Neuronal Network Activity Research Articles

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations invitro.

SummaryMicro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution, and data analysis. Therefore, we benchmarked the robustness of MEA-derived neuronal activity patterns from ten healthy individual control lines, and uncover comparable network phenotypes. To achieve standardization, we provide recommendations on experimental design and analysis. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field toward the use of MEAs for disease phenotyping and drug discovery.

Sonlicromanol improves neuronal network dysfunction and transcriptome changes linked to m.3243A>G heteroplasmy in iPSC-derived neurons.

SummaryMitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.

Neuron-astrocyte networking: astrocytes orchestrate and respond to changes in neuronal network activity across brain states and behaviors.

Astrocytes are known to play many important roles in brain function. However, research underscoring the extent to which astrocytes modulate neuronal activity is still underway. Here we review the latest evidence regarding the contribution of astrocytes to neuronal oscillations across the brain, with a specific focus on how astrocytes respond to changes in brain state (e.g., sleep, arousal, stress). We then discuss the general mechanisms by which astrocytes signal to neurons to modulate neuronal activity, ultimately driving changes in behavior, followed by a discussion of how astrocytes contribute to respiratory rhythms in the medulla. Finally, we contemplate the possibility that brain stem astrocytes could modulate brainwide oscillations by communicating the status of oxygenation to higher cortical areas.

The neurogenic niche in Alzheimer’s disease

Adult hippocampal neurogenesis is the process of generation and functional incorporation of new neurons, formed by adult neural stem cells in the dentate gyrus. Adult hippocampal neurogenesis is highly dependent upon the integration of dynamic external stimuli and is instrumental in the formation of new spatial memories. Adult hippocampal neurogenesis is therefore uniquely sensitive to the summation of neuronal circuit and neuroimmune environments that comprise the neurogenic niche, and has powerful implications in diseases of aging and neurological disorders. This sensitivity underlies the neurogenic niche alterations commonly observed in Alzheimer’s disease, the most common form of dementia. This review summarizes Alzheimer’s disease associated changes in neuronal network activity, neuroinflammatory processes, and adult neural stem cell fate choice that ultimately result in neurogenic niche dysfunction and impaired adult hippocampal neurogenesis. A more comprehensive understanding of the complex changes mediating neurogenic niche disturbances in Alzheimer’s disease will aid development of future therapies targeting adult neurogenesis.

Tonic Endocannabinoid Signaling Gates Synaptic Plasticity in Dorsal Raphe Nucleus Serotonin Neurons Through Peroxisome Proliferator-Activated Receptors.

Endocannabinoids (eCBs), which include 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are lipid signaling molecules involved in the regulation of an array of behavioral and physiological functions. Released by postsynaptic neurons, eCBs mediate both phasic and tonic signaling at central synapses. While the roles of phasic eCB signaling in modulating synaptic functions and plasticity are well characterized, very little is known regarding the physiological roles and mechanisms regulating tonic eCB signaling at central synapses. In this study, we show that both 2-AG and AEA are constitutively released in the dorsal raphe nucleus (DRN), where they exert tonic control of glutamatergic synaptic transmission onto serotonin (5-HT) neurons. The magnitude of this tonic eCB signaling is tightly regulated by the overall activity of neuronal network. Thus, short term in vitro neuronal silencing or blockade of excitatory synaptic transmission abolishes tonic eCB signaling in the DRn. Importantly, in addition to controlling basal synaptic transmission, this study reveals that tonic 2-AG, but not AEA signaling, modulates synaptic plasticity. Indeed, short-term increase in tonic 2-AG signaling impairs spike-timing dependent potentiation (tLTP) of glutamate synapses. This tonic 2-AG-mediated homeostatic control of DRN glutamate synapses is not signaled by canonical cannabinoid receptors, but by intracellular peroxisome proliferator-activated receptor gamma (PPARγ). Further examination reveals that 2-AG mediated activation of PPARγ blocks tLTP by inhibiting nitric oxide (NO), soluble guanylate cyclase, and protein kinase G (NO/sGC/PKG) signaling pathway. Collectively, these results unravel novel mechanisms by which tonic 2-AG signaling integrates network activities and controls the synaptic plasticity in the brain.

Inhibitory control in neuronal networks relies on the extracellular matrix integrity

Inhibitory control is essential for the regulation of neuronal network activity, where excitatory and inhibitory synapses can act synergistically, reciprocally, and antagonistically. Sustained excitation-inhibition (E-I) balance, therefore, relies on the orchestrated adjustment of excitatory and inhibitory synaptic strength. While growing evidence indicates that the brain’s extracellular matrix (ECM) is a crucial regulator of excitatory synapse plasticity, it remains unclear whether and how the ECM contributes to inhibitory control in neuronal networks. Here we studied the simultaneous changes in excitatory and inhibitory connectivity after ECM depletion. We demonstrate that the ECM supports the maintenance of E-I balance by retaining inhibitory connectivity. Quantification of synapses and super-resolution microscopy showed that depletion of the ECM in mature neuronal networks preferentially decreases the density of inhibitory synapses and the size of individual inhibitory postsynaptic scaffolds. The reduction of inhibitory synapse density is partially compensated by the homeostatically increasing synaptic strength via the reduction of presynaptic GABAB receptors, as indicated by patch-clamp measurements and GABAB receptor expression quantifications. However, both spiking and bursting activity in neuronal networks is increased after ECM depletion, as indicated by multi-electrode recordings. With computational modelling, we determined that ECM depletion reduces the inhibitory connectivity to an extent that the inhibitory synapse scaling does not fully compensate for the reduced inhibitory synapse density. Our results indicate that the brain’s ECM preserves the balanced state of neuronal networks by supporting inhibitory control via inhibitory synapse stabilization, which expands the current understanding of brain activity regulation.Graphic abstract

Analysing Intercellular Communication in Astrocytic Networks Using "Astral".

Astrocytic networks are critically involved in regulating the activity of neuronal networks. However, a comprehensive and ready-to-use data analysis tool for investigating functional interactions between the astrocytes is missing. We developed the novel software package named “Astral” to analyse intercellular communication in astrocytic networks based on live-cell calcium imaging. Our method for analysing calcium imaging data does not require the assignment of regions of interest. The package contains two applications: the core processing pipeline for detecting and quantifying Ca++ events, and the auxiliary visualization tool for controlling data quality. Our method allows for the network-wide quantification of Ca++ events and the analysis of their intercellular propagation. In a set of proof-of-concept experiments, we examined Ca++ events in flat monolayers of primary astrocytes and confirmed that inter-astrocytic interactions depend on the permeability of gap junctions and connexin hemichannels. The Astral tool is particularly useful for studying astrocyte-neuronal interactions on the network level. We demonstrate that compared with purely astrocytic cultures, spontaneous generation of Ca++ events in astrocytes that were co-cultivated with neurons was significantly increased. Interestingly, the increased astrocytic Ca++ activity after long-term co-cultivation with neurons was driven by the enhanced formation of gap junctions and connexin hemichannels but was not affected by silencing neuronal activity. Our data indicate the necessity for systematic investigation of astrocyte-neuronal interactions at the network level. For this purpose, the Astral software offers a powerful tool for processing and quantifying calcium imaging data.

Spike signal transmission between modules and the predictability of spike activity in modular neuronal networks

Modularity is a common feature of the nervous system across species and scales. Although it has been qualitatively investigated in network science, very little is known about how it affects spike signal transmission in neuronal networks at the mesoscopic level. Here, a neuronal network model is built to simulate dynamic interactions among different modules of neuronal networks. This neuronal network model follows the organizational principle of modular structure. The neurons can generate spikes like biological neurons, and changes in the strength of synaptic connections conform to the STDP learning rule. Based on this neuronal network model, we first quantitatively studied whether and to what extent the connectivity within and between modules can affect spike signal transmission, and found that spike signal transmission heavily depends on the connectivity between modules, but has little to do with the connectivity within modules. More importantly, we further found that the spike activity of a module can be predicted according to the spike activities of its adjacent modules through building a resting-state functional connectivity matrix.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Sex-Specific Cannabidiol- and Iloperidone-Induced Neuronal Activity Changes in an In Vitro MAM Model System of Schizophrenia.

Cortical circuit dysfunction is thought to be an underlying mechanism of schizophrenia (SZ) pathophysiology with normalization of aberrant circuit activity proposed as a biomarker for antipsychotic efficacy. Cannabidiol (CBD) shows potential as an adjunctive antipsychotic therapy; however, potential sex effects in these drug interactions remain unknown. In the present study, we sought to elucidate sex effects of CBD coadministration with the atypical antipsychotic iloperidone (ILO) on the activity of primary cortical neuron cultures derived from the rat methylazoxymethanol acetate (MAM) model used for the study of SZ. Spontaneous network activity measurements were obtained using a multielectrode array at baseline and following administration of CBD or ILO alone, or combined. At baseline, MAM male neurons displayed increased bursting activity whereas MAM female neurons exhibited no difference in bursting activity compared to sex-matched controls. CBD administered alone showed a rapid but transient increase in neuronal activity in the MAM networks, an effect more pronounced in females. Furthermore, ILO had an additive effect on CBD-induced elevations in activity in the MAM male neurons. In the MAM female neurons, CBD or ILO administration resulted in time-dependent elevations in neuronal activity, but the short-term CBD-induced increases in activity were lost when CBD and ILO were combined. Our findings indicate that CBD induces rapid increases in cortical neuronal activity, with sex-specific drug interactions upon ILO coadministration. This suggests that sex should be a consideration when implementing adjunct therapy for treatment of SZ.

16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.

Structural basis for positive allosteric modulation of AMPA and kainate receptors.

This paper summarizes the present knowledge on how positive allosteric modulators (PAMs) interact with the ligand-binding domain (LBD) of AMPA and kainate receptors, based on structure determinations. AMPA and kainate receptors belong to the family of ionotropic glutamate receptors that are responsible for mediating the majority of fast excitatory neurotransmission. These receptors have been related to brain disorders, e.g. Alzheimer's disease and attention deficit hyperactivity disorder. PAMs are small molecules that potentiate AMPA and kainate receptor currents by interfering with receptor desensitization. Therefore, PAMs are considered to be of interest for the development of pharmacological tools. Whereas PAMs for AMPA receptors have been known for several years, only recently have PAMs for kainate receptors been reported. Today, >80 structures are available for AMPA receptors with PAMs. These PAMs bind at the interface between two LBD subunits in the vicinity of residue 775, which is important for functional differences between flip and flop isoforms of AMPA receptors. PAMs can be divided into five classes based on their binding mode. The most potent PAM reported to date belongs to class 3, which comprises dimerized PAMs. Three structures of the kainate receptor GluK1 were determined with PAMs belonging to class 2. One PAM enhances kainate receptor currents 5- to 59-fold but shows 100-fold lower potency compared to AMPA receptors. Selective PAMs for kainate receptors will be of great use as pharmacological tools for functional investigations in vivo and might potentially prove useful as drugs in controlling the activity of neuronal networks.

Emergent population activity in metric-free and metric networks of neurons with stochastic spontaneous spikes and dynamic synapses

We show that networks of excitatory neurons with stochastic spontaneous spiking activity and short-term synaptic plasticity can exhibit spontaneous repetitive synchronization in so-called population spikes. The major reason for this is that synaptic plasticity nonlinearly modulates the interaction between neurons. For large-scale two-dimensional networks, where the connection probability decreases exponentially with increasing distance between the neurons resulting in a small-world network connectome, a population spike occurs in the form of circular traveling waves diverging from seemingly non-stationary nucleation sites. The latter is in drastic contrast to the case of networks with a fixed fraction of steady pacemaker neurons, where the set of a few spontaneously formed nucleation sites is stationary. Despite the spatial non-stationarity of their nucleation, population spikes may occur surprisingly regularly. From a theoretical viewpoint, these findings show that the regime of nearly-periodic population spikes, which mimics respiratory rhythm, can occur strictly without stochastic resonance. In addition, the observed spatiotemporal effects serve as an example of transient chimera patterns.

Differential acute impact of therapeutically effective and overdose concentrations of lithium on human neuronal single cell and network function

Lithium salts are used as mood-balancing medication prescribed to patients suffering from neuropsychiatric disorders, such as bipolar disorder and major depressive disorder. Lithium salts cross the blood-brain barrier and reach the brain parenchyma within few hours after oral application, however, how lithium influences directly human neuronal function is unknown. We applied patch–clamp and microelectrode array technology on human induced pluripotent stem cell (iPSC)-derived cortical neurons acutely exposed to therapeutic (<1 mM) and overdose concentrations (>1 mM) of lithium chloride (LiCl) to assess how therapeutically effective and overdose concentrations of LiCl directly influence human neuronal electrophysiological function at the synapse, single-cell, and neuronal network level. We describe that human iPSC-cortical neurons exposed to lithium showed an increased neuronal activity under all tested concentrations. Furthermore, we reveal a lithium-induced, concentration-dependent, transition of regular synchronous neuronal network activity using therapeutically effective concentration (<1 mM LiCl) to epileptiform-like neuronal discharges using overdose concentration (>1 mM LiCl). The overdose concentration lithium-induced epileptiform-like activity was similar to the epileptiform-like activity caused by the GABAA-receptor antagonist. Patch–clamp recordings reveal that lithium reduces action potential threshold at all concentrations, however, only overdose concentration causes increased frequency of spontaneous AMPA-receptor mediated transmission. By applying the AMPA-receptor antagonist and anti-epileptic drug Perampanel, we demonstrate that Perampanel suppresses lithium-induced epileptiform-like activity in human cortical neurons. We provide insights in how therapeutically effective and overdose concentration of lithium directly influences human neuronal function at synapse, a single neuron, and neuronal network levels. Furthermore, we provide evidence that Perampanel suppresses pathological neuronal discharges caused by overdose concentrations of lithium in human neurons.

Astrocytes control the spiking of mouse visual cortex layer 5 pyramidal neurons.

Astrocytes control the spiking of mouse visual cortex layer 5 pyramidal neurons.

Temporal Relations between Cortical Network Oscillations and Breathing Frequency during REM Sleep.

Nasal breathing generates a rhythmic signal which entrains cortical network oscillations in widespread brain regions on a cycle-to-cycle time scale. It is unknown, however, how respiration and neuronal network activity interact on a larger time scale: are breathing frequency and typical neuronal oscillation patterns correlated? Is there any directionality or temporal relationship? To address these questions, we recorded field potentials from the posterior parietal cortex of mice together with respiration during REM sleep. In this state, the parietal cortex exhibits prominent θ and γ oscillations while behavioral activity is minimal, reducing confounding signals. We found that the instantaneous breathing frequency strongly correlates with the instantaneous frequency and amplitude of both θ and γ oscillations. Cross-correlograms and Granger causality revealed specific directionalities for different rhythms: changes in θ activity precede and Granger-cause changes in breathing frequency, suggesting control by the functional state of the brain. On the other hand, the instantaneous breathing frequency Granger causes changes in γ frequency, suggesting that γ is influenced by a peripheral reafference signal. These findings show that changes in breathing frequency temporally relate to changes in different patterns of rhythmic brain activity. We hypothesize that such temporal relations are mediated by a common central drive likely to be located in the brainstem.

Intermittent Fasting Alters Cognition, Anxiety‐like Behavior, and Hippocampal Norepinephrine Content in Aged Mice

Intermittent fasting (IMF) is associated with many health benefits in animal models and humans. Yet, little is known if an IMF diet affects mood and cognitive processing. We have previously identified that IMF in diet-induced obese males increases norepinephrine content in the hypothalamus and increase arcuate neuropeptide Y (NPY) gene expression more than in ad libitum males. This suggests that IMF may improve cognition through activation of the hindbrain norepinephrine neuronal network and reverse the age-dependent decline in NPY expression. Less is known about the association between anxiety and IMF. In humans, IMF during Ramadan may alleviate anxiety. Here, we address the impact of IMF on hippocampal-dependent memory using the Y-maze and spatial object recognition (SOR), hippocampal-independent memory using novel object recognition (NOR), and anxiety-like behavior using the open field task (OFT) in middle-aged male (12 mo) and aged female (18 mo) mice. Y-maze data indicate that IMF males perform worse in the identification of the unknown arm than same-sex controls, suggesting an IMF-induced deficit in interpreting spatial navigation routes. Conversely, no difference was detected in females on this task. In the SOR task, which evaluates hippocampal-dependent object orientation, IMF males were not affected; however, IMF females exhibited a decrease in performance. This suggests that, in females, IMF impairs spatial information about the arrangement of objects. In the NOR, which detect differences in hippocampal-independent memory, IMF males performed better than their same-sex counterparts; however, we did not detect an effect in females. IMF treated males displayed and increase in hippocampal norepinephrine content, suggesting IMF may alter hindbrain norepinephrine signaling. Our overall cognitive results suggest that, in males, IMF results in deficits only in hippocampal-dependent tasks, yet an enhancement in performance when hippocampal-independent tasks are conducted. In females, only the hippocampal-dependent task of SOR was affected by IMF, suggesting IMF disrupts spatial object configuration. In the OFT, IMF male and female mice spent more time in the center zone, indicative of an anxiolytic phenotype. Collectively our research suggests that IMF is enough to produce both an impairment and enhancement in memory dependent upon age, sex, and hippocampal involvement, as well as an anxiolytic phenotype in both male and female aged mice.

Characterization of neuronal viability and network activity under microfluidic flow

BackgroundMicrofluidics technology has the potential to allow precise control of the temporal and spatial aspects of solute concentration, making it highly relevant for the study of volume transmission mechanisms in neural tissue. However, full utilization of this technology depends on understanding how microfluidic flow at the rates needed for rapid solution exchange affects neuronal viability and network activity. New methodWe designed a tape-based pressurized microfluidic flow system that is simple to fabricate and can be attached to commercial microelectrode arrays. The device is multi-layered, allowing the inclusion of a porous polycarbonate membrane to isolate neuronal cultures from shear forces while maintaining diffusive exchange of solutes. We used this system to investigate how flow affected survival and spiking patterns of cultured hippocampal neurons. ResultsViability and network activity of the cultures were reduced in proportion to flow rate. However, shear reduction measures did not improve survival or spiking activity; media conditioning in conjunction with culture age proved to be the critical factors for network stability. Diffusion simulations indicate that dilution of a small molecule accounts for the deleterious effects of flow on neuronal cultures. Comparison with existing methodsThis work establishes the experimental conditions for real time measurement of network activity during rapid solution exchange, using multi-layered chambers with reversible bonding that allow for reuse of microelectrode arrays. ConclusionsWith correct media conditioning, the microfluidic flow system allows drug delivery on a subsecond timescale without disruption of network activity or viability, enabling in vitro reproduction of volume transmission mechanisms.

A novel methodology to describe neuronal networks activity reveals spatiotemporal recruitment dynamics of synchronous bursting states.

We propose a novel phase based analysis with the purpose of quantifying the periodic bursts of activity observed in various neuronal systems. The way bursts are intiated and propagate in a spatial network is still insufficiently characterized. In particular, we investigate here how these spatiotemporal dynamics depend on the mean connection length. We use a simplified description of a neuron's state as a time varying phase between firings. This leads to a definition of network bursts, that does not depend on the practitioner's individual judgment as the usage of subjective thresholds and time scales. This allows both an easy and objective characterization of the bursting dynamics, only depending on system's proper scales. Our approach thus ensures more reliable and reproducible measurements. We here use it to describe the spatiotemporal processes in networks of intrinsically oscillating neurons. The analysis rigorously reveals the role of the mean connectivity length in spatially embedded networks in determining the existence of "leader" neurons during burst initiation, a feature incompletely understood observed in several neuronal cultures experiments. The precise definition of a burst with our method allowed us to rigorously characterize the initiation dynamics of bursts and show how it depends on the mean connectivity length. Although presented with simulations, the methodology can be applied to other forms of neuronal spatiotemporal data. As shown in a preliminary study with MEA recordings, it is not limited to in silico modeling.

The Roles of Calmodulin and CaMKII in Cx36 Plasticity.

Anatomical and electrophysiological evidence that gap junctions and electrical coupling occur between neurons was initially confined to invertebrates and nonmammals and was thought to be a primitive form of synaptic transmission. More recent studies revealed that electrical communication is common in the mammalian central nervous system (CNS), often coexisting with chemical synaptic transmission. The subsequent progress indicated that electrical synapses formed by the gap junction protein connexin-36 (Cx36) and its paralogs in nonmammals constitute vital elements in mammalian and fish synaptic circuitry. They govern the collective activity of ensembles of coupled neurons, and Cx36 gap junctions endow them with enormous adaptive plasticity, like that seen at chemical synapses. Moreover, they orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie the fundamental integrative processes, such as memory and learning. Here, we review the available mechanistic evidence and models that argue for the essential roles of calcium, calmodulin, and the Ca2+/calmodulin-dependent protein kinase II in integrating calcium signals to modulate the strength of electrical synapses through interactions with the gap junction protein Cx36.