Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

GABAergic circuits drive focal seizures.

Epilepsy is based on abnormal neuronal activities that have historically been suggested to arise from an excess of excitation and a defect of inhibition, or in other words from an excessive glutamatergic drive not balanced by GABAergic activity. More recent data however indicate that GABAergic signaling is not defective at focal seizure onset and may even be actively involved in seizure generation by providing excitatory inputs. Recordings of interneurons revealed that they are active at seizure initiation and that their selective and time-controlled activation using optogenetics triggers seizures in a more general context of increased excitability. Moreover, GABAergic signaling appears to be mandatory at seizure onset in many models. The main pro-ictogenic effect of GABAergic signaling is the depolarizing action of GABAA conductance which may occur when an excessive GABAergic activity causes Cl- accumulation in neurons. This process may combine with background dysregulation of Cl-, well described in epileptic tissues. Cl- equilibrium is maintained by (Na+)/K+/Cl- co-transporters, which can be defective and therefore favor the depolarizing effects of GABA. In addition, these co-transporters further contribute to this effect as they mediate K+ outflow together with Cl- extrusion, a process that is responsible for K+ accumulation in the extracellular space and subsequent increase of local excitability. The role of GABAergic signaling in focal seizure generation is obvious but its complex dynamics and balance between GABAA flux polarity and local excitability still remain to be established, especially in epileptic tissues where receptors and ion regulators are disrupted and in which GABAergic signaling rather plays a 2 faces Janus role.

GluN2C selective inhibition is a target to develop new antiepileptic compounds.

OBJECTIVE: Many early-onset epilepsies present as developmental and epileptic encephalopathy associated with refractory seizures, altered psychomotor development, and disorganized interictal cortical activity. Abnormal upregulation of specific N-methyl-d-aspartate receptor (NMDA-R) subunits is being disentangled as one of the mechanisms of severe early-onset epilepsies. In tuberous sclerosis complex (TSC), upregulation of the GluN2C subunit of the NMDA-R with slow deactivation kinetic results in increased neuronal excitation and synchronization. METHODS: Starting from an available GluN2C/D antagonist, NMDA-R-modulating compounds were developed and screened using a patch clamp on neuronal culture to select those with the strongest inhibitory effect on glutamatergic NMDA currents. For these selected compounds, blood pharmacokinetics and passage through the blood-brain barrier were studied. We tested the effect of the most promising compounds on epileptic activity in Tsc1+/- mice brain slices with multielectrode array, and then in vivo at postnatal ages P14-P17, comparable with the usual age at epilepsy onset in human TSC. RESULTS: Using a double-electrode voltage clamp on isolated NMDA currents, we identified the most prominent antagonists of the GluN2C subunit with no effect on GluN2A as a means of preventing side effects. The best compound passing through the blood-brain barrier was selected. Applied in vivo in six Tsc1+/- mice at P14-P17, this compound reduced or completely stopped spontaneous seizures in four of them, and decreased the background activity disorganization. Furthermore, ictal-like discharges stopped on a human brain sample from an infant with epilepsy due to TSC. INTERPRETATION: Subunit-selective inhibition is a valuable target for developing drugs for severe epilepsies resulting from an upregulation of NMDA-R subunit-mediated transmission.

Pannexin 1 channels and ATP release in epilepsy: two sides of the same coin : The contribution of pannexin-1, connexins, and CALHM ATP-release channels to purinergic signaling.

Purinergic signaling mediated by ATP and its metabolites contributes to various brain physiological processes as well as to several pathological conditions, including neurodegenerative and neurological disorders, such as epilepsy. Among the different ATP release pathways, pannexin 1 channels represent one of the major conduits being primarily activated in pathological contexts. Investigations on in vitro and in vivo models of epileptiform activity and seizures in mice and human tissues revealed pannexin 1 involvement in aberrant network activity and epilepsy, and highlighted that pannexin 1 exerts a complex role. Pannexin 1 can indeed either sustain seizures through release of ATP that can directly activate purinergic receptors, or tune down epileptic activity via ATP-derived adenosine that decreases neuronal excitability. Interestingly, in-depth analysis of the literature unveils that this dichotomy is only apparent, as it depends on the model of seizure induction and the type of evoked epileptiform activity, two factors that can differentially activate pannexin 1 channels and trigger distinct intracellular signaling cascades. Here, we review the general properties and ATP permeability of pannexin 1 channels, and discuss their impact on acute epileptiform activity and chronic epilepsy according to the regime of activity and disease state. These data pave the way for the development of new antiepileptic strategies selectively targeting pannexin 1 channels in a context-dependent manner.

Gamma-aminobutyric acidergic transmission underlies interictal epileptogenicity in pediatric focal cortical dysplasia.

OBJECTIVE: Dysregulation of γ-aminobutyric acidergic (GABAergic) transmission has been reported in lesional acquired epilepsies (gliomas, hippocampal sclerosis). We investigated its involvement in a developmental disorder, human focal cortical dysplasia (FCD), focusing on chloride regulation driving GABAergic signals. METHODS: In vitro recordings of 47 human cortical acute slices from 11 pediatric patients who received operations for FCD were performed on multielectrode arrays. GABAergic receptors and chloride regulators were pharmacologically modulated. Immunostaining for chloride cotransporter KCC2 and interneurons were performed on recorded slices to correlate electrophysiology and expression patterns. RESULTS: FCD slices retain intrinsic epileptogenicity. Thirty-six of 47 slices displayed spontaneous interictal discharges, along with a pattern specific to the histological subtypes. Ictal discharges were induced in proepileptic conditions in 6 of 8 slices in the areas generating spontaneous interictal discharges, with a transition to seizure involving the emergence of preictal discharges. Interictal discharges were sustained by GABAergic signaling, as a GABAA receptor blocker stopped them in 2 of 3 slices. Blockade of NKCC1 Cl- cotransporters further controlled interictal discharges in 9 of 12 cases, revealing a Cl- dysregulation affecting actions of GABA. Immunohistochemistry highlighted decreased expression and changes in KCC2 subcellular localization and a decrease in the number of GAD67-positive interneurons in regions generating interictal discharges. INTERPRETATION: Altered chloride cotransporter expression and changes in interneuron density in FCD may lead to paradoxical depolarization of pyramidal cells. Spontaneous interictal discharges are consequently mediated by GABAergic signals, and targeting chloride regulation in neurons may be considered for the development of new antiepileptic drugs. Ann Neurol 2019; 1-14 ANN NEUROL 2019;85:204-217.

Astroglial Cx30 sustains neuronal population bursts independently of gap-junction mediated biochemical coupling.

Astroglial networks mediated by gap junction channels contribute to neurotransmission and promote neuronal coordination. Connexin 30, one of the two main astroglial gap junction forming protein, alters at the behavioral level the reactivity of mice to novel environment and at the synaptic level excitatory transmission. However, the role and function of Cx30 at the neuronal network level remain unclear. We thus investigated whether Cx30 regulates neuronal population bursts and associated convulsive behavior. We found in vivo that Cx30 is upregulated by kainate-induced seizures and that it regulates in turn the severity of associated behavioral seizures. Using electrophysiology ex vivo, we report that Cx30 regulates aberrant network activity via control of astroglial glutamate clearance independently of gap-junction mediated biochemical coupling. Altogether, our results indicate that astroglial Cx30 is an important player in orchestrating neuronal network activity.

Neuron-glia cross talk revealed in reverberating networks by simultaneous extracellular recording of spikes and astrocytes' glutamate transporter and K+ currents.

Astrocytes uptake synaptically released glutamate with electrogenic transporters (GluT) and buffer the spike-dependent extracellular K+ excess with background K+ channels. We studied neuronal spikes and the slower astrocytic signals on reverberating neocortical cultures and organotypic slices from mouse brains. Spike trains and glial responses were simultaneously captured from individual sites of multielectrode arrays (MEA) by splitting the recorded traces into appropriate filters and reconstructing the original signal by deconvolution. GluT currents were identified by using dl-threo-ß-benzyloxyaspartate (TBOA). K+ currents were blocked by 30 µM Ba2+, suggesting a major contribution of inwardly rectifying K+ currents. Both types of current were tightly correlated with the spike rate, and their astrocytic origin was tested in primary cultures by blocking glial proliferation with cytosine ß-d-arabinofuranoside (AraC). The spike-related, time-locked inward and outward K+ currents in different regions of the astrocyte syncytium were consistent with the assumptions of the spatial K+ buffering model. In organotypic slices from ventral tegmental area and prefrontal cortex, the GluT current amplitudes exceeded those observed in primary cultures by several orders of magnitude, which allowed to directly measure transporter currents with a single electrode. Simultaneously measuring cell signals displaying widely different amplitudes and kinetics will help clarify the neuron-glia interplay and make it possible to follow the cross talk between different cell types in excitable as well as nonexcitable tissue.

A Highly Selective Potassium Sensor for the Detection of Potassium in Living Tissues.

The development of highly selective sensors for potassium is of great interest in biology. Two new hydrosoluble potassium sensors (Calix-COU-Alkyne and Calix-COU-Am) based on a calix[4]arene bis(crown-6) and an extended coumarin were synthesized and characterized. The photophysical properties and complexation studies of these compounds have been investigated and show high molar extinction coefficients and high fluorescence quantum yields. Upon complexation with potassium in the millimolar concentration range, an increase of one- and two-photon fluorescence emission is detected. A twofold fluorescence enhancement is observed upon excitation at λ=405 nm. The ligands present excellent selectivity for potassium in the presence of various competitive cations in water and in a physiological medium. The photophysical properties are not affected by the presence of a large amount of competing cations (Na+ , Ca2+ , Mg2+ , etc.). Ex vivo measurements on mouse hippocampal slices show that Calix-COU-Alkyne accumulates extracellularly and does not alter the neuronal activity. Furthermore, the sensor can be utilized to monitor slow extracellular K+ increase induced by inhibition of K+ entry into the cells.

Functional regeneration of the ex-vivo reconstructed mesocorticolimbic dopaminergic system.

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

Astroglial gap junctions strengthen hippocampal network activity by sustaining afterhyperpolarization via KCNQ channels.

Throughout the brain, astrocytes form networks mediated by gap junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecular gap junction channels shape neuronal network patterns remains unclear. Here, using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we show that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a facilitation-depression model, we identify neuronal afterhyperpolarization as the key parameter underlying bursting pattern regulation by extracellular potassium in mice with disconnected astrocytes. We confirm this prediction experimentally and reveal that astroglial network control of extracellular potassium sustains neuronal afterhyperpolarization via KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap junctions mechanistically strengthen neuronal population bursts and point to approaches for controlling aberrant activity in neurological diseases.

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

Aberrant survival of hippocampal Cajal-Retzius cells leads to memory deficits, gamma rhythmopathies and susceptibility to seizures in adult mice.

Cajal-Retzius cells (CRs) are transient neurons, disappearing almost completely in the postnatal neocortex by programmed cell death (PCD), with a percentage surviving up to adulthood in the hippocampus. Here, we evaluate CR's role in the establishment of adult neuronal and cognitive function using a mouse model preventing Bax-dependent PCD. CRs abnormal survival resulted in impairment of hippocampus-dependent memory, associated in vivo with attenuated theta oscillations and enhanced gamma activity in the dorsal CA1. At the cellular level, we observed transient changes in the number of NPY+ cells and altered CA1 pyramidal cell spine density. At the synaptic level, these changes translated into enhanced inhibitory currents in hippocampal pyramidal cells. Finally, adult mutants displayed an increased susceptibility to lethal tonic-clonic seizures in a kainate model of epilepsy. Our data reveal that aberrant survival of a small proportion of postnatal hippocampal CRs results in cognitive deficits and epilepsy-prone phenotypes in adulthood.

Spatio-temporal characterization of causal electrophysiological activity stimulated by single pulse focused ultrasound: anex vivostudy on hippocampal brain slices.

Objective.The brain operates via generation, transmission and integration of neuronal signals and most neurological disorders are related to perturbation of these processes. Neurostimulation by focused ultrasound (FUS) is a promising technology with potential to rival other clinically used techniques for the investigation of brain function and treatment of numerous neurological diseases. The purpose of this study was to characterize spatial and temporal aspects of causal electrophysiological signals directly stimulated by short, single pulses of FUS onex vivomouse hippocampal brain slices.Approach.Microelectrode arrays (MEAs) are used to study the spatio-temporal dynamics of extracellular neuronal activities both at the single neuron and neural networks scales. Hence, MEAs provide an excellent platform for characterization of electrical activity generated, modulated and transmitted in response to FUS exposure. In this study, a novel mixed FUS/MEA platform was designed for the spatio-temporal description of the causal responses generated by single 1.78 MHz FUS pulses inex vivomouse hippocampal brain slices.Main results.Our results show that FUS pulses can generate local field potentials (LFPs), sustained by synchronized neuronal post-synaptic potentials, and reproducing network activities. LFPs induced by FUS stimulation were found to be repeatable to consecutive FUS pulses though exhibiting a wide range of amplitudes (50-600µV), durations (20-200 ms), and response delays (10-60 ms). Moreover, LFPs were spread across the hippocampal slice following single FUS pulses thus demonstrating that FUS may be capable of stimulating different neural structures within the hippocampus.Significance.Current knowledge on neurostimulation by ultrasound describes neuronal activity generated by trains of repetitive ultrasound pulses. This novel study details the causal neural responses produced by single-pulse FUS neurostimulation while illustrating the distribution and propagation properties of this neural activity along major neural pathways of the hippocampus.

Human astrocytes in the diseased brain.

Astrocytes are key active elements of the brain that contribute to information processing. They not only provide neurons with metabolic and structural support, but also regulate neurogenesis and brain wiring. Furthermore, astrocytes modulate synaptic activity and plasticity in part by controlling the extracellular space volume, as well as ion and neurotransmitter homeostasis. These findings, together with the discovery that human astrocytes display contrasting characteristics with their rodent counterparts, point to a role for astrocytes in higher cognitive functions. Dysfunction of astrocytes can thereby induce major alterations in neuronal functions, contributing to the pathogenesis of several brain disorders. In this review we summarize the current knowledge on the structural and functional alterations occurring in astrocytes from the human brain in pathological conditions such as epilepsy, primary tumours, Alzheimer's disease, major depressive disorder and Down syndrome. Compelling evidence thus shows that dysregulations of astrocyte functions and interplay with neurons contribute to the development and progression of various neurological diseases. Targeting astrocytes is thus a promising alternative approach that could contribute to the development of novel and effective therapies to treat brain disorders.

Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy.

Epilepsies are characterized by recurrent seizures, which disrupt normal brain function. Alterations in neuronal excitability and excitation-inhibition balance have been shown to promote seizure generation, yet molecular determinants of such alterations remain to be identified. Pannexin channels are nonselective, large-pore channels mediating extracellular exchange of neuroactive molecules. Recent data suggest that these channels are activated under pathological conditions and regulate neuronal excitability. However, whether pannexin channels sustain or counteract chronic epilepsy in human patients remains unknown. We studied the impact of pannexin-1 channel activation in postoperative human tissue samples from patients with epilepsy displaying epileptic activity ex vivo. These samples were obtained from surgical resection of epileptogenic zones in patients suffering from lesional or drug-resistant epilepsy. We found that pannexin-1 channel activation promoted seizure generation and maintenance through adenosine triphosphate signaling via purinergic 2 receptors. Pharmacological inhibition of pannexin-1 channels with probenecid or mefloquine-two medications currently used for treating gout and malaria, respectively-blocked ictal discharges in human cortical brain tissue slices. Genetic deletion of pannexin-1 channels in mice had anticonvulsant effects when the mice were exposed to kainic acid, a model of temporal lobe epilepsy. Our data suggest a proepileptic role of pannexin-1 channels in chronic epilepsy in human patients and that pannexin-1 channel inhibition might represent an alternative therapeutic strategy for treating lesional and drug-resistant epilepsies.

Human astrocytes: structure and functions in the healthy brain.

Data collected on astrocytes' physiology in the rodent have placed them as key regulators of synaptic, neuronal, network, and cognitive functions. While these findings proved highly valuable for our awareness and appreciation of non-neuronal cell significance in brain physiology, early structural and phylogenic investigations of human astrocytes hinted at potentially different astrocytic properties. This idea sparked interest to replicate rodent-based studies on human samples, which have revealed an analogous but enhanced involvement of astrocytes in neuronal function of the human brain. Such evidence pointed to a central role of human astrocytes in sustaining more complex information processing. Here, we review the current state of our knowledge of human astrocytes regarding their structure, gene profile, and functions, highlighting the differences with rodent astrocytes. This recent insight is essential for assessment of the relevance of findings using animal models and for comprehending the functional significance of species-specific properties of astrocytes. Moreover, since dysfunctional astrocytes have been described in many brain disorders, a more thorough understanding of human-specific astrocytic properties is crucial for better-adapted translational applications.

Astroglial networks promote neuronal coordination.

Astrocytes interact with neurons to regulate network activity. Although the gap junction subunits connexin 30 and connexin 43 mediate the formation of extensive astroglial networks that cover large functional neuronal territories, their role in neuronal synchronization remains unknown. Using connexin 30- and connexin 43-deficient mice, we showed that astroglial networks promoted sustained population bursts in hippocampal slices by setting the basal active state of neurons. Astroglial networks limited excessive neuronal depolarization induced by spontaneous synaptic activity, increased neuronal release probability, and favored the recruitment of neurons during bursting, thus promoting the coordinated activation of neuronal networks. In vivo, this sustained neuronal coordination translated into increased severity of acutely evoked epileptiform events and convulsive behavior. These results revealed that connexin-mediated astroglial networks synchronize bursting of neuronal assemblies, which can exacerbate pathological network activity and associated behavior. Our data thus provide molecular and biophysical evidence predicting selective astroglial gap junction inhibitors as anticonvulsive drugs.

Multi-electrode array recordings of human epileptic postoperative cortical tissue.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

Ventral tegmental area/substantia nigra and prefrontal cortex rodent organotypic brain slices as an integrated model to study the cellular changes induced by oxygen/glucose deprivation and reperfusion: effect of neuroprotective agents.

Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of "alternatively" activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment.

Novel modulatory effects of neurosteroids and benzodiazepines on excitatory and inhibitory neurons excitability: a multi-electrode array recording study.

The balance between glutamate- and GABA-mediated neurotransmission in the brain is fundamental in the nervous system, but it is regulated by the "tonic" release of a variety of endogenous factors. One such important group of molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in vivo physiologically relevant concentrations, the effects of NSs and BDZs as GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 µM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after wash-out, the excitability of inhibitory neurons continued to be depressed, leading to a network long-term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex vivo networks show a sensitivity to NSs and BDZs comparable to that expressed in vivo, but also provide a new global in vitro description that can help in understanding their activity in more complex systems.