Spontaneous Neuronal Network Activity Research Articles

In‐vitro exposure of neuronal networks to the GSM‐1800 signal

The central nervous system is the most likely target of mobile telephony radiofrequency (RF) field exposure in terms of biological effects. Several electroencephalography (EEG) studies have reported variations in the alpha-band power spectrum during and/or after RF exposure, in resting EEG and during sleep. In this context, the observation of the spontaneous electrical activity of neuronal networks under RF exposure can be an efficient tool to detect the occurrence of low-level RF effects on the nervous system. Our research group has developed a dedicated experimental setup in the GHz range for the simultaneous exposure of neuronal networks and monitoring of electrical activity. A transverse electromagnetic (TEM) cell was used to expose the neuronal networks to GSM-1800 signals at a SAR level of 3.2 W/kg. Recording of the neuronal electrical activity and detection of the extracellular spikes and bursts under exposure were performed using microelectrode arrays (MEAs). This work provides the proof of feasibility and preliminary results of the integrated investigation regarding exposure setup, culture of the neuronal network, recording of the electrical activity, and analysis of the signals obtained under RF exposure. In this pilot study on 16 cultures, there was a 30% reversible decrease in firing rate (FR) and bursting rate (BR) during a 3 min exposure to RF. Additional experiments are needed to further characterize this effect.

The emergence of spontaneous activity in neuronal cultures, coherence from noise

When neurons are grown in vitro and left unperturbed, they create their own network of connections and eventually reach a state where they fire simultaneously in a strikingly regular collective pattern of nearly periodic bursts [1]. Even though the spontaneous activity of neuronal networks is widely recognized as a fundamental problem in neuroscience, the mechanisms governing these bursts of spontaneous activity are not well understood. In neuronal cultures many conflicting views coexist, from burst initiation zones in 1D systems [2], to leading neurons [3] and network interactions [4] amongst others. Here we give new insights on the emergence of spontaneous activity in two-dimensional neuronal cultures and tackle this problem with two complementary approaches. Using high-speed calcium imaging techniques in cultures derived from cortical neurons of embryonic rat brains, we show that the collective bursts of activity are in fact propagating waves of activity that are locally nucleated. The nucleation develops in specific sites of the cultures that are independent of each other and have a well defined size. With in silico modelling, we elucidate the origin of the bursting activity in terms of a new phenomenon that we call noise focusing, a fast nucleation mechanism that relies both on detailed network topology and on the neurons' integrate-and-fire dynamics. The phenomenon consists of a selective, highly directional noise amplification mechanism that drives activity avalanches towards a few focal points that act as noise attractors. The resulting scenario challenges common understanding of neuronal cultures while providing a simple physical explanation of all the existing data with no appeal to any biological guidance of the network architecture.

Concurrent scale-free and small-world networks support criticality in cortical ensembles

Nowadays, it is widely accepted that structural features of cortical networks are tightly linked to aspects of brain function, playing a crucial role in determining which electrophysiological patterns (and thus, brain states) can and cannot occur. In this work, we investigated the interplay between network topology and spontaneous dynamics within the framework of neuronal avalanches and self-organized criticality [1]. The aim of this study is to sustain the hypothesis that the emergence of critical states, which in their turn would optimize functional properties in the cortex, is supported by specific complex network topologies. We followed a computational approach by developing large-scale network models mimicking the electrophysiological patterns (i.e., spiking and bursting activity) displayed by in vitro cortical networks coupled to Micro-Electrode Arrays (MEAs). In this preparation, neurons are able to freely re-create networks that exhibit complex and highly variable spatio-temporal patterns of activity [2]. As neurons grow without external constraints and self-organize depending on many parameters, we do not have any clue about the resulting morphological connectivity. Moreover, some cultures actually exhibit scale-free distributions of neuronal avalanches, a hallmark of SOC, thus demonstrating that they preserve self-organization properties featured by in vivo-formed cell assemblies [3]. Therefore, cortical networks actually show different states (critical, subcritical or supercritical), but their relationship with the underlying connectivity remains unknown [4]. Due to the difficulties of determining the network topology of our cultures from a limited number of recording sites (60 microelectrodes), we took advantage of a computational model consisting of a neuronal network made up of 1024 Izhikevich neurons [5]. Network topologies were designed following the canonical architectures scale-free, random, and small-world [6]. We simulated the spontaneous activity of such neuronal networks, by sweeping the most common parameters used to characterize these graphs, such as clustering coefficient, connection density, etc. [7]. The main finding which emerges is that although all the network configurations determine a mix of spiking, and bursting activity, the scale-free with small-world features display a critical behavior.

NeuVision: A novel simulation environment to model spontaneous and stimulus-evoked activity of large-scale neuronal networks

NeuVision is a novel simulation environment tailored to simulate the dynamics of large-scale neuronal networks. It has been developed using .NET programming languages and allows the user to implement networks of neurons made up of thousands of synaptically connected elements. This simulator (available at the website http://www.neuvisionsimulator.org) offers the user several visualization tools. At the same time, a rich repertoire of data analysis algorithms to process both spontaneous and stimulus-evoked activity has been introduced to create a complete package for the generation and analysis of synthetic data. In this paper, we provide a detailed description of the simulation environment, its features, and an estimation of the performance. In addition, we present some results related to the simulation of highly connected large-scale neuronal networks focusing on the stimulus-evoked activity and the propagation of network activity. We found that the simulated results qualitatively fit experimental recordings obtained by recently developed high density multi-electrode transducers for in vitro applications. In this context, NeuVision can be utilized along with multi-site recordings to understand neural networks dynamics.

Differential responses to ω-agatoxin IVA in murine frontal cortex and spinal cord derived neuronal networks

ω-Agatoxin-IVA is a well known P/Q-type Ca2+ channel blocker and has been shown to affect presynaptic Ca2+ currents as well postsynaptic potentials. P/Q-type voltage gated Ca2+ channels play a vital role in presynaptic neurotransmitter release and thus play a role in action potential generation. Monitoring spontaneous activity of neuronal networks on microelectrode arrays (MEAs) provides an important tool for examining this neurotoxin. Changes in extracellular action potentials are readily observed and are dependent on synaptic function. Given the efficacy of murine frontal cortex and spinal cord networks to detect neuroactive substances, we investigated the effects of ω-agatoxin on spontaneous action potential firing within these networks. We found that networks derived from spinal cord are more sensitive to the toxin than those from frontal cortex; a concentration of only 10nM produced statistically significant effects on activity from spinal cord networks whereas 50nM was required to alter activity in frontal cortex networks. Furthermore, the effects of the toxin on frontal cortex are more complex as unit specific responses were observed. These manifested as either a decrease or increase in action potential firing rate which could be statistically separated as unique clusters. Administration of bicuculline, a GABAA inhibitor, isolated a single response to ω-agatoxin, which was characterized by a reduction in network activity. These data support the notion that the two clusters detected with ω-agatoxin exposure represent differential responses from excitatory and inhibitory neuronal populations.

Seizure-like activity in hyaluronidase-treated dissociated hippocampal cultures

The extracellular matrix (ECM) plays an important role in use-dependent synaptic plasticity. Hyaluronic acid (HA) is the backbone of the neural ECM, which has been shown to modulate α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor mobility, paired-pulse depression, L-type voltage-dependent Ca2+ channel (L-VDCC) activity, long-term potentiation and contextual fear conditioning. To investigate the role of HA in the development of spontaneous neuronal network activity, we used microelectrode array recording and Ca2+ imaging in hippocampal cultures enzymatically treated with hyaluronidase. Our findings revealed an appearance of epileptiform activity 9 days after hyaluronidase treatment. The treatment transformed the normal network firing bursts and Ca2+ oscillations into long-lasting “superbursts” and “superoscillations” with durations of 11–100 s. The changes in Ca2+ transients in hyaluronidase-treated neurons were more prominent then in astrocytes and preceded changes in electrical activity. The Ca2+ superoscillations could be suppressed by applying the L-VDCC blocker diltiazem, whereas the neuronal firing superbursts could be additionally suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione as an antagonist of AMPA/kainate receptors. These results suggest that changes in the expression of HA can be epileptogenic and that hyaluronidase treatment in vitro provides a robust model for the dissection of the underlying mechanisms.

Non-normal amplification in random balanced neuronal networks

In dynamical models of cortical networks, the recurrent connectivity can amplify the input given to the network in two distinct ways. One is induced by the presence of near-critical eigenvalues in the connectivity matrix W, producing large but slow activity fluctuations along the corresponding eigenvectors (dynamical slowing). The other relies on W not being normal, which allows the network activity to make large but fast excursions along specific directions. Here we investigate the trade-off between non-normal amplification and dynamical slowing in the spontaneous activity of large random neuronal networks composed of excitatory and inhibitory neurons. We use a Schur decomposition of W to separate the two amplification mechanisms. Assuming linear stochastic dynamics, we derive an exact expression for the expected amount of purely non-normal amplification. We find that amplification is very limited if dynamical slowing must be kept weak. We conclude that, to achieve strong transient amplification with little slowing, the connectivity must be structured. We show that unidirectional connections between neurons of the same type together with reciprocal connections between neurons of different types, allow for amplification already in the fast dynamical regime. Finally, our results also shed light on the differences between balanced networks in which inhibition exactly cancels excitation and those where inhibition dominates.

Electrical activity patterns and the functional maturation of the neocortex

At the earliest developmental stages, sensory neocortical areas in various species reveal distinct patterns of spontaneous neuronal network activity. These activity patterns either propagate over large neocortical areas or synchronize local neuronal ensembles. In vitro and in vivo experiments indicate that these spontaneous activity patterns are generated from neuronal networks in the cerebral cortex, in subcortical structures or in the sensory periphery (retina, cochlea, whiskers). At early stages spontaneous periphery-driven and also sensory evoked activity is relayed to the developing cerebral cortex via the thalamus and the neocortical subplate, which amplifies the afferent sensory input. These early local and large-scale neuronal activity patterns influence a variety of developmental processes during corticogenesis, such as neurogenesis, apoptosis, neuronal migration, differentiation and network formation. The experimental data also indicate that disturbances in early neuronal patterns may have an impact on the development of cortical layers, columns and networks. In this article we review our current knowledge on the origin of early electrical activity patterns in neocortical sensory areas and their functional implications on shaping developing cortical networks.

Interdependence between network dynamics and connectivity in dissociated cortical cultures: a theoretical and experimental approach

In this work we aimed at investigating the interdependence between connectivity and dynamics in large-scale cortical networks cultured in vitro onto planar Micro-Electrode Arrays (MEAs). In this experimental model, neurons are free of predefined constraints and thus able to re-create networks that exhibit complex and highly variable spatio-temporal patterns of activity composed of synchronized bursts, mixed with random spikes. Starting from this experimental evidence, here we address the questions: does a particular network architecture promote such dynamics? Is it possible to predict the activity of a neuronal network on the basis of its connectivity map? We determined the dynamic state of the network according to the statistical distribution of neuronal avalanches (namely critical, sub-critical or super-critical) [1,2]. Due to the difficulties of determining the network topology of our cultures from a limited number of recording sites (60 microelectrodes), we took advantage of a computational model consisting of a neuronal network made up of 1024 Izhikevich neurons [3]. Network topologies were designed following the canonical architectures scale-free, random, and small-world [4]. Within this approach, the network is dealt as a graph, where each neuron corresponds to a node, and each synaptically connections to an edge. We simulated the spontaneous activity of such neuronal networks, by sweeping the most common parameters used to characterize these graphs, such as clustering coefficient, connection density, etc. [5]. The main finding which emerges is that although all the network configurations determine a mix of spiking, and bursting activity, the scale-free and partially small-world architectures display a critical behavior, and thus self-organization could be influenced by the network architecture.

Enhanced synaptic activity and epileptiform events in the embryonic KCC2 deficient hippocampus

The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for protein) and Kcc2 (for gene)] is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2+/+) and Kcc2 deficient (Kcc2−/−) mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when KCC2 is thought not to be functional in the hippocampus. Embryonic Kcc2−/− hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABA-ergic postsynaptic currents than naïve age matched neurons. However, intracellular chloride concentration ([Cl−]i) and the reversal potential of GABA-mediated currents (EGABA) were similar in embryonic Kcc2+/+ and Kcc2−/− CA3 neurons. In addition, KCC2 immunolabeling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, KCC2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABA-ergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which KCC2 operates early as a chloride exporter or by transporter independent actions of KCC2 that are instrumental in synapse formation and networks construction.

Spatial-temporal dynamics of chaotic behavior in cultured hippocampal networks

Using multiple nonlinear techniques, we revealed the existence of chaos in the spontaneous activity of neuronal networks in vitro. The spatial-temporal dynamics of these networks indicated that emergent transition between chaotic behavior and superburst occurred periodically in low-frequency oscillations. An analysis of network-wide activity indicated that chaos was synchronized among different sites. Moreover, we found that the degree of chaos increased as the number of active sites in the network increased during long-term development (over three months in vitro). The chaotic behavior of the dissociated networks had similar spatial-temporal characteristics (rapid transition, periodicity, and synchronization) as the intact brain; however, the degree of chaos depended on the number of active sites at the mesoscopic level. This work could provide insight into neural coding and neurocybernetics.

Calmodulin Controls Synaptic Strength via Presynaptic Activation of Calmodulin Kinase II

Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by approximately 70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIalpha (CaMKIIalpha); in contrast, in control neurons, expression of constitutively active CaMKIIalpha had no effect on presynaptic release. Viewed together, these data suggest that calmodulin performs a major function in boosting synaptic strength via direct activation of presynaptic calmodulin-dependent kinase II.

Glycine Release from Radial Cells Modulates the Spontaneous Activity and Its Propagation during Early Spinal Cord Development

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.

Transient reduction of spontaneous neuronal network activity by sublethal amyloid β (1–42) peptide concentrations

Soluble amyloid beta(1-42) (A beta(1-42)) peptide has recently been assigned a key role in early Alzheimer's disease (AD) pathophysiology accounting for synaptic dysfunction before amyloid plaque formation and neurodegeneration can occur. Following sublethal A beta(1-42) administration, we observed an acute but transient reduction of the spike and burst rate of spontaneously active cortical networks cultured on microelectrode arrays. This simple experimental system appears suitable for future long-term pharmacological and genetic studies of A beta(1-42) signaling, thus providing a valuable new tool in AD research.

EARLY EFFECTS OF CLOZAPINE TREATMENT ON THE GLUCOSE-INSULINHOMEOSTASIS IN PATIENTS WITH SCHIZOPHRENIA

The therapy of patients suffering the psychiatric disorder schizophrenia requires the usage of antipsychotic drugs that are classified into two different groups, the first-generation (FGAs) and the second-generation antipsychotics (SGAs).This study compares the effects of the two FGAs haloperidol and flupentixol with those of the SGA olanzapine on synapse formation and synaptic activity of embryonic rat hippocampal neurons. To this end, the development of perineuronal nets (PNNs), the formation of synapses and the resulting spontaneous network activity under control and treatment conditions were studied using an indirect co-culture system of neurons and astrocytes in completely defined media.The number and extent of PNNs that consist of extracellular matrix superstructures surrounding synapses was not altered in hippocampal neurons by exposure to antipsychotic drugs. In contrast treatment of hippocampal neurons with haloperidol led to a slight decrease whereas olanzapine induced a significant increase of the number of structural synapses after 13 days. This differential effect concerning synapse numbers was also reflected in the spontaneous activity of neuronal networks, as monitored on multielectrode arrays (MEAs). In that context, application of haloperidol reduced while olanzapine significantly enhanced network activity. Unexpectedly, flupentixol that is regarded as an FGA caused similar effects than the SGA olanzapine in that it augmented synapse number as well as network activity. Our pilot study provides a proof of concept that the neuron-astrocyte co-culture model can be used to investigate the impact of antipsychotics on pivotal parameters of neuronal cell biology. Thereby, it may support the comparative analysis of antipsychotics applied in the therapy of schizophrenia.

Low-Frequency Stimulation Induces Stable Transitions in Stereotypical Activity in Cortical Networks

Reverberating spontaneous synchronized brain activity is believed to play an important role in neural information processing. Whether and how external stimuli can influence this spontaneous activity is poorly understood. Because periodic synchronized network activity is also prominent in in vitro neuronal cultures, we used cortical cultures grown on multielectrode arrays to examine how spontaneous activity is affected by external stimuli. Spontaneous network activity before and after low-frequency electrical stimulation was quantified in several ways. Our results show that the initially stable pattern of stereotypical spontaneous activity was transformed into another activity pattern that remained stable for at least 1h. The transformations consisted of changes in single site and culture-wide network activity as well as in the spatiotemporal dynamics of network bursting. We show for the first time that low-frequency electrical stimulation can induce long-lasting alterations in spontaneous activity of cortical neuronal networks. We discuss whether the observed transformations in network activity could represent a switch in attractor state.

Complete inhibition of spontaneous activity in neuronal networks in vitro by deltamethrin and permethrin

Types I and II pyrethroid insecticides cause temporally distinct decreases in voltage-gated sodium channel (VGSC) inactivation rates that are proposed to underlie their characteristic differences in toxicity signs. How alterations in VGSC channel function give rise to the characteristic differences in signs of pyrethroid intoxication is not completely understood, particularly those changes that occur in functional networks of interconnected neurons. To characterize better pyrethroid actions at the network level, effects of the Type I pyrethroid permethrin (PM) and the Type II pyrethroid deltamethrin (DM) on spontaneous glutamate network-dependent spikes and bursts were investigated in primary cultures of frontal cortex or spinal cord neurons grown on microelectrode arrays (MEAs). Fast GABAergic transmission was blocked by BIC, and concentration-dependent effects of DM (1 nM to 5 μM) and PM (10 nM to 50 μM) were examined. Both compounds caused concentration-dependent reductions in the network spike and burst rates. DM was more potent than PM, with IC 50 values of ∼0.13 and ∼4 μM for inhibition of spike rate in cortical and spinal cord neurons, respectively. Both compounds decreased the percentage of spikes that occurred within a burst and increased the interspike interval within bursts. Onset of effects was rapid, but recovery from total activity loss was not readily achievable. Individual neurons responded heterogeneously; activity of most declined monophasically, but activity in others exhibited biphasic responses with increases followed by decreases in activity. In spinal cord, DM caused a greater number of biphasic responses (29%) than PM (10%). These results demonstrate that both DM and PM inhibit activity of glutamatergic networks, but with different potencies.

Coemergence of regularity and complexity during neural network development

With the growing recognition that rhythmic and oscillatory patterns are widespread in the brain and play important roles in all aspects of the function of our nervous system, there has been a resurgence of interest in neuronal synchronized bursting activity. Here, we were interested in understanding the development of synchronized bursts as information-bearing neuronal activity patterns. For that, we have monitored the morphological organization and spontaneous activity of neuronal networks cultured on multielectrode-arrays during their self-executed evolvement from a mixture of dissociated cells into an active network. Complex collective network electrical activity evolved from sporadic firing patterns of the single neurons. On the system (network) level, the activity was marked by bursting events with interneuronal synchronization and nonarbitrary temporal ordering. We quantified these individual-to-collective activity transitions using newly-developed system level quantitative measures of time series regularity and complexity. We found that individual neuronal activity before synchronization was characterized by high regularity and low complexity. During neuronal wiring, there was a transient period of reorganization marked by low regularity, which then leads to coemergence of elevated regularity and functional (nonstochastic) complexity. We further investigated the morphology-activity interplay by modeling artificial neuronal networks with different topological organizations and connectivity schemes. The simulations support our experimental results by showing increased levels of complexity of neuronal activity patterns when neurons are wired up and organized in clusters (similar to mature real networks), as well as network-level activity regulation once collective activity forms.

On the Dynamics of the Spontaneous Activity in Neuronal Networks

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABAA receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics.

Substance identification by quantitative characterization of oscillatory activity in murine spinal cord networks on microelectrode arrays.

This paper presents a novel and comprehensive method to identify substances on the basis of electrical activity and is a substantial improvement for drug screening. The spontaneous activity of primary neuronal networks is influenced by neurotransmitters, ligands, and other substances in a similar fashion as known from in vivo pharmacology. However, quantitative methods for the identification of substances through their characteristic effects on network activity states have not yet been reported. We approached this problem by creating a database including native activity and five drug-induced oscillatory activity states from extracellular multisite recordings from microelectrode arrays. The response profiles consisted of 30 activity features derived from the temporal distribution of action potentials, integrated burst properties, calculated coefficients of variation, and features of Gabor fits to autocorrelograms. The different oscillatory states were induced by blocking neurotransmitter receptors for: (i) GABA(A); (ii) glycine; (iii) GABA(A) and glycine; (iv) all major synaptic types except AMPA, and (v) all major synapses except NMDA. To test the identification capability of the six substance-specific response profiles, five blind experiments were performed. The response features from the unknown substances were compared to the database using proximity measures using the normalized Euclidian distance to each activity state. This process created six identification coefficients where the smallest correctly identified the unknown substances. Such activity profiles are expected to become substance-specific 'finger prints' that classify unique responses to known and unknown substances. It is anticipated that this kind of approach will help to quantify pharmacological responses of networks used as biosensors.