Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.     

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.     

Activity-dependent clustering of functional synaptic inputs on developing hippocampal dendrites

During brain development, before sensory systems become functional, neuronal networks spontaneously generate repetitive bursts of neuronal activity, which are typically synchronized across many neurons. Such activity patterns have been described on the level of networks and cells, but the fine-structure of inputs received by an individual neuron during spontaneous network activity has not been studied. Here, we used calcium imaging to record activity at many synapses of hippocampal pyramidal neurons simultaneously to establish the activity patterns in the majority of synapses of an entire cell. Analysis of the spatiotemporal patterns of synaptic activity revealed a fine-scale connectivity rule: neighboring synapses (<16?μm intersynapse distance) are more likely to be coactive than synapses that are farther away from each other. Blocking spiking activity or NMDA receptor activation revealed that the clustering of synaptic inputs required neuronal activity, demonstrating a role of developmentally expressed spontaneous activity for connecting neurons with subcellular precision.     

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.     

Management of synchronized network activity by highly active neurons

Increasing evidence supports the idea that spontaneous brain activity may have an important functional role. Cultured neuronal networks provide a suitable model system to search for the mechanisms by which neuronal spontaneous activity is maintained and regulated. This activity is marked by synchronized bursting events (SBEs)--short time windows (hundreds of milliseconds) of rapid neuronal firing separated by long quiescent periods (seconds). However, there exists a special subset of rapidly firing neurons whose activity also persists between SBEs. It has been proposed that these highly active (HA) neurons play an important role in the management (i.e. establishment, maintenance and regulation) of the synchronized network activity. Here, we studied the dynamical properties and the functional role of HA neurons in homogeneous and engineered networks, during early network development, upon recovery from chemical inhibition and in response to electrical stimulations. We found that their sequences of inter-spike intervals (ISI) exhibit long time correlations and a unimodal distribution. During the network's development and under intense inhibition, the observed activity follows a transition period during which mostly HA neurons are active. Studying networks with engineered geometry, we found that HA neurons are precursors (the first to fire) of the spontaneous SBEs and are more responsive to electrical stimulations.     

NAD causes dissociation of neural networks into subpopulations of neurons by inhibiting the network synchronous hyperactivity evoked by ammonium ions

The mechanisms of hyperexcitability of neuronal networks by ammonium ions and inhibition of this activity by coenzyme NAD were investigated on mixed neuro-glial cultures of rat hippocampus. Ammonium ions cause activation of silent or spontaneously active neuronal networks inducing a bursting electrical activity of neurons and high-frequency synchronous calcium oscillations. In control conditions NAD completely inhibits spontaneous activity of the neuronal network. NAD added after NH₄Cl disrupts synchronous oscillation in neurons and splits the network into five populations of neurons. In 4% of cells NAD decreased the amplitude of Ca²⁺ oscillations, preserving initial mode of oscillations. In 32% of cells, a transient suppression of the neuronal oscillations was observed: inhibition was followed by restoration of the synchronous periodic activity. In 10% of cells, NAD produced a gradual decrease of Ca²⁺ oscillations down to a complete termination of the initial periodic activity induced by ammonium. Fast and total inhibition of Ca²⁺ oscillations by NAD was observed in two small groups of neurons. First group (A) participated in the initial spontaneous network activity (5% of cells) with a period of 66–100 s. Second group (B), on the contrary, did not participate in the spontaneous activity. This group of neurons began to pulse with a high frequency (with a period of 6–8 s) synchronously with other neurons in the network after the addition of NH₄Cl. Based on the comparison of calcium responses of different cell groups to the depolarization caused by KCl and NH₄Cl and to the application of domoic acid, as well as on the results obtained in experiments with fluorescent antibodies against GAD 65/67, parvalbumin, calretinin, and calbindin, we propose that neurons of populations (A) and (B) may belong to GABAergic neurons containing calbindin and parvalbumin, respectively. Further analysis of specificity of the NAD effect on these neuronal groups may allow identification of the main targets of the ammonium toxic action in the brain. Thus, we have shown that NAD selectively inhibits neuronal activity and high-frequency synchronous Ca²⁺ oscillations in GABAergic neurons containing calcium-binding proteins. The inhibition is accompanied by desynchronization of oscillations and dissociation of neuronal network into several populations.     

Modulation of neural network activity by patterning.

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.     

Activity-dependent regulation of dendritic spine density on cortical pyramidal neurons in organotypic slice cultures

In order to examine the effects of activity on spine production and/or maintenance in the cerebral cortex, we have compared the number of dendritic spines on pyramidal neurons in slices of PO mouse somatosensory cortex maintained in organotypic slice cultures under conditions that altered basal levels of spontaneous electrical activity. Cultures chronically exposed to 100 μM picrotoxin (PTX) for 14 days exhibited significantly elevated levels of electrical activity when compared to neurons in control cultures. Pyramidal neurons raised in the presence of PTX showed significantly densities of dendritic spines on primary apical, secondary apical, and secondary basal dendrites when compared to control cultures. The PTX-induced increase in spine density was dose dependent and appeared to saturate at 100 μM. Cultures exhibiting little or no spontaneous activity, as a result of growth in a combination of PTX and tetrodotoxin (TTx), showed significantly fewer dendritic spines compared to cultures maintained in PTX alone. These results demonstrate that the density of spines on layers V and VI pyramidal neurons can be modulated by growth conditions that alter the levels of spontaneous electrical activity. 1994 John Wiley & Sons, Inc.     

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 GABA(A) 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.     

Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus

The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.     

An integrate-and-fire model for synchronized bursting in a network of cultured cortical neurons

It has been suggested that spontaneous synchronous neuronal activity is an essential step in the formation of functional networks in the central nervous system. The key features of this type of activity consist of bursts of action potentials with associated spikes of elevated cytoplasmic calcium. These features are also observed in networks of rat cortical neurons that have been formed in culture. Experimental studies of these cultured networks have led to several hypotheses for the mechanisms underlying the observed synchronized oscillations. In this paper, bursting integrate-and-fire type mathematical models for regular spiking (RS) and intrinsic bursting (IB) neurons are introduced and incorporated through a small-world connection scheme into a two-dimensional excitatory network similar to those in the cultured network. This computer model exhibits spontaneous synchronous activity through mechanisms similar to those hypothesized for the cultured experimental networks. Traces of the membrane potential and cytoplasmic calcium from the model closely match those obtained from experiments. We also consider the impact on network behavior of the IB neurons, the geometry and the small world connection scheme. Action Editor: David Golomb     

Effects of Isoflurane Anesthesia on Ensemble Patterns of Ca2+ Activity in Mouse V1: Reduced Direction Selectivity Independent of Increased Correlations in Cellular Activity

Anesthesia affects brain activity at the molecular, neuronal and network level, but it is not well-understood how tuning properties of sensory neurons and network connectivity change under its influence. Using in vivo two-photon calcium imaging we matched neuron identity across episodes of wakefulness and anesthesia in the same mouse and recorded spontaneous and visually evoked activity patterns of neuronal ensembles in these two states. Correlations in spontaneous patterns of calcium activity between pairs of neurons were increased under anesthesia. While orientation selectivity remained unaffected by anesthesia, this treatment reduced direction selectivity, which was attributable to an increased response to the null-direction. As compared to anesthesia, populations of V1 neurons coded more mutual information on opposite stimulus directions during wakefulness, whereas information on stimulus orientation differences was lower. Increases in correlations of calcium activity during visual stimulation were correlated with poorer population coding, which raised the hypothesis that the anesthesia-induced increase in correlations may be causal to degrading directional coding. Visual stimulation under anesthesia, however, decorrelated ongoing activity patterns to a level comparable to wakefulness. Because visual stimulation thus appears to ‘break’ the strength of pairwise correlations normally found in spontaneous activity under anesthesia, the changes in correlational structure cannot explain the awake-anesthesia difference in direction coding. The population-wide decrease in coding for stimulus direction thus occurs independently of anesthesia-induced increments in correlations of spontaneous activity.     

Targeted optical probing of neuronal circuit dynamics using fluorescent protein sensors

Interest in non-invasive methods for optical probing of neuronal electrical activity has been ongoing for several decades and methods for imaging the activity of single or multiple individual neurons in networks composed of thousands of neurons have been developed. Most widely used are techniques that use organic chemistry-based dyes as indicators of calcium and membrane potential. More recently a new generation of probes, genetically encoded fluorescent protein sensors, have emerged for use by physiologists studying the operation of neuronal circuits. In this review we describe the advance of these emerging optical techniques and compare them with more conventional approaches.     

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.     

Spontaneous coordinated activity in cultured networks: Analysis of multiple ignition sites,primary circuits,and burst phase delay distributions

All higher order central nervous systems exhibit spontaneous neural activity, though the purpose and mechanistic origin of such activity remains poorly understood. We quantitatively analyzed the ignition and spread of collective spontaneous electrophysiological activity in networks of cultured cortical neurons growing on microelectrode arrays. Leader neurons, which form a mono-synaptically connected primary circuit, and initiate a majority of network bursts were found to be a small subset of recorded neurons. Leader/follower firing delay times formed temporally stable positively skewed distributions. Blocking inhibitory synapses usually resulted in shorter delay times with reduced variance. These distributions are characterizations of general aspects of internal network dynamics and provide estimates of pair-wise synaptic distances. The resulting analysis produced specific quantitative constraints and insights into the activation patterns of collective neuronal activity in self-organized cortical networks, which may prove useful for models emulating spontaneously active systems.     

Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurons in chronic epilepsy. Discerning the mechanisms underlying signal integration within EC neurons is essential for understanding network excitability alterations involving the hippocampus during epilepsy. Twenty-four hours following a single seizure episode when there were no behavioral or electrographic seizures, we found enhanced spontaneous activity still present in the rat EC in vivo and in vitro. The increased excitability was accompanied by a profound reduction in I(h) in EC layer III neurons and a significant decline in HCN1 and HCN2 subunits that encode for h channels. Consequently, dendritic excitability was enhanced, resulting in increased neuronal firing despite hyperpolarized membrane potentials. The loss of I(h) and the increased neuronal excitability persisted for 1 week following seizures. Our results suggest that dendritic I(h) plays an important role in determining the excitability of EC layer III neurons and their associated neural networks.     

Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations

For the analysis of neuronal networks it is an important yet unresolved task to relate the neurons' activities to their morphology. Here we introduce activity correlation imaging to simultaneously visualize the activity and morphology of populations of neurons. To this end we first stain the network's neurons using a membrane-permeable [Ca²⁺] indicator (e.g., Fluo-4/AM) and record their activities. We then exploit the recorded temporal activity patterns as a means of intrinsic contrast to visualize individual neurons' dendritic morphology. The result is a high-contrast, multicolor visualization of the neuronal network. Taking the Xenopus olfactory bulb as an example we show the activities of the mitral/tufted cells of the olfactory bulb as well as their projections into the olfactory glomeruli. This method, yielding both functional and structural information of neuronal populations, will open up unprecedented possibilities for the investigation of neuronal networks.     

Shaping Neuronal Network Activity by Presynaptic Mechanisms

Neuronal microcircuits generate oscillatory activity, which has been linked to basic functions such as sleep, learning and sensorimotor gating. Although synaptic release processes are well known for their ability to shape the interaction between neurons in microcircuits, most computational models do not simulate the synaptic transmission process directly and hence cannot explain how changes in synaptic parameters alter neuronal network activity. In this paper, we present a novel neuronal network model that incorporates presynaptic release mechanisms, such as vesicle pool dynamics and calcium-dependent release probability, to model the spontaneous activity of neuronal networks. The model, which is based on modified leaky integrate-and-fire neurons, generates spontaneous network activity patterns, which are similar to experimental data and robust under changes in the model''s primary gain parameters such as excitatory postsynaptic potential and connectivity ratio. Furthermore, it reliably recreates experimental findings and provides mechanistic explanations for data obtained from microelectrode array recordings, such as network burst termination and the effects of pharmacological and genetic manipulations. The model demonstrates how elevated asynchronous release, but not spontaneous release, synchronizes neuronal network activity and reveals that asynchronous release enhances utilization of the recycling vesicle pool to induce the network effect. The model further predicts a positive correlation between vesicle priming at the single-neuron level and burst frequency at the network level; this prediction is supported by experimental findings. Thus, the model is utilized to reveal how synaptic release processes at the neuronal level govern activity patterns and synchronization at the network level.