Spontaneous Network Activity Research Articles

In vitro Oscillation Patterns Throughout the Hippocampal Formation in a Rodent Model of Epilepsy

Specific oscillatory patterns are considered biomarkers of pathological neuronal network in brain diseases, such as epilepsy. However, the dynamics of underlying oscillations during the epileptogenesis throughout the hippocampal formation in the temporal lobe epilepsy is not clear. Here, we characterized in vitro oscillatory patterns within the hippocampal formation of epileptic rats, under 4-aminopyridine (4-AP)-induced hyperexcitability and during the spontaneous network activity, at two periods of epileptogenesis. First, at the beginning of epileptic chronic phase, 30 days post-pilocarpine-induced Status Epilepticus (SE). Second, at the established epilepsy, 60 days post-SE. The 4-AP-bathed slices from epileptic rats had increased susceptibility to ictogenesis in CA1 at 30 days post-SE, and in entorhinal cortex and dentate gyrus at 60 days post-SE. Higher power and phase coherence were detected mainly for gamma and/or high frequency oscillations (HFOs), in a region- and stage-specific manner. Interestingly, under spontaneous network activity, even without 4-AP-induced hyperexcitability, slices from epileptic animals already exhibited higher power of gamma and HFOs in different areas of hippocampal formation at both periods of epileptogenesis, and higher phase coherence in fast ripples at 60 days post-SE. These findings reinforce the critical role of gamma and HFOs in each one of the hippocampal formation areas during ongoing neuropathological processes, tuning the neuronal network to epilepsy.

Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity

Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits.1-3 However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered intrinsic muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA.4-6 These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Neural activity begins well before it becomes patterned, emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input, as well as inhibition of muscle contractions, results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, transient mechanosensory inhibition during PaSNA, followed by quantitative modeling of larval behavior, shows that mechanosensory modulation during development is required for proper larval foraging. This work provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.

The Structural E/I Balance Constrains the Early Development of Cortical Network Activity.

Neocortical networks have a characteristic constant ratio in the number of glutamatergic projection neurons (PN) and GABAergic interneurons (IN), and deviations in this ratio are often associated with developmental neuropathologies. Cultured networks with defined cellular content allowed us to ask if initial PN/IN ratios change the developmental population dynamics, and how different ratios impact the physiological excitatory/inhibitory (E/I) balance and the network activity development. During the first week in vitro, the IN content modulated PN numbers, increasing their proliferation in networks with higher IN proportions. The proportion of INs in each network set remained similar to the initial plating ratio during the 4 weeks cultivation period. Results from additional networks generated with more diverse cellular composition, including early-born GABA neurons, suggest that a GABA-dependent mechanism may decrease the survival of additional INs. A large variation of the PN/IN ratio did not change the balance between isolated spontaneous glutamatergic and GABAergic postsynaptic currents charge transfer (E/I balance) measured in PNs or INs. In contrast, the E/I balance of multisynaptic bursts reflected differences in IN content. Additionally, the spontaneous activity recorded by calcium imaging showed that higher IN ratios were associated with increased frequency of network bursts combined with a decrease of participating neurons per event. In the 4th week in vitro, bursting activity was stereotypically synchronized in networks with very few INs but was more desynchronized in networks with higher IN proportions. These results suggest that the E/I balance of isolated postsynaptic currents in single cells may be regulated independently of PN/IN proportions, but the network bursts E/I balance and the maturation of spontaneous network activity critically depends upon the structural PN/IN ratio.

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.

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.

Entropy estimation within in vitro neural-astrocyte networks as a measure of development instability.

The brain demands a significant fraction of the energy budget in an organism; in humans, it accounts for 2% of the body mass, but utilizes 20% of the total energy metabolized. This is due to the large load required for information processing; spiking demands from neurons are high but are a key component to understanding brain functioning. Astrocytic brain cells contribute to the healthy functioning of brain circuits by mediating neuronal network energy and facilitating the formation and stabilization of synaptic connectivity. During development, spontaneous activity influences synaptic formation, shaping brain circuit construction, and adverse astrocyte mutations can lead to pathological processes impacting cognitive impairment due to inefficiencies in network spiking activity. We have developed a measure that quantifies information stability within in vitro networks consisting of mixed neural-astrocyte cells. Brain cells were harvested from mice with mutations to a gene associated with the strongest known genetic risk factor for Alzheimer's disease, APOE. We calculate energy states of the networks and using these states, we present an entropy-based measure to assess changes in information stability over time. We show that during development, stability profiles of spontaneous network activity are modified by exogenous astrocytes and that network stability, in terms of the rate of change of entropy, is allele dependent.

Flexible Frequency Switching in Adult Mouse Visual Cortex Is Mediated by Competition Between Parvalbumin and Somatostatin Expressing Interneurons.

Neuronal networks in rodent primary visual cortex (V1) can generate oscillations in different frequency bands depending on the network state and the level of visual stimulation. High-frequency gamma rhythms, for example, dominate the network's spontaneous activity in adult mice but are attenuated upon visual stimulation, during which the network switches to the beta band instead. The spontaneous local field potential (LFP) of juvenile mouse V1, however, mainly contains beta rhythms and presenting a stimulus does not elicit drastic changes in network oscillations. We study, in a spiking neuron network model, the mechanism in adult mice allowing for flexible switches between multiple frequency bands and contrast this to the network structure in juvenile mice that lack this flexibility. The model comprises excitatory pyramidal cells (PCs) and two types of interneurons: the parvalbumin-expressing (PV) and the somatostatinexpressing (SOM) interneuron. In accordance with experimental findings, the pyramidal-PV and pyramidal-SOM cell subnetworks are associated with gamma and beta oscillations, respectively. In our model, they are both generated via a pyramidal-interneuron gamma (PING) mechanism, wherein the PCs drive the oscillations. Furthermore, we demonstrate that large but not small visual stimulation activates SOM cells, which shift the frequency of resting-state gamma oscillations produced by the pyramidal-PV cell subnetwork so that beta rhythms emerge. Finally, we show that this behavior is obtained for only a subset of PV and SOM interneuron projection strengths, indicating that their influence on the PCs should be balanced so that they can compete for oscillatory control of the PCs. In sum, we propose a mechanism by which visual beta rhythms can emerge from spontaneous gamma oscillations in a network model of the mouse V1; for this mechanism to reproduce V1 dynamics in adult mice, balance between the effective strengths of PV and SOM cells is required.

Mechanosensory Input Shapes Drosophila Motor Behavior Through Patterned Spontaneous Network Activity

Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits. However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered spontaneous muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA. These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Consistent with previous observations, we observe neural activity well before it becomes patterned, initially emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input results in excessive PaSNA, demonstrating that muscle movement serves as a brake. Finally, using an optogenetic strategy to selectively disrupt mechanosensory inputs during PaSNA, followed by quantitative modeling of larval behavior, we demonstrate that mechanosensory modulation during development is required for proper larval foraging. This work thus provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior . These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

Poly I:C Activated Microglia Disrupt Perineuronal Nets and Modulate Synaptic Balance in Primary Hippocampal Neurons in vitro.

Perineuronal nets (PNNs) are specialized, reticular structures of the extracellular matrix (ECM) that can be found covering the soma and proximal dendrites of a neuronal subpopulation. Recent studies have shown that PNNs can highly influence synaptic plasticity and are disrupted in different neuropsychiatric disorders like schizophrenia. Interestingly, there is a growing evidence that microglia can promote the loss of PNNs and contribute to neuropsychiatric disorders. Based on this knowledge, we analyzed the impact of activated microglia on hippocampal neuronal networks in vitro. Therefore, primary cortical microglia were cultured and stimulated via polyinosinic-polycytidylic acid (Poly I:C; 50 μg/ml) administration. The Poly I:C treatment induced the expression and secretion of different cytokines belonging to the CCL- and CXCL-motif chemokine family as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, the expression of matrix metalloproteinases (MMPs) could be verified via RT-PCR analysis. Embryonic hippocampal neurons were then cultured for 12 days in vitro (DIV) and treated for 24 h with microglial conditioned medium. Interestingly, immunocytochemical staining of the PNN component Aggrecan revealed a clear disruption of PNNs accompanied by a significant increase of glutamatergic and a decrease of γ-aminobutyric acid-(GABA)ergic synapse numbers on PNN wearing neurons. In contrast, PNN negative neurons showed a significant reduction in both, glutamatergic and GABAergic synapses. Electrophysiological recordings were performed via multielectrode array (MEA) technology and unraveled a significantly increased spontaneous network activity that sustained also 24 and 48 h after the administration of microglia conditioned medium. Taken together, we could observe a strong impact of microglial secreted factors on PNN integrity, synaptic plasticity and electrophysiological properties of cultured neurons. Our observations might enhance the understanding of neuron-microglia interactions considering the ECM.

Spontaneous synchronous network activity in the neonatal development of mPFC in mice.

Spontaneous Synchronous Network Activity (SSA) is a hallmark of neurodevelopment found in numerous central nervous system structures, including neocortex. SSA occurs during restricted developmental time‐windows, commonly referred to as critical periods in sensory neocortex. Although part of the neocortex, the critical period for SSA in the medial prefrontal cortex (mPFC) and the underlying mechanisms for generation and propagation are unknown. Using Ca2+ imaging and whole‐cell patch‐clamp in an acute mPFC slice mouse model, the development of spontaneous activity and SSA was investigated at cellular and network levels during the two first postnatal weeks. The data revealed that developing mPFC neuronal networks are spontaneously active and exhibit SSA in the first two postnatal weeks, with peak synchronous activity at postnatal days (P)8–9. Networks remain active but are desynchronized by the end of this 2‐week period. SSA was driven by excitatory ionotropic glutamatergic transmission with a small contribution of excitatory GABAergic transmission at early time points. The neurohormone oxytocin desynchronized SSA in the first postnatal week only without affecting concurrent spontaneous activity. By the end of the second postnatal week, inhibiting GABAA receptors restored SSA. These findings point to the emergence of GABAA receptor‐mediated inhibition as a major factor in the termination of SSA in mouse mPFC.

A simple Ca2+-imaging approach to neural network analyses in cultured neurons

BackgroundCa2+-imaging is a powerful tool to measure neuronal dynamics and network activity. To monitor network-level changes in cultured neurons, neuronal activity is often evoked by electrical or optogenetic stimulation and assessed using multi-electrode arrays or sophisticated imaging. Although such approaches allow detailed network analyses, multi-electrode arrays lack single-cell precision, whereas optical physiology generally requires advanced instrumentation that may not be universally available. New methodHere we developed a simple, stimulation-free protocol with associated Matlab algorithms that enables scalable analyses of spontaneous network activity in cultured human and mouse neurons. The approach allows analysis of the overall network activity and of single-neuron dynamics, and is amenable to screening purposes. ResultsWe validated the new protocol by assessing human neurons with a heterozygous conditional deletion of Munc18-1, and mouse neurons with a homozygous conditional deletion of neurexins. The approach described enabled identification of differential changes in these mutant neurons, allowing quantifications of the synchronous firing rate at the network level and of the amplitude and frequency of Ca2+-spikes at the single-neuron level. These results demonstrate the utility of the approach. Comparision with existing methodsCompared with current imaging platforms, our method is simple, scalable, accessible, and easy to implement. It enables quantification of more detailed parameters than multi-electrode arrays, but does not have the resolution and depth of more sophisticated yet labour-intensive methods, such as patch-clamp electrophysiology. ConclusionThe method reported here is scalable for a rapid direct assessment of neuronal function in culture, and can be applied to both human and mouse neurons. Thus, the method can serve as a basis for phenotypical analysis of mutations and for drug discovery efforts.

Functional Abnormalities of Cerebellum and Motor Cortex in Spinal Muscular Atrophy Mice

Spinal muscular atrophy (SMA) is a devastating genetic neuromuscular disease. Diffuse neuropathology has been reported in SMA patients and mouse models, however, functional changes in brain regions have not been studied. In the SMNΔ7 mouse model, we identified three types of differences in neuronal function in the cerebellum and motor cortex from two age groups: P7-9 (P7) and P11-14 (P11). Microelectrode array studies revealed significantly lower spontaneous firing and network activity in the cerebellum of SMA mice in both age groups, but it was more profound in the P11 group. In the motor cortex, however, neural activity was not different in either age group. Whole-cell patch-clamp was used to study the function of output neurons in both brain regions. In cerebellar Purkinje cells (PCs) of SMA mice, the input resistance was larger at P7, while capacitance was smaller at P11. In the motor cortex, no difference was observed in the passive membrane properties of layer V pyramidal neurons (PN5s). The action potential threshold of both types of output neurons was depolarized in the P11 group. We also observed lower spontaneous excitatory and inhibitory synaptic activity in PN5s and PCs respectively from P11 SMA mice. Overall, these differences suggest functional alterations in the neural network in these motor regions that change during development. Our results also suggest that neuronal dysfunction in these brain regions may contribute to the pathology of SMA. Comprehensive treatment strategies may consider motor regions outside of the spinal cord for better outcomes.

Synthetic cannabinoids enhanced ethanol-induced motor impairments through reduction of central glutamate neurotransmission

Marijuana or synthetic cannabinoids and alcohol are often used together, with these combinations causing motor impairments that can subsequently lead to motor vehicle accidents. This study investigated the combined use of both synthetic cannabinoids and ethanol and their effect on motor coordination in mice in addition to examining the neurochemical changes in the cerebellum. Ethanol (2 g/kg, i.p.) significantly induced motor impairment in the accelerating rotarod test in mice. Furthermore, ethanol-induced motor impairments were further accentuated when combined with the synthetic cannabinoid, JWH-018 or AB-CHMINACA. The enhancement effects of the synthetic cannabinoids were completely antagonized by pretreatment with the selective CB1 receptor antagonist AM251, but not by the selective CB2 receptor antagonist AM630. Neurochemical study results showed that ethanol caused a reduction in the extracellular glutamate levels in the cerebellum during periods of ethanol-induced motor impairment. In addition to the enhanced motor impairment seen when ethanol was combined with JWH-018, these combinations also enhanced the reduction of the extracellular glutamate levels in the cerebellum. We additionally used microelectrode array recordings to examine the effects of ethanol and/or JWH-018 on the spontaneous network activity in primary cultures from mouse cerebellum. Results showed that ethanol combined with JWH-018 significantly reduced spontaneous neuronal network activity in the primary cerebellar culture. Our findings demonstrate that ethanol-induced motor impairments are enhanced by synthetic cannabinoids, with these effects potentially mediated by CB1 receptors. An accentuated reduction of neurotransmissions in the cerebellum may play an important role in motor impairments caused by ethanol combined with synthetic cannabinoids.

Effects of Homocysteine and its Derivatives on Spontaneous Network Activity in the Hippocampus of Neonatal Rat Pups

Homocysteine is a sulfur-containing amino acid, which at high concentrations has neurotoxic effects and induces impairments to the development of the nervous system. Homocysteine is rapidly oxidized in the plasma, forming disulfide bonds with proteins and other low molecular weight thiols; it also undergoes transformation into the into homocysteine thiolactone. On chronic exposure, the neurotoxicity of homocysteine is therefore mediated mainly by its derivatives. The aim of the present work was to investigate the effects of homocysteine and its derivatives – homocystine and homocysteine thiolactone – on spontaneous network activity in the hippocampus of rats in the first week after birth. Giant depolarizing potentials (GDP) and multiple action potentials (MAP) were recorded using an extracellular electrode in hippocampal field CA3. All three study compounds were found to induce increases in the frequency of GDP and MAP at concentrations of 100 and 500 μM, homocystine producing the most significant increase in neuron network activity. The effects of homocysteine, homocystine, and homocysteine thiolactone on the spontaneous network activity of neurons were completely eliminated on blockade of NMDA and AMPA receptors. Thus, homocysteine and its derivatives lead to increased spontaneous network activity of hippocampal neurons in neonatal rats, which can induce impairments to the formation of the neural networks of the hippocampus in conditions of chronic hyperhomocysteinemia, and could also induce hyperexcitability and the risk of developing epilepsy in the postnatal period.

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 μm to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

Poly I:C‐induced maternal immune challenge reduces perineuronal net area and raises spontaneous network activity of hippocampal neurons in vitro

Activation of the maternal immune system (MIA) during gestation is linked to neuropsychiatric diseases like schizophrenia. While many studies address behavioural aspects, less is known about underlying cellular mechanisms. In the following study, BALB/c mice received intraperitoneal injections of polyinosinic-polycytidylic acid (Poly I:C) (20µg/ml) or saline (0.9%) at gestation day (GD) 9.5 before hippocampal neurons were isolated and cultured from embryonic mice for further analysis. Interestingly, strongest effects were observed when the perineuronal net (PNN) wearing subpopulation of neurons was analysed. Here, a significant reduction of aggrecan staining intensity, area and soma size could be detected. Alterations of PNNs are often linked to neuropsychiatric diseases, changes in synaptic plasticity and in electrophysiology. Utilizing multielectrode array analysis (MEA), we observed a remarkable increase of the spontaneous network activity in neuronal networks after 21days in vitro (DIV) when mother mice suffered a prenatal immune challenge. As PNNs are associated with GABAergic interneurons, our data indicate that this neuronal subtype might be stronger affected by a prenatal MIA. Degradation or damage of this subtype might cause the hyperexcitability observed in the whole network. In addition, embryonic neurons of the Poly I:C condition developed significantly shorter axons after five days in culture, while dendritic parameters and apoptosis rate remained unchanged. Structural analysis of synapse numbers revealed an increase of postsynaptic density 95 (PSD-95) puncta after 14 DIV and an increase of presynaptic vesicular glutamate transporter (vGlut) puncta after 21 DIV, while inhibitory synaptic proteins were not altered.

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

A modular brain-on-a-chip for modelling epileptic seizures with functionally connected human neuronal networks

Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.

Brain-Derived Neurotrophic Factor (BDNF) Preserves the Functional Integrity of Neural Networks in the β-Amyloidopathy Model in vitro.

Alzheimer’s disease (AD) is a widespread chronic neurodegenerative pathology characterized by synaptic dysfunction, partial neuronal death, cognitive decline and memory impairments. The major hallmarks of AD are extracellular senile amyloid plaques formed by various types of amyloid proteins (Aβ) and the formation and accumulation of intracellular neurofibrillary tangles. However, there is a lack of relevant experimental models for studying changes in neural network activity, the features of intercellular signaling or the effects of drugs on the functional activity of nervous cells during AD development. In this work, we examined two experimental models of amyloidopathy using primary hippocampal cultures. The first model involves the embryonic brains of 5xFAD mice; the second uses chronic application of amyloid beta 1-42 (Aβ1-42). The model based on primary hippocampal cells obtained from 5xFAD mice demonstrated changes in spontaneous network calcium activity characterized by a decrease in the number of cells exhibiting Ca2+ activity, a decrease in the number of Ca2+ oscillations and an increase in the duration of Ca2+ events from day 21 of culture development in vitro. Chronic application of Aβ1-42 resulted in the rapid establishment of significant neurodegenerative changes in primary hippocampal cultures, leading to marked impairments in neural network calcium activity and increased cell death. Using this model and multielectrode arrays, we studied the influence of amyloidopathy on spontaneous bioelectrical neural network activity in primary hippocampal cultures. It was shown that chronic Aβ application decreased the number of network bursts and spikes in a burst. The spatial structure of neural networks was also disturbed that characterized by reduction in both the number of key network elements (hubs) and connections between network elements. Moreover, application of brain-derived neurotrophic factor (BDNF) recombinant protein and BDNF hyperexpression by an adeno-associated virus vector partially prevented these amyloidopathy-induced neurodegenerative phenomena. BDNF maintained cell viability and spontaneous bioelectrical and calcium network activity in primary hippocampal cultures.