigerian universities with limited educational and research resources. The workshop's goal was to introduce low cost equipment for student lab exercises and computational tutorials that could enhance the teaching and research capabilities of local neuroscience educators. Participant assessment of the workshop's activities was very positive and suggested that similar workshops for other faculty from institutions with limited resources could have a great impact on the quality of both the undergraduate and faculty experience.

Long-Term Dynamical Constraints on Pharmacologically Evoked Potentiation Imply Activity Conservation within In Vitro Hippocampal Networks.

This paper describes a long-term study of network dynamics from in vitro, cultured hippocampal neurons after a pharmacological induction of synaptic potentiation. We plate a suspension of hippocampal neurons on an array of extracellular electrodes and record electrical activity in the absence of the drugs several days after treatment. While previous studies have reported on potentiation lasting up to a few hours after treatment, to the best of our knowledge, this is the first report to characterize the network effects of a potentiating mechanism several days after treatment. Using this reduced, two-dimensional in vitro network of hippocampal neurons, we show that the effects of potentiation are persistent over time but are modulated under a conservation of spike principle. We suggest that this conservation principle might be mediated by the appearance of a resonant inter-spike interval that prevents the network from advancing towards a state of hyperexcitability.

Synaptic potentiation facilitates memory-like attractor dynamics in cultured in vitro hippocampal networks.

Collective rhythmic dynamics from neurons is vital for cognitive functions such as memory formation but how neurons self-organize to produce such activity is not well understood. Attractor-based computational models have been successfully implemented as a theoretical framework for memory storage in networks of neurons. Additionally, activity-dependent modification of synaptic transmission is thought to be the physiological basis of learning and memory. The goal of this study is to demonstrate that using a pharmacological treatment that has been shown to increase synaptic strength within in vitro networks of hippocampal neurons follows the dynamical postulates theorized by attractor models. We use a grid of extracellular electrodes to study changes in network activity after this perturbation and show that there is a persistent increase in overall spiking and bursting activity after treatment. This increase in activity appears to recruit more "errant" spikes into bursts. Phase plots indicate a conserved activity pattern suggesting that a synaptic potentiation perturbation to the attractor leaves it unchanged. Lastly, we construct a computational model to demonstrate that these synaptic perturbations can account for the dynamical changes seen within the network.

Estimating the structure of small dynamical networks from the state time evolution of one node.

We consider small dynamical networks of coupled oscillators for which the network topology is unknown and try to use partial knowledge of the oscillators' dynamics to estimate both the network couplings and the states of the nodes. We focus on the case where the state time evolution from only one oscillator is available. We propose an adaptive strategy that uses synchronization between the true network and a replica network in order to estimate both the couplings and the states. The adaptive scheme is tested with several modules of coupled oscillators. We consider the effects of small mismatches in the parameters of the individual oscillators and we propose an alternative version of the strategy that is suitable to handle noise in the received signal.

Hippocampal neuron firing and local field potentials in the in vitro 4-aminopyridine epilepsy model.

Excessive synchronous neuronal activity is a defining feature of epileptic activity. We previously characterized the properties of distinct glutamatergic and GABAergic transmission-dependent synchronous epileptiform discharges in mouse hippocampal slices using the 4-aminopyridine model of epilepsy. In the present study, we sought to identify the specific hippocampal neuronal populations that initiate and underlie these local field potentials (LFPs). A perforated multielectrode array was used to simultaneously record multiunit action potential firing and LFPs during spontaneous epileptiform activity. LFPs had distinct components based on the initiation site, extent of propagation, and pharmacological sensitivity. Individual units, located in different hippocampal subregions, fired action potentials during these LFPs. A specific neuron subgroup generated sustained action potential firing throughout the various components of the LFPs. The activity of this subgroup preceded the LFPs observed in the presence of antagonists of ionotropic glutamatergic synaptic transmission. In the absence of ionotropic glutamatergic and GABAergic transmission, LFPs disappeared, but units with shorter spike duration and high basal firing rates were still active. These spontaneously active units had an increased level of activity during LFPs and consistently preceded all LFPs recorded before blockade of synaptic transmission. Our findings reveal that neuronal subpopulations with interneuron properties are likely responsible for initiating synchronous activity in an in vitro model of epileptiform discharges.

MMPs and soluble ICAM-5 increase neuronal excitability within in vitro networks of hippocampal neurons.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are released from neurons in an activity dependent manner. Published studies suggest their activity is important to varied forms of learning and memory. At least one MMP can stimulate an increase in the size of dendritic spines, structures which represent the post synaptic component for a large number of glutamatergic synapses. This change may be associated with increased synaptic glutamate receptor incorporation, and an increased amplitude and/or frequency of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) mini excitatory post-synaptic currents (EPSCs). An associated increase in the probability of action potential occurrence would be expected. While the mechanism(s) by which MMPs may influence synaptic structure and function are not completely understood, MMP dependent shedding of specific cell adhesion molecules (CAMs) could play an important role. CAMs are ideally positioned to be cleaved by synaptically released MMPs, and shed N terminal domains could potentially interact with previously unengaged integrins to stimulate dendritic actin polymerization with spine expansion. In the present study, we have used multielectrode arrays (MEAs) to investigate MMP and soluble CAM dependent changes in neuronal activity recorded from hippocampal cultures. We have focused on intercellular adhesion molecule-5 (ICAM-5) in particular, as this CAM is expressed on glutamatergic dendrites and shed in an MMP dependent manner. We show that chemical long-term potentiation (cLTP) evoked changes in recorded activity, and the dynamics of action potential bursts in particular, are altered by MMP inhibition. A blocking antibody to ß(1) integrins has a similar effect. We also show that the ectodomain of ICAM-5 can stimulate ß(1) integrin dependent increases in spike counts and burst number. These results support a growing body of literature suggesting that MMPs have important effects on neuronal excitability. They also support the possibility that MMP dependent shedding of specific synaptic CAMs can contribute to these effects.

Cellular mechanisms of desynchronizing effects of hypothermia in an in vitro epilepsy model.

Hypothermia can terminate epileptiform discharges in vitro and in vivo epilepsy models. Hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy, and it is gaining ground as a potential treatment in patients with drug resistant epilepsy. However, the exact mechanism of action of cooling the brain tissue is unclear. We have studied the 4-aminopyridine model of epilepsy in mice using single- and dual-patch clamp and perforated multi-electrode array recordings from the hippocampus and cortex. Cooling consistently terminated 4-aminopyridine induced epileptiform-like discharges in hippocampal neurons and increased input resistance that was not mimicked by transient receptor potential channel antagonists. Dual-patch clamp recordings showed significant synchrony between distant CA1 and CA3 pyramidal neurons, but less so between the pyramidal neurons and interneurons. In CA1 and CA3 neurons, hypothermia blocked rhythmic action potential discharges and disrupted their synchrony; however, in interneurons, hypothermia blocked rhythmic discharges without abolishing action potentials. In parallel, multi-electrode array recordings showed that synchronized discharges were disrupted by hypothermia, whereas multi-unit activity was unaffected. The differential effect of cooling on transmitting or secreting γ-aminobutyric acid interneurons might disrupt normal network synchrony, aborting the epileptiform discharges. Moreover, the persistence of action potential firing in interneurons would have additional antiepileptic effects through tonic γ-aminobutyric acid release.

EphA4 expression promotes network activity and spine maturation in cortical neuronal cultures.

BACKGROUND: Neurons form specific connections with targets via synapses and patterns of synaptic connectivity dictate neural function. During development, intrinsic neuronal specification and environmental factors guide both initial formation of synapses and strength of resulting connections. Once synapses form, non-evoked, spontaneous activity serves to modulate connections, strengthening some and eliminating others. Molecules that mediate intercellular communication are particularly important in synaptic refinement. Here, we characterize the influences of EphA4, a transmembrane signaling molecule, on neural connectivity. RESULTS: Using multi-electrode array analysis on in vitro cultures, we confirmed that cortical neurons mature and generate spontaneous circuit activity as cells differentiate, with activity growing both stronger and more patterned over time. When EphA4 was over-expressed in a subset of neurons in these cultures, network activity was enhanced: bursts were longer and were composed of more spikes than in control-transfected cultures. To characterize the cellular basis of this effect, dendritic spines, the major excitatory input site on neurons, were examined on transfected neurons in vitro. Strikingly, while spine number and density were similar between conditions, cortical neurons with elevated levels of EphA4 had significantly more mature spines, fewer immature spines, and elevated colocalization with a mature synaptic marker. CONCLUSIONS: These results demonstrate that experimental elevation of EphA4 promotes network activity in vitro, supporting spine maturation, producing more functional synaptic pairings, and promoting more active circuitry.

The 4-aminopyridine in vitro epilepsy model analyzed with a perforated multi-electrode array.

Epileptiform discharges recorded in the 4-aminopyridine (4-AP) in vitro epilepsy model are mediated by glutamatergic and GABAergic signaling. Using a 60-channel perforated multi-electrode array (pMEA) on corticohippocampal slices from 2 to 3 week old mice we recorded interictal- and ictal-like events. When glutamatergic transmission was blocked, interictal-like events no longer initiated in the hilus or CA3/CA1 pyramidal layers but originated from the dentate gyrus granule and molecular layers. Furthermore, frequencies of interictal-like events were reduced and durations were increased in these regions while cortical discharges were completely blocked. Following GABA(A) receptor blockade interictal-like events no longer propagated to the dentate gyrus while their frequency in CA3 increased; in addition, ictal-like cortical events became shorter while increasing in frequency. Lastly, drugs that affect tonic and synaptic GABAergic conductance modulated the frequency, duration, initiation and propagation of interictal-like events. These findings confirm and expand on previous studies indicating that multiple synaptic mechanisms contribute to synchronize neuronal network activity in forebrain structures. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Observed network dynamics from altering the balance between excitatory and inhibitory neurons in cultured networks.

Complexity in the temporal organization of neural systems may be a reflection of the diversity of their neural constituents. These constituents, excitatory and inhibitory neurons, comprise a well-defined ratio in vivo and form the substrate for rhythmic oscillatory activity. To begin to elucidate the dynamical implications that underlie this balance, we construct neural circuits not ordinarily found in nature and study the resulting temporal patterns. We culture several networks of neurons composed of varying fractions of excitatory and inhibitory cells and use a multielectrode array to study their temporal dynamics as this balance is modulated. We use the electrode burst as the temporal imprimatur to signify the presence of network activity. Burst durations, interburst intervals, and the number of spikes participating within a burst are used to illustrate the vivid differences in the temporal organization between the various cultured networks. When the network consists largely of excitatory neurons, no network temporal structure is apparent. However, the addition of inhibitory neurons evokes a temporal order. Calculation of the temporal autocorrelation shows that when the number of inhibitory neurons is a major fraction of the network, a striking network pattern materializes when none was previously present.

Causal entropies--a measure for determining changes in the temporal organization of neural systems.

We propose a novel measure to detect temporal ordering in the activity of individual neurons in a local network, which is thought to be a hallmark of activity-dependent synaptic modifications during learning. The measure, called causal entropy, is based on the time-adaptive detection of asymmetries in the relative temporal patterning between neuronal pairs. We characterize properties of the measure on both simulated data and experimental multiunit recordings of hippocampal neurons from the awake, behaving rat, and show that the metric can more readily detect those asymmetries than standard cross correlation-based techniques, especially since the temporal sensitivity of causal entropy can detect such changes rapidly and dynamically.

Measuring asymmetric temporal interdependencies in simulated and biological networks.

We use a newly developed metric to characterize asymmetric temporal interdependencies in networks of coupled dynamical elements. We studied the formation of temporal ordering in a system of coupled Rossler oscillators for different connectivity ratios and network topologies and also applied the metric to investigate the functional structure of a biological network (cerebral ganglia of Helix snail). In the former example we show how the local ordering evolves to the global one as a function of structural parameters of the network, while in the latter we show spontaneous emergence of functional interdependence between two groups of electrodes.

Transition from local to global phase synchrony in small world neural network and its possible implications for epilepsy.

Temporal correlations in the brain are thought to have very dichotomous roles. On one hand they are ubiquitously present in the healthy brain and are thought to underlie feature binding during information processing. On the other hand, large-scale synchronization is an underlying mechanism of epileptic seizures. In this paper we show a potential mechanism for the transition to pathological coherence underlying seizure generation. We show that properties of phase synchronization in a two-dimensional lattice of nonidentical coupled Hindmarsh-Rose neurons change radically depending on the connectivity structure of the network. We modify the connectivity using the small world network paradigm and measure properties of phase synchronization using a previously developed measure based on assessment of the distributions of relative interspike intervals. We show that the temporal ordering undergoes a dramatic change as a function of topology of the network from local coherence strongly dependent on the distance between two neurons, to global coherence exhibiting a larger degree of ordering and spanning the whole network.

Adaptation through minimization of the phase lag in coupled nonidentical systems.

We show that the internal control of adaptation can be obtained from the properties of the phase lag that results from phase synchronization of two nonidentical chaotic oscillators. The direction and magnitude of the phase lag depend upon the relative internal properties of the coupled units, and they can be used as indicators during the adjustment of dynamics, i.e., adaptation of the target unit to match that of the control. The properties of the phase lag are obtained using a method based on the estimation of properties of the distributions of relative event times of both (target and control) units. The phase lag dependent mechanism to control the adaptation process was applied to a system of nonidentical Rössler oscillators and a system of nonidentical Lorenz oscillators. We also elucidate its importance as a control mechanism of the changes of neuronal activity showing its application to neural adaptation.

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. II: Experimental applications.

An experimental verification of an optical microscope technique to create spatial map images of dynamically scattered light fluctuation decay rates is presented. The dynamic light scattering microscopy technique is demonstrated on polystyrene beads and living macrophage cells. With a slow progressive scan charge-coupled device camera employed in a streak-like mode, rapid intensity fluctuations with timescales the order of milliseconds can be recorded from these samples. From such streak images, the autocorrelation function of these fluctuations can be computed at each location in the sample. The characteristic decay times of the autocorrelation functions report the rates of motion of scattering centers. These rates show reasonable agreement to theoretically expected values for known samples with good signal/noise ratio. The rates can be used to construct an image-like spatial map of the rapidity of submicroscopic motions of scattering centers.

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. I: theory.

The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy.