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![Follower neuron time-locked to the stimulation of a trigger cell. (A) Whole-cell recording of neuron 1 (trigger). In response to five depolarizing current pulses (3 nA; 5 msec), the neuron fires five APs. (B) Simultaneous fluorescence measurements with a photodiode of the somatic region of neuron 1 show discrete calcium accumulations that correspond to the five APs. The sign of the fluorescence signals in all figures has been inverted. ADU, analog-digital voltage units. (C) Simultaneous fluorescence measurements of neuron 2 (follower), showing an intracellular calcium concentration ([Ca 2 ]i) transient phase-locked with the fourth AP of neuron 1. The onset of the calcium signal in neuron 2 occurs coincident (0.6 msec) with the peak of the AP in neuron 1. Neuron 2 has subsequent [Ca 2 ]i increases. The experiment was carried out in a P18 mouse cortical slice under ACSF with 2 mM Ca 2 1 mM Mg 2 .](profile/Areti-Tsiola/publication/12578074/figure/fig3/AS:667825500856324@1536233415379/Follower-neuron-time-locked-to-the-stimulation-of-a-trigger-cell-A-Whole-cell.png)
Follower neuron time-locked to the stimulation of a trigger cell. (A) Whole-cell recording of neuron 1 (trigger). In response to five depolarizing current pulses (3 nA; 5 msec), the neuron fires five APs. (B) Simultaneous fluorescence measurements with a photodiode of the somatic region of neuron 1 show discrete calcium accumulations that correspond to the five APs. The sign of the fluorescence signals in all figures has been inverted. ADU, analog-digital voltage units. (C) Simultaneous fluorescence measurements of neuron 2 (follower), showing an intracellular calcium concentration ([Ca 2 ]i) transient phase-locked with the fourth AP of neuron 1. The onset of the calcium signal in neuron 2 occurs coincident (0.6 msec) with the peak of the AP in neuron 1. Neuron 2 has subsequent [Ca 2 ]i increases. The experiment was carried out in a P18 mouse cortical slice under ACSF with 2 mM Ca 2 1 mM Mg 2 .
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An experimental difficulty in unraveling circuits in the mammalian nervous system is the identification of postsynaptic targets of a given neuron. Besides ultrastructural reconstructions, simultaneous recordings from pairs of cells in brain slices have been used to identify connected neurons. We describe in this paper a technique using calcium imag...
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... stimulated the trigger neuron to fire a train of APs by injecting pulses of depolarizing current ( Fig. 2A). Each AP produced a calcium transient in the soma of the neuron with peak amplitudes of 1-5% FF, time-to-peak of 10 msec, and decays that lasted for several seconds (Fig. 2B). Depolarizing pulses that did not reach threshold did not result in any detect- able somatic calcium accumulations. Because the onset of the calcium transient ...
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... stimulated the trigger neuron to fire a train of APs by injecting pulses of depolarizing current ( Fig. 2A). Each AP produced a calcium transient in the soma of the neuron with peak amplitudes of 1-5% FF, time-to-peak of 10 msec, and decays that lasted for several seconds (Fig. 2B). Depolarizing pulses that did not reach threshold did not result in any detect- able somatic calcium accumulations. Because the onset of the calcium transient occurred within 1 msec from the peak of the AP, by using a photodiode array with high temporal resolution (0.6 msec), we could accurately follow the occurrence of each AP in the ...
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... our initial experiments, we found that stimulation of the trigger cell with a train of APs produced time-locked increases of the intracellular Ca 2 concentration in other neurons (''fol- lowers''; Fig. 2C). Because of the similarity in amplitude and kinetics of the responses in the follower cells to those elicited by a single AP in the trigger cell, we reasoned that they were also produced by single APs and that the follower neurons were brought to AP threshold by the stimulation of the trigger cell. The latency of the trigger's AP to ...
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... (followers) from any given neuron (trigger) by using calcium imaging of populations of neurons in brain slices. We show five different lines of evidence indicating that the trigger and follower cells can be monosyn- aptically connected. First, the onset of the response of the follower can occur within 1 msec of the peak of an AP of the trigger (Fig. 2). Second, in Mg 2 -free ACSF, followers occur time-locked to the stimulus trains at probabilities that are statistically different from spontaneous activation. Third, fol- lowers can be activated in more than one trial (Fig. 3). Fourth, the axon of the trigger can be traced to the dendrites of the follower, and contacts are found at the ...
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... 21, and 22; Fig. 5). At the same time, we have detected followers with activation hundreds of milliseconds after the end of the train of APs in the trigger cell (Figs. 2 and 3). It is possible that in those cases, the follower remained close to threshold and was brought to spike by background EPSPs. ...
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Citations
... Neuronal action potentials (AP) are tightly regulated through the influx and efflux of sodium and potassium ions that regulate the transmembrane potential of the cells rapidly on timescales of milliseconds. Other ions also transit across the membrane during the AP including Ca 2+ , which moves out of the cell in tens to hundreds of milliseconds making it a useful tool for monitoring neuronal activity in real-time without the need for millisecond timescale imaging [14][15][16] . Several technologies take advantage of these slower Ca 2+ transients, namely calcium-sensitive dyes (like Fluo-4, X-Rhod, and CalBryte) and genetically encoded calcium indicators (like the GCaMP family of proteins) 17,18 . ...
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... Second, the Ca 2+ response had to begin within a window of 300 ms or less after photostimulation of the targeted neuron. This delay window is based on the finding of Peterlin et al. (2000) that large increases of Ca 2+ in a neuron monosynaptically connected to a patch-clamped neuron typically occurred about 300 ms after the first action potential in the patch-clamped neuron. A longer delay between LSPS and a Ca 2+ response might indicate a polysynaptic connection or spontaneous activity unrelated to functional links between neurons. ...
A technique combining fluorescence imaging with Ca²⁺ indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allows identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg²⁺ concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. A GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.
... Second, the Ca 2+ response had to begin within a window of 300 ms or less after photostimulation of the targeted neuron. This delay window is based on the finding of Peterlin et al. (2000) that large increases of Ca 2+ in a neuron monosynaptically connected to a patch-clamped neuron typically occurred about 300 ms after the first action potential in the patch-clamped neuron. A longer delay between LSPS and a Ca 2+ response might indicate a polysynaptic connection or spontaneous activity unrelated to functional links between neurons. ...
A technique combining fluorescence imaging with Ca ²⁺ indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allows identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg ²⁺ concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. A GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.
... Most of the recent work on calcium imaging is focused on identifying the sources (neurons and axonal or dendritic processes) and denoising/deconvolving the neural activity from calcium signals (Apthorpe et al., 2016;Giovannucci et al., 2017;Inan, Erdogdu, & Schnitzer, 2017;Speiser et al., 2017). Moreover, while recovery of action potential timing and circuit structure and dynamics using CaI modality of recording has an established history in the field (Peterlin, Kozloski, Mao, Tsiola, & Yuste, 2000), it was not until recently that this problem was approached at whole brain scales (Betzel, 2020;Naumann et al., 2016;Vladimirov et al., 2018), which is necessary in order to gain scientific insight into the macroscale brain function, but at the same time poses computational and algorithmic challenges. It is in this direction that our work attempts to make a contribution. ...
Many natural systems, especially biological ones, exhibit complex multivariate nonlinear dynamical behaviors that can be hard to capture by linear autoregressive models. On the other hand, generic nonlinear models such as deep recurrent neural networks often require large amounts of training data, not always available in domains such as brain imaging; also, they often lack interpretability. Domain knowledge about the types of dynamics typically observed in such systems, such as a certain type of dynamical systems models, could complement purely data-driven techniques by providing a good prior. In this work, we consider a class of ordinary differential equation (ODE) models known as van der Pol (VDP) oscil lators and evaluate their ability to capture a low-dimensional representation of neural activity measured by different brain imaging modalities, such as calcium imaging (CaI) and fMRI, in different living organisms: larval zebrafish, rat, and human. We develop a novel and efficient approach to the nontrivial problem of parameters estimation for a network of coupled dynamical systems from multivariate data and demonstrate that the resulting VDP models are both accurate and interpretable, as VDP's coupling matrix reveals anatomically meaningful excitatory and inhibitory interactions across different brain subsystems. VDP outperforms linear autoregressive models (VAR) in terms of both the data fit accuracy and the quality of insight provided by the coupling matrices and often tends to generalize better to unseen data when predicting future brain activity, being comparable to and sometimes better than the recurrent neural networks (LSTMs). Finally, we demonstrate that our (generative) VDP model can also serve as a data-augmentation tool leading to marked improvements in predictive accuracy of recurrent neural networks. Thus, our work contributes to both basic and applied dimensions of neuroimaging: gaining scientific insights and improving brain-based predictive models, an area of potentially high practical importance in clinical diagnosis and neurotechnology.
... The initiation of sequential activity by single neurons results from few very strong pairwise connections to both E and I neuronsreminiscent of ''detonator synapses'' in mammalian hippocampal CA3 (Henze et al., 2002) and neocortex (Lefort et al., 2009;Li et al., 2009;Peterlin et al., 2000). Once triggered, this activity spread across neurons and space, resulting in distributed E and I influence. ...
Recent studies reveal the occasional impact of single neurons on surround firing statistics and even simple behaviors. Exploiting the advantages of a simple cortex, we examined the influence of single pyramidal neurons on surrounding cortical circuits. Brief activation of single neurons triggered reliable sequences of firing in tens of other excitatory and inhibitory cortical neurons, reflecting cascading activity through local networks, as indicated by delayed yet precisely timed polysynaptic subthreshold potentials. The evoked patterns were specific to the pyramidal cell of origin, extended over hundreds of micrometers from their source, and unfolded over up to 200 ms. Simultaneous activation of pyramidal cell pairs indicated balanced control of population activity, preventing paroxysmal amplification. Single cortical pyramidal neurons can thus trigger reliable postsynaptic activity that can propagate in a reliable fashion through cortex, generating rapidly evolving and non-random firing sequences reminiscent of those observed in mammalian hippocampus during "replay" and in avian song circuits.
... OGB-1 and other calcium-sensitive dyes, such as fura-2, have been used in previous studies to detect neural activity in the neural tissue 12 . OGB-1 can detect changes in calcium concentration in small dendritic compartments [13][14][15] and therefore has been proposed as a tool for tracking activity that is not visible using traditional electrophysiology methods 16 . Finally, we developed a computational neuronal network model to simulate the two traveling waves simultaneously and compare their properties to test the possible mechanisms of generation and propagation of these waves. ...
Fast and slow neural waves have been observed to propagate in the human brain during seizures. Yet the nature of these waves is difficult to study in a surgical setting. Here, we report an observation of two different traveling waves propagating in the in-vitro epileptic hippocampus at speeds similar to those in the human brain. A fast traveling spike and a slow moving wave were recorded simultaneously with a genetically encoded voltage sensitive fluorescent protein (VSFP Butterfly 1.2) and a high speed camera. The results of this study indicate that the fast traveling spike is NMDA-sensitive but the slow moving wave is not. Image analysis and model simulation demonstrate that the slow moving wave is moving slowly, generating the fast traveling spike and is, therefore, a moving source of the epileptiform activity. This slow moving wave is associated with a propagating neural calcium wave detected with calcium dye (OGB-1) but is independent of NMDA receptors, not related to ATP release, and much faster than those previously recorded potassium waves. Computer modeling suggests that the slow moving wave can propagate by the ephaptic effect like epileptiform activity. These findings provide an alternative explanation for slow propagation seizure wavefronts associated with fast propagating spikes.
... With the development of fluorescent calcium indicators and fast imaging technology, calcium imaging has enabled us to optically detect neuronal activity within local neuronal populations [1][2][3], which is essential for understanding the neural information processing. Reconstruction methods could recover spike patterns from the recorded calcium traces, and play an important role in analyzing neuronal activity from optical imaging [4,5]. ...
Calcium imaging is becoming an increasingly popular technology to indirectly measure activity patterns in local neuronal networks. Based on the dependence of calcium fluorescence on neuronal spiking, two-photon calcium imaging affords single-cell resolution of neuronal population activity. However, it is still difficult to reconstruct neuronal activity from complex calcium fluorescence traces, particularly for traces contaminated by noise. Here, we describe a robust and efficient neuronal-activity reconstruction method that utilizes Lq minimization and interval screening (IS), which we refer to as LqIS. The simulation results show that LqIS performs satisfactorily in terms of both accuracy and speed of reconstruction. Reconstruction of simulation and experimental data also shows that LqIS has advantages in terms of the recall rate, precision rate, and timing error. Finally, LqIS is demonstrated to effectively reconstruct neuronal burst activity from calcium fluorescence traces recorded from large-size neuronal population.
... Fluorescent synthetic dyes, derived from the EGTA homolog BAPTA, such as Fluo-4, Oregon Green BAPTA, and Indo-1 are frequently used for calcium imaging (Tsien, 1980;Uematsu et al., 1991;Kreitzer et al., 2000;McDonough et al., 2000;Stosiek et al., 2003;Kuga et al., 2011;Lillis et al., 2012), but have several drawbacks. Synthetic dye loading techniques are particularly difficult to employ in mature or pathological tissue, limiting their use to a subset of experimental preparations-those in healthy young tissue (Peterlin et al., 2000;Reeves et al., 2011). Additionally, synthetic dyes cannot be targeted to specific cell types or intracellular regions (Mank and Griesbeck, 2008;Mao et al., 2008) except in special cases (Ding, 2012). ...
... For technical reasons, most published calcium imaging data have been collected from perinatal or adolescent animals. Because of the vast differences in cellular function, connectivity and gene expression between the young and adult brain , it is important to develop methods for recording activity in tissue obtained from animals of a wide range of ages (Yuste and Katz, 1991;Schwartz et al., 1998;Peterlin et al., 2000;Reeves et al., 2011). To test GCaMP6f function and expression stability in the rat brain following IUE, we prepared ABS from older animals (P125-P127; "old" group) electroporated with the pPBC-LG6f plasmid during gestation. ...
Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb). In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.
... For measurements of KCC2 activity, slices were loaded with BCECF AM [2′, 7′ -bis-(2carboxyethyl)-5-(and-6-) carboxyfluorescein AM] (25 μM, TefLabs) for 15 min in the presence of 0.02% pluronic acid as described previously (Beierlein et al. 2002;Besser et al. 2009). Slices from mice at P15-P20 were used as loading of fluorescent dyes is substantially more efficient by bulk loading at this age, when compared to slices obtained from older animals, likely due to the extensive neuropil development (Peterlin et al. 2000). At two weeks of age, KCC2 as well as Zn 2+ are found in the hippocampus at levels similar to those of adult neurons (Slomianka et al. 1997;Rivera et al. 1999;Nitzan et al. 2002;Stein et al. 2004), thus allowing measurements of the effects of kainate-dependent Zn 2+ release on KCC2 activity in those tissues. ...
The aim of this study was to investigate the role of the synaptic metabotropic zinc receptor mZnR/GPR39 in physiological adaptation to epileptic seizures. We previously demonstrated that synaptic activation of mZnR/GPR39 enhances inhibitory drive in the hippocampus by upregulating neuronal K(+)/Cl(-) co-transporter 2 (KCC2) activity. Here, we first show that mZnR/GPR39 knockout (KO) adult mice have dramatically enhanced susceptibility to seizures triggered by a single intraperitoneal injection of kainic acid, when compared to wild type (WT) littermates. Kainate also substantially enhances seizure-associated gamma oscillatory activity in juvenile mZnR/GPR39 KO hippocampal slices, a phenomenon that can be reproduced in WT tissue by extracellular Zn(2+) chelation. Importantly, kainate-induced synaptic Zn(2+) release enhances surface expression and transport activity of KCC2 in WT, but not mZnR/GPR39 KO hippocampal neurons. Kainate-dependent upregulation of KCC2 requires mZnR/GPR39 activation of the Gαq/phospholipase C/extracellular regulated kinase (ERK1/2) signaling cascade. We suggest that mZnR/GPR39-dependent upregulation of KCC2 activity provides homeostatic adaptation to an excitotoxic stimulus by increasing inhibition. As such, mZnR/GPR39 may provide a novel pharmacological target for dampening epileptic seizure activity.
Copyright © 2014. Published by Elsevier Inc.
... To circumvent these issues, membrane voltage or intracellular Ca 2? concentrations can instead be used as a proxy for Ca 2? -dependent hormone release [47][48][49][50]. To this end, functional multicellular Ca 2? imaging (fMCI), originally used to map activity in cortical circuits [51][52][53], has recently been adapted for use in beta cells [27,28]. By coupling a laser bank to a Nipkow spinning disk, the millisecond organization of beta cell population Ca 2? -spiking activity can be captured in near real time with reduced phototoxicity and photobleaching. ...
Beta cell connectivity describes the phenomenon whereby the islet context improves insulin secretion by providing a three-dimensional platform for intercellular signaling processes. Thus, the precise flow of information through homotypically interconnected beta cells leads to the large-scale organization of hormone release activities, influencing cell responses to glucose and other secretagogues. Although a phenomenon whose importance has arguably been underappreciated in islet biology until recently, a growing number of studies suggest that such cell-cell communication is a fundamental property of this micro-organ. Hence, connectivity may plausibly be targeted by both environmental and genetic factors in type 2 diabetes mellitus (T2DM) to perturb normal beta cell function and insulin release. Here, we review the mechanisms that contribute to beta cell connectivity, discuss how these may fail during T2DM, and examine approaches to restore insulin secretion by boosting cell communication.
