Characterization of synchronized bursts in cultured hippocampal neuronal networks with learning training on microelectrode arrays

Spontaneous synchronized bursts seem to play a key role in brain functions such as learning and memory. Still controversial is the characterization of spontaneous synchronized bursts in neuronal networks after learning training, whether depression or promotion. By taking advantages of the main features of the microelectrode array (MEA) technology (i.e. multisite recordings, stable and long-term coupling with the biological preparation), we analyzed changes of spontaneous synchronized bursts in cultured hippocampal neuronal networks after learning training. And for this purpose, a learning model at networking level on MEA system was constructed, and analysis of spontaneous synchronized burst activity modulation was presented. Preliminary results show that, the number of burst was increased by 154%, burst duration was increased by 35%, and the number of spikes per burst was increased by 124%, while interburst interval decreased by 44% with learning. In particular, correlation and synchrony of neuronal activities in networks were enhanced by 51% and 36%, respectively, with learning. In contrast, dynamic properties of neuronal networks were not changed much when the network was under “non-learning” condition. These results indicate that firing, association and synchrony of spontaneous bursts in neuronal networks were promoted by learning. Furthermore, from these observations, we are encouraged to think of a more engineered system based on in vitro hippocampal neurons, as a novel sensitive system for electrophysiological evaluations.     

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.     

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     

MEA-based recording of neuronal activity in vitro

Based on the advantages of MEA-based recording, developmental changes of spontaneous activity and tetanus-induced modification of evoked activity were studied. Rat cortical neurons were cultured on MEAs and the spontaneous activity was continuously monitored for two months. The activity started a few days after plating. During the second week, the cultures generated periodic synchronized bursts, which were the characteristic properties of cortical neurons in vitro. In about one month, the cultured networks reached a steady state. Between these two, we found a critical period during which only weak activities were generated. This critical period might reflect the transition from immature networks to mature networks including precisely controlled excitatory and inhibitory synapses. We could elicit clear evoked responses with high reproducibility in mature cultures. A focal tetanic stimulation was applied to the mature cultures and how the tetanus affects 64 kinds of evoked activity was studied. The evoked responses showed bi-directional changes in their propagation patterns, potentiation and depression. These induced changes reflected the correlation properties with the tetanized activity pattern. The next step will be the combination of long-term recording and multi-site stimulation. How long does the induced change last, as well as how additional strong activity affects the previously induced changes, will be studied.     

Homeostatically regulated synchronized oscillations induced by short-term tetrodotoxin treatment in cultured neuronal network

Homeostatic plasticity plays a critical role in the stability of neuronal activities. Here, with high-density hippocampal networks cultured on multi-electrode arrays (MEAs), the transformation of spontaneous neuronal firing patterns induced by 1microM tetrodotoxin was clarified. Once tetrodotoxin was washed out after a 4-h treatment, spontaneous activities rose significantly with spike rate increasing approximately three times, and synchronized burst oscillations appeared throughout the network, with the cross-correlation coefficient between the active sites rising from 0.06+/-0.03 to 0.27+/-0.05. The long-term recording showed that the oscillations lasted for more than 4h before the network recovered. These results suggest that short-term treatment by tetrodotoxin may induce the homeostatically enhanced neuronal excitability, and that the spontaneous synchronized oscillations should be an indicator of homeostatic plasticity in cultured neuronal network. Furthermore, the non-invasive and long-term recording with MEAs as a novel sensing system is identified to be appropriate for pharmacological investigations of neuronal plasticity at the network level.     

Patterns of spontaneous activity in single rat olfactory receptor neurons are different in normally breathing and tracheotomized animals

Spontaneous firing of olfactory receptor neurons (ORNs) was recently shown to be required for the survival of ORNs and the maintenance of their appropriate synaptic connections with mitral cells in the olfactory bulb. ORN spontaneous activity has never been described or characterized quantitatively in mammals. To do so we have made extracellular single unit recordings from ORNs of freely breathing (FB) and tracheotomized (TT) rats. We show that the firing behavior of TT neurons was relatively simple: they tended to fire spikes at the same average frequency according to purely random (Poisson) or simple (Gamma or Weibull) statistical laws. A minority of them were bursting with relatively infrequent and short bursts. The activity of FB neurons was less simple: their firing rates were more diverse, some of them showed trends or were driven by breathing. Although more of them were regular, only a minority could be described by simple laws; the majority displayed random bursts with more spikes than the bursts of TT neurons. In both categories bursts and isolated spikes (outside bursts) occurred completely at random. The spontaneous activity of ORNs in rats resembles that of frogs, but is higher, which may be due to a difference in body temperature. These results suggest that, in addition to the intrinsic thermal noise, spontaneous activity is provoked in part by mechanical, thermal, or chemical (odorant molecules) effects of air movements due to respiration, this extrinsic part being naturally larger in FB neurons. It is suggested that spontaneous activity may be modulated by respiration. Because natural sampling of odors is synchronized with breathing, such modulation may prepare and keep olfactory bulb circuits tuned to process odor stimuli.     

Plasticity of recurring spatiotemporal activity patterns in cortical networks

How do neurons encode and store information for long periods of time? Recurring patterns of activity have been reported in various cortical structures and were suggested to play a role in information processing and memory. To study the potential role of bursts of action potentials in memory mechanisms, we investigated patterns of spontaneous multi-single-unit activity in dissociated rat cortical cultures in vitro. Spontaneous spikes were recorded from networks of approximately 50 000 neurons and glia cultured on a grid of 60 extracellular substrate- embedded electrodes (multi-electrode arrays). These networks expressed spontaneous culture- wide bursting from approximately one week in vitro. During bursts, a large portion of the active electrodes showed elevated levels of firing. Spatiotemporal activity patterns within spontaneous bursts were clustered using a correlation-based clustering algorithm, and the occurrences of these burst clusters were tracked over several hours. This analysis revealed spatiotemporally diverse bursts occurring in well-defined patterns, which remained stable for several hours. Activity evoked by strong local tetanic stimulation resulted in significant changes in the occurrences of spontaneous bursts belonging to different clusters, indicating that the dynamical flow of information in the neuronal network had been altered. The diversity of spatiotemporal structure and long-term stability of spontaneous bursts together with their plastic nature strongly suggests that such network patterns could be used as codes for information transfer and the expression of memories stored in cortical networks.     

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.     

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.     

Leptin counteracts the hypoxia-induced inhibition of spontaneously firing hippocampal neurons: a microelectrode array study

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O(2)). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca(2+)-activated K(+) channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 μM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons.     

Network bursts in cortical cultures are best simulated using pacemaker neurons and adaptive synapses

One of the most specific and exhibited features in the electrical activity of dissociated cultured neural networks (NNs) is the phenomenon of synchronized bursts, whose profiles vary widely in shape, width and firing rate. On the way to understanding the organization and behavior of biological NNs, we reproduced those features with random connectivity network models with 5,000 neurons. While the common approach to induce bursting behavior in neuronal network models is noise injection, there is experimental evidence suggesting the existence of pacemaker-like neurons. In our simulations noise did evoke bursts, but with an unrealistically gentle rising slope. We show that a small subset of ‘pacemaker’ neurons can trigger bursts with a more realistic profile. We found that adding pacemaker-like neurons as well as adaptive synapses yield burst features (shape, width, and height of the main phase) in the same ranges as obtained experimentally. Finally, we demonstrate how changes in network connectivity, transmission delays, and excitatory fraction influence network burst features quantitatively.     

The origin of spontaneous synchronized burst in cultured neuronal networks based on multi-electrode arrays

Many neural networks in mammalian central nervous system (CNS) fire single spike and complex spike burst. In fact, the conditions for triggering burst are not well understood. In the paper multi-electrode arrays (MEA) are used to record the spontaneous electrophysiological activities of cultured rat hippocampal neuronal network for a long time. After about 3 weeks culture, a transition from single spike to burst is observed in several networks. All of these spikes fire quickly before burst begins. The firing rate during the burst is lower than that just before the burst, but differences of inter-spike intervals (ISIs) between two firing patterns are not clear. Moreover, the electrical activities on neighboring electrodes show strong synchrony during the burst activities. In a word, the generation of the burst requires that network should have a sufficient level of excitation as well as a balance of synaptic inhibition.     

Cortical network modeling: analytical methods for firing rates and some properties of networks of LIF neurons.

The circuitry of cortical networks involves interacting populations of excitatory (E) and inhibitory (I) neurons whose relationships are now known to a large extent. Inputs to E- and I-cells may have their origins in remote or local cortical areas. We consider a rudimentary model involving E- and I-cells. One of our goals is to test an analytic approach to finding firing rates in neural networks without using a diffusion approximation and to this end we consider in detail networks of excitatory neurons with leaky integrate-and-fire (LIF) dynamics. A simple measure of synchronization, denoted by S(q), where q is between 0 and 100 is introduced. Fully connected E-networks have a large tendency to become dominated by synchronously firing groups of cells, except when inputs are relatively weak. We observed random or asynchronous firing in such networks with diverse sets of parameter values. When such firing patterns were found, the analytical approach was often able to accurately predict average neuronal firing rates. We also considered several properties of E-E networks, distinguishing several kinds of firing pattern. Included were those with silences before or after periods of intense activity or with periodic synchronization. We investigated the occurrence of synchronized firing with respect to changes in the internal excitatory postsynaptic potential (EPSP) magnitude in a network of 100 neurons with fixed values of the remaining parameters. When the internal EPSP size was less than a certain value, synchronization was absent. The amount of synchronization then increased slowly as the EPSP amplitude increased until at a particular EPSP size the amount of synchronization abruptly increased, with S(5) attaining the maximum value of 100%. We also found network frequency transfer characteristics for various network sizes and found a linear dependence of firing frequency over wide ranges of the external afferent frequency, with non-linear effects at lower input frequencies. The theory may also be applied to sparsely connected networks, whose firing behaviour was found to change abruptly as the probability of a connection passed through a critical value. The analytical method was also found to be useful for a feed-forward excitatory network and a network of excitatory and inhibitory neurons.     

Some properties of auditory neuron’s model trained by firing caused by tones modulated by low-frequency noise

In many sensory systems the formation of burst firing can be observed along a way from the periphery to the central nuclei. We investigate the putative transformation of spontaneous activity in the auditory pathway using a neuron model trained by real firing recorded in the auditory nuclei of the frog. The model has 200 separate inputs (neuronal spines). It is supposed that every spine is a coincidence detector. Its output (synaptic potential) sharply increases at emergence of the precisely certain interpulse interval in an input pulse sequence. If the total synaptic potentials excess a threshold, the model generates output spike, which changes weight of all spines according to the simplified Hebb principle. The model was trained by real firing caused in the auditory nuclei of the frog by tones modulated by low-frequency noise in the frequency ranges of 0–15 Hz, 0–50 Hz or 0–150 Hz. After that training the synaptic weights of every spine essentially changed. Thus, along with some increase of weights of spines tuned to boundary frequencies of modulating noise, the most characteristic change was the emphasizing weights of spines tuned to short interpulse intervals. As a result the spontaneous activity passed through the trained model became much more bursting. Efficiency of a signal transmission in model was higher when input spontaneous activity of real cells contains bursts of spikes. Results of modeling are discussed in connection with modern physiological data demonstrating the functional advantage of bursting.     

帕金森病大鼠中缝背核5-羟色胺能神经元电活动的变化

本实验采用玻璃微电极细胞外记录法,观察了帕金森病(Parkinson’s disease,PD)大鼠中缝背核(dorsal raphe nucleus, DRN)5-羟色胺(5-hydroxytryptamine,5-HT)能神经元电活动的变化。在大鼠右侧中脑黑质致密部内微量注射6-羟多巴胺(6- hydroxydopamine,6-OHDA)制作PD模型。结果显示,对照组和PD组大鼠DRN中5-HT能神经元的放电频率分别是(1．76±0．11)spikes／s(n=24)和(2．43±0．17)spikes／(n=21),PD组大鼠的放电频率显著高于对照组(P<0．001)。在对照组大鼠,92％(22／24)的神经元呈规则放电,8％(2／24)为爆发式放电;在PD组大鼠,具有规则、不规则和爆发式放电的神经元比例分别为9％(2／21)、43％(9／21)和48％(10／21),爆发式放电的5-HT能神经元比例明显高于对照组(P<0．001)。在对照组大鼠,DRN内局部注射5-HT1A拮抗剂WAY-100635(3μg/200nL)显著增加5-HT能神经元的放电频率而不影响其放电形式(n=19,P<0．002);而WAY-100635不改变PD组大鼠5-HT能神经元的放电频率和放电形式(n=17,P>0．05)。结果提示,用6-OHDA损毁黑质致密部造成的PD模型大鼠中神经元5-HT1A受体功能失调,并且DRN参与PD的病理生理学机制。     

Effect of O-superfamily conotoxin SO3 on synchronized spontaneous calcium spikes in cultured hippocampal networks

SO3 belongs to the O-superfamily of conotoxins and is known to have analgesic effects in experimental animals. In order to explore the mechanism of its potential pharmacological actions, the effect of SO3 on synchronized spontaneous calcium spikes was examined in cultured hippocampal networks by calcium imaging. Spontaneous oscillations of intracellular concentrations of calcium (Ca²⁺) in the form of waves and spikes are found in cultured hippocampal networks. Exposure to increasing concentrations of SO3 resulted in a progressive decrease in synchronized spontaneous calcium spikes. The higher concentrations (0.1 μmol/L and 1 μmol/L) of SO3 showed the strongest inhibition. The rank order of inhibition was 1 μmol/L > 0.1 μmol/L > 10 μmol/L > 0.01 μmol/L. This action of SO3 in reducing synchronized calcium spikes suggests a possible application for therapeutic treatment of epilepsy.     

Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels

The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25–0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.     

Postnatal changes in the dorsal root reflex in the isolated spinal cord of the hamster,Mesocricetus auratus

Spontaneous activity has been demonstrated in the lumbar dorsal roots of isolated spinal cord preparations taken from animals ranging in age from 2 to 65 days. Peaks of activity were recorded at 2 and 5 weeks of age, with mean firing frequencies of 33 Hz and 28 Hz respectively. The firing frequency in weeks 3 and 4 was lower (15 Hz) as was the frequency in cords taken from animals older than 6 weeks. The pattern of the spontaneous dorsal root activity changed during the first 5 weeks of life. In cords taken from animals less than 10 days old, the roots fired single action potentials, producing a single broad peak in Inter Spike Interval plots (ISI). Dorsal root recordings made from cords taken from animals in weeks 2 and 3 of life exhibited both single spikes and bursts of action potentials. By the end of the third week of life, individual spike activityhad declined and the bursts of action potentials characteristic of the adult pattern had become dominant, producing a bimodal ISI plot. Cross correlation analysis of dorsal root and dorsal horn activity in lumbar segments up to five segments apart, revealed an increasing degree of correlation developing over the first 4 weeks of postnatal life. Dorsal horn responses to dorsal root stimulation in cords taken from young animals were prolonged, lasting in excess of 250 msec. In the third week of life, the duration of the excitatory component of the response was reduced to approximately 50 msec by the development of an inhibitory phase.     

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 1 h. 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.