Spontaneous Development of Synchronous Oscillatory Activity During Maturation of Cortical Networks In Vitro

2002 ◽  
Vol 88 (5) ◽  
pp. 2196-2206 ◽  
Author(s):  
Thoralf Opitz ◽  
Ana D. De Lima ◽  
Thomas Voigt
Keyword(s):  
Large Scale ◽  
Reversal Potential ◽  
Neuronal Population ◽  
Network Activity ◽  
Oscillatory Activity ◽  
Pharmacological Intervention ◽  
Resting Membrane Potential ◽  
Cortical Networks ◽  
Synchronous Network

Recent studies have focused attention on mechanisms of spontaneous large-scale wavelike activity during early development of the neocortex. In this study, we describe and characterize synchronous neuronal activity that occurs in cultured cortical networks naturally without pharmacological intervention. The synchronous activity that can be detected by means of Fluo-3 fluorescence imaging starts to develop at the beginning of the second week in culture and eventually includes the entire neuronal population about 1 wk later. A synchronous increase of [Ca2+]i in the neuronal population is associated with a burst of action potentials riding on a long-lasting depolarization recorded in a single cell. It is suggested that this depolarization results directly from synaptic current, which was comprised of at least three different components mediated by AMPA, N-methyl-d-aspartate (NMDA), and GABAA receptors. We never observed a gradually depolarizing pacemaker potential and found no evidence for a change of excitability during inter-burst periods. However, we found evidence for a period of synaptic depression after bursts. Network excitability recovers gradually over seconds from this depression that can explain the episodic nature of spontaneous network activity. Using pharmacological manipulation to investigate the propagation of activity in the network, we show that synchronous network activity depends on both glutamatergic and GABAAergic neurotransmission during a brief period. Reversal potential of GABAA receptor-mediated current was found to be significantly more positive than resting membrane potential both at 1 and 2 wk in culture, suggesting depolarizing action of GABA. However, in cultures older than 2 wk, inhibition of GABAAreceptors does not result in block of synchronous network activity but in modulation of burst width and frequency.

scholarly journals Excitatory GABA in Rodent Developing Neocortex In Vitro

2008 ◽  
Vol 100 (2) ◽  
pp. 609-619 ◽  
Author(s):  
Sylvain Rheims ◽  
Marat Minlebaev ◽  
Anton Ivanov ◽  
Alfonso Represa ◽  
Rustem Khazipov ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Pyramidal Neurons ◽  
Brain Slices ◽  
Reversal Potential ◽  
Pyramidal Cells ◽  
Oscillatory Activity ◽  
Resting Membrane Potential ◽  
Network Oscillations ◽  
Excitatory Gaba

GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential ( Em), reversal potential of GABA ( EGABA), and threshold of action potential generation ( Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and EGABA in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2–P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5–P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl−]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

scholarly journals Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mouhamed Alsaqati ◽  
Vivi M. Heine ◽  
Adrian J. Harwood
Keyword(s):  
Neuronal Network ◽  
Electrode Array ◽  
Network Connectivity ◽  
Autism Spectrum ◽  
Network Activity ◽  
Inhibitory Synapses ◽  
Pharmacological Intervention ◽  
Spatial Connectivity ◽  
Neuronal Network Activity

Abstract Background Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear. Methods Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network. Results We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory–excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes. Limitations Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients. Conclusions Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.

scholarly journals Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tanja Hyvärinen ◽  
Anu Hyysalo ◽  
Fikret Emre Kapucu ◽  
Laura Aarnos ◽  
Andrey Vinogradov ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Microelectrode Array ◽  
Functional Characterization ◽  
Principal Component ◽  
Network Activity ◽  
Human Pluripotent Stem Cell ◽  
Cortical Networks ◽  
Cortical Cultures

AbstractHuman pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.

Membrane Properties Underlying Patterns of GABA-Dependent Action Potentials in Developing Mouse Hypothalamic Neurons

2001 ◽  
Vol 86 (3) ◽  
pp. 1252-1265 ◽  
Author(s):  
Yu-Feng Wang ◽  
Xiao-Bing Gao ◽  
Anthony N. van den Pol
Keyword(s):  
Membrane Potential ◽  
Action Potentials ◽  
Brain Slices ◽  
Reversal Potential ◽  
Membrane Properties ◽  
Resting Membrane Potential ◽  
Hypothalamic Neurons ◽  
Spike Threshold ◽  
Spike Patterns

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons ( n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2–9( P2- 9) mice. With conventional patch pipettes (containing 29 mM Cl−), action potentials were also elicited by GABA from neurons of 2–13 days in vitro (2–13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 μM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 μM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd2+ (200 μM) and Ni2+(300 μM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl−] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl−]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current ( E GABA) was positive to the resting membrane potential, suggesting a high intracellular [Cl−] in developing mouse neurons. Varying the holding potential from −80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca2+ from the extracellular solution did not block spikes, indicating GABA-evoked Na+-based spikes. Although E GABA was more positive within 2–5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E GABA is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl−, not bicarbonate, was primarily responsible for generatingmultiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.

scholarly journals Regional heterogeneity of developing GABAergic interneuron excitation in vivo

2019 ◽  
Author(s):  
Yasunobu Murata ◽  
Matthew T. Colonnese
Keyword(s):  
Synapse Formation ◽  
Critical Role ◽  
Reversal Potential ◽  
Pyramidal Cells ◽  
Gabaergic Neurons ◽  
Network Activity ◽  
Gabaergic Interneuron ◽  
Intact Brain

AbstractGABAergic interneurons are proposed to be critical for early activity and synapse formation by directly exciting, rather than inhibiting, neurons in developing hippocampus and neocortex. However, the role of GABAergic neurons in the generation of neonatal network activity has not been tested in vivo, and recent studies have challenged the excitatory nature of early GABA. By locally manipulating interneuron activity in unanesthetized neonatal mice, we show that GABAergic neurons are indeed excitatory in hippocampus at postnatal-day 3 (P3), and responsible for most of the spontaneous firing of pyramidal cells at that age. Hippocampal interneurons become inhibitory by P7, whereas cortical interneurons are inhibitory at P3 and remain so throughout development. This regional and age heterogeneity is the result of a change in chloride reversal potential as activation of light-gated anion channels expressed in glutamatergic neurons causes firing in hippocampus at P3, but silences it at P7. This study in the intact brain reveals a critical role for GABAergic interneuron excitation in neonatal hippocampus, and a surprising heterogeneity of interneuron function in cortical circuits that was not predicted from in vitro studies.

scholarly journals Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSCs with a TSC2 mutation

2020 ◽  
Author(s):  
Mouhamed Alsaqati ◽  
Vivi M Heine ◽  
Adrian J. Harwood
Keyword(s):  
Neuronal Network ◽  
Electrode Array ◽  
Network Connectivity ◽  
Autism Spectrum ◽  
Network Activity ◽  
Inhibitory Synapses ◽  
Pharmacological Intervention ◽  
Spatial Connectivity ◽  
Neuronal Network Activity

Abstract Background Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system is currently unclear.MethodsHere we apply Multi-electrode array (MEA)-based assays to study the effects of TSC2 loss on neuronal network activity using Autism Spectrum Disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting, and spatial connectivity between electrodes across the network. Results We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory-excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK), and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604 increases the network behaviour, shortens the network burst lengths, and reduces the number of uncorrelated spikes.LimitationsAlthough a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients.ConclusionsOur observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.

Inhibitory effect of somatostatin on vagal motoneurons in the rat brain stem in vitro

1989 ◽  
Vol 256 (1) ◽  
pp. C155-C159 ◽  
Author(s):  
J. Nabekura ◽  
Y. Mizuno ◽  
Y. Oomura
Keyword(s):  
Brain Stem ◽  
Rat Brain ◽  
Reversal Potential ◽  
Input Resistance ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Resting Membrane Potential ◽  
Motor Nucleus ◽  
Dorsal Motor Nucleus

Effects of somatostatin-14 (SRIF) on membrane electrical properties were studied in rat brain stem slice preparations maintained in vitro. SRIF hyperpolarized the resting membrane potential and decreased the input resistance of more than two-thirds of the 85 vagal motoneurons tested in the dorsal motor nucleus of the vagus. These effects persisted under synaptic blockade caused by perfusion with a solution containing tetrodotoxin or a Ca2+-free/high-Mg2+ solution and were dependent on the extracellular SRIF concentration (5 X 10(-8) to 1 X 10(-8) M). The Hill coefficient was estimated to be 2. The reversal potential of SRIF-induced hyperpolarization was affected by changing external K+ concentration. The results suggest that, in addition to its well-known peripheral action, SRIF may inhibit secretomotor functions of visceral organs by reducing vagal output in the central nervous system.

scholarly journals Modulation of Network Oscillatory Activity and GABAergic Synaptic Transmission by CB1 Cannabinoid Receptors in the Rat Medial Entorhinal Cortex

2008 ◽  
Vol 2008 ◽  
pp. 1-12 ◽  
Author(s):  
Nicola H. Morgan ◽  
Ian M. Stanford ◽  
Gavin L. Woodhall
Keyword(s):  
Synaptic Transmission ◽  
Cannabinoid Receptors ◽  
Brain Regions ◽  
Synaptic Activity ◽  
Network Activity ◽  
Oscillatory Activity ◽  
Gabaergic Neurotransmission ◽  
Network Oscillations ◽  
Gabaergic Synaptic Transmission

Cannabinoids modulate inhibitory GABAergic neurotransmission in many brain regions. Within the temporal lobe, cannabinoid receptors are highly expressed, and are located presynaptically at inhibitory terminals. Here, we have explored the role of type-1 cannabinoid receptors (CB1Rs) at the level of inhibitory synaptic currents and field-recorded network oscillations. We report that arachidonylcyclopropylamide (ACPA; 10 M), an agonist at CB1R, inhibits GABAergic synaptic transmission onto both superficial and deep medial entorhinal (mEC) neurones, but this has little effect on network oscillations in beta/gamma frequency bands. By contrast, the CB1R antagonist/inverse agonist LY320135 (500 nM), increased GABAergic synaptic activity and beta/gamma oscillatory activity in superficial mEC, was suppressed, whilst that in deep mEC was enhanced. These data indicate that cannabinoid-mediated effects on inhibitory synaptic activity may be constitutively active in vitro, and that modulation of CB1R activation using inverse agonists unmasks complex effects of CBR function on network activity.

Ionic mechanism of substance P actions on neurons in trigeminal root ganglia

1990 ◽  
Vol 64 (1) ◽  
pp. 273-281 ◽  
Author(s):  
I. Spigelman ◽  
E. Puil
Keyword(s):  
Substance P ◽  
Voltage Clamp ◽  
Reversal Potential ◽  
Input Resistance ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Ionic Mechanism ◽  
Trigeminal Root

1. Responses of primary sensory neurons to substance P applications by perfusion were studied with intracellular recording techniques in in vitro slice preparations of trigeminal root ganglia (guinea pigs). Application of substance P in micromolar doses produced reversible depolarizations of 2–47 mV in 48 out of 64 neurons. The depolarizing influence facilitated repetitive spike discharge evoked by current-pulse injection. Evidence of desensitization was observed during prolonged or repeated applications of the peptide. 2. The responses to substance P were associated with decreased input resistance, although increased input resistance was observed in neurons where the resting membrane potential was compensated with DC injection. In single-electrode voltage-clamp (SEVC) recordings, substance P evoked an inward shift in the holding current and reduced an outwardly rectifying component in the I-V relationships. The reversal potential for the substance P response could not be determined. These results suggested that the perikaryal response to substance P has a complex ionic mechanism involving activation and deactivation of membrane conductances. 3. Substance P-induced depolarizations were greatly attenuated during perfusion with solutions that were deficient in [Na+] or [Mg2+] and were not significantly affected during perfusion with low-[Ca2+]-, CO2(+)-containing solutions. 4. In the voltage-clamp investigations, an inward current contributed to the substance P responses during combined application with the K(+)-channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). This current was not abolished by the inclusion of CsCl in the perfusing solution or by internal Cs+ application from the recording electrode, suggesting that an anomalous inward rectifier was not involved in the responses to substance P.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Rapid fluctuations in functional connectivity of cortical networks encode spontaneous behavior

2021 ◽  
Author(s):  
Hadas Benisty ◽  
Andrew H Moberly ◽  
Sweyta Lohani ◽  
Daniel Barson ◽  
Ronald R Coifman ◽  
...  
Keyword(s):  
Functional Connectivity ◽  
Neural Activity ◽  
Large Scale ◽  
Analytical Framework ◽  
Network Activity ◽  
Wide Field ◽  
Time Varying ◽  
Cortical Networks ◽  
Novel Approach ◽  
Spontaneous Behavior

Experimental work across a variety of species has demonstrated that spontaneously generated behaviors are robustly coupled to variation in neural activity within the cerebral cortex. Indeed, functional magnetic resonance imaging (fMRI) data suggest that functional connectivity in cortical networks varies across distinct behavioral states, providing for the dynamic reorganization of patterned activity. However, these studies generally lack the temporal resolution to establish links between cortical signals and the continuously varying fluctuations in spontaneous behavior typically observed in awake animals. Here, we took advantage of recent developments in wide-field, mesoscopic calcium imaging to monitor neural activity across the neocortex of awake mice. Applying a novel approach to quantifying time-varying functional connectivity, we show that spontaneous behaviors are more accurately represented by fast changes in the correlational structure versus the magnitude of large-scale network activity. Moreover, dynamic functional connectivity reveals subnetworks that are not predicted by traditional anatomical atlas-based parcellation of the cortex. These results provide insight into how behavioral information is represented across the mammalian neocortex and demonstrate a new analytical framework for investigating time-varying functional connectivity in neural networks.

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