Increase In Neuronal Excitability Research Articles

ML-Based Models as a Strategy to Discover Novel Antiepileptic Drugs Targeting Sodium Receptor Channel.

Epilepsy remains the most common and chronic disorder demanding longterm management. The impact of epilepsy disease is a cause of great concern and has resulted in efforts to develop treatment for epilepsy. It occurs due to an increase in neuronal excitability produced by changes affecting the voltage-dependent properties of Voltage-gated Sodium Channels (VGSCs). Weka, a popular suite for machine learning techniques, was used on a dataset comprising 1781 chemical compounds, showing inhibition activity for sodium channel protein IX alpha subunit. After the analysis of the dataset obtained from ChEMBL, molecular fingerprints were computed for the molecules by the ChemDes server. Different classifiers available in the Weka software were explored to find out the algorithm that could be more suitable for the dataset or produce the highest accuracy for the classification of molecules as active or inactive. In this work, a comprehensive comparison of different classifiers in the Weka suite for the prediction of active, inactive, and intermediate classes of molecules showing inhibition against human NaV1.7 protein was made. The prediction accuracy of these classifiers was assessed based on performance measures, including accuracy, Root Mean Squared Error (RMSE), Receiver Operating Characteristic (ROC), precision, Mathews Correlation Coefficient (MCC), recall, and Fmeasure. The comparison of results for model performance demonstrated that the OneR classifier performed best over others when validated using percentage split, cross-validation, and supplied test methods. J48 and Bagging also performed equally well in the prediction of different classes with an MCC value of 1, ROC area equal to 1, and RMSE close to 0. Machine Learning (ML) tools provide a fast, reliable, and cost-effective approach required to identify or predict inhibitory molecules for the treatment of a disease. This study shows that the ML methods, particularly OneR, J48, and Bagging have the ability to identify active and inactive classes of compounds for the human NaV1.7 protein target. Such predictive models may provide a reliable and time-saving approach that can aid in the design of potential inhibitors for the treatment of epilepsy disease.

Seizures in small brains

Neonatal seizures are the most common neurological emergency in newborns; often signalling significant neurological dysfunction. Their pathophysiology is complex and relates to the imbalance between excitatory and inhibitory neurotransmitters in the immature brain. During the neonatal period, excitatory circuits mediated via neuro-transmitters like glutamate predominate while inhibitory circuits mediated via gamma-aminobutyric acid (GABA) signalling are underdeveloped. Additionally, immature ion channels, such as sodium, chloride, potassium, and calcium, increase neuronal excitability. The most common aetiology of seizures is attributed to neonatal hypoxic cerebral injury. However, metabolic vulnerabilities, such as hypoglycaemia and electrolyte imbalances, can also precipitate seizures. Identifying the underlying cause, which in a large majority is acute symptomatic, is critical for prompt treatment. Early intervention is essential to prevent long-term neurodevelopmental consequences.Diagnosing neonatal seizures is challenging due to their subtle presentations. Distinguishing them from non-seizure events is crucial. Accurate diagnosis is essential for appropriate treatment and management. Continuous electroencephalography (cEEG) is the gold standard for diagnosis, but an amplitude-integrated EEG (aEEG) is a useful alternative tool in neonatal intensive care units (NICUs), for high-risk infants. This facilitates diagnosis of electroclinical as well as electrical seizures; commonly encountered in the very sick newborns and those born prematurely. Neuroimaging, like magnetic resonance imaging (MRI) and computed tomography (CT) is required to identify structural causes, while laboratory and genetic tests check for infectious, metabolic and genetic aetiologies. The new classification proposed by the International League Against Epilepsy (ILAE), underscores the importance of recognition of electrical seizures and highlights the value of recognising the seizure semiology for the diagnosis of the underlying aetiology.

Association Of IL-6, And IL-10 Gene Snps In Childhood Febrile Seizure

Background: Febrile seizure are typically defined as convulsions=that-occur in children, between/6 months to 5years, who have a fever of more,,than”38 degrees Celsius, that is not associated with an intracranial’ reason such as an infection, ,head .injury, or epilepsy. It is also known as the immature brain's response to fever, which is age-dependent. As a child’s brain develops, there is an increase in neuronal excitability which puts the child at the risk of febrile seizures. Aims: the aim of the present study is to find out the association of Inflammatory cytokines (IL-6, IL-10) SNP with the onset of childhood febrile seizure. Method- Blood samples from patients with childhood febrile seizure will be collected in a sterile condition, and the association of SNP for both IL-6 (-597) G/A, and IL-10 (-819) C/T with disease susceptibility will be studied by using allele specific PCR. Results: The case-control study of 40 patients with Febrile seizure and 40 control without Febrile seizure has revealed that a substantial difference in the frequency distribution of IL-6 genotypes between the patient group and the control group where (p =0.041). Besides, there was no discernible variation in the frequency distribution of IL-10 genotypes and alleles. (p > 0.05); therefore, none of genotypes or alleles can be regarded as risk factor or protective factor. Conclusion: The present study has concluded that IL-6(-597) G/A, (rs:1800797) single nucleotide polymorphism (SNP) was associated with Febrile seizure susceptibility, and GG, genotype considered as, risk factor, while genotype GA act as protective factor. However, it refers that the IL-10-819C/T (rs:1800871), genes may not represent the Febrile-seizure-associated genetic risk factor.

Acute ketamine induces neuronal hyperexcitability and deficits in prepulse inhibition by upregulating IL-6

Acute ketamine administration results in psychotic symptoms similar to those observed in schizophrenia and is regarded as a pharmacological model of schizophrenia. Accumulating evidence suggests that patients with schizophrenia show increased IL-6 levels in the blood and cerebrospinal fluid and that IL-6 levels are associated with the severity of psychotic symptoms. In the present study, we found that a single ketamine exposure led to increased expression of IL-6 and IL-6Rα, decreased dendritic spine density, increased expression and currents of T-type calcium channels, and increased neuron excitability in the hippocampal CA1 area 12 h after exposure. Acute ketamine administration also led to impaired prepulse inhibition (PPI) 12 h after administration. Additionally, we found that the expression of signaling molecules IKKα/β, NF-κB, JAK2, and STAT3 was upregulated 12 h after a single ketamine injection. The decreases in dendritic spine density, the increases in calcium currents and neuron excitability, and the impairments in PPI were ameliorated by blocking IL-6 or IL-6Rα. Our findings show that blocking IL-6 or its receptor may protect hippocampal neurons from hyperexcitability, thereby ameliorating ketamine-induced psychotic effects. Our study provides additional evidence that targeting IL-6 and its receptor is a potential strategy for treating psychotic symptoms in acute ketamine-induced psychosis and schizophrenia.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Structural-functional properties of direct-pathway striatal neurons at early and chronic stages of dopamine denervation.

The dendritic arbour of striatal projection neurons (SPNs) is the primary anatomical site where dopamine and glutamate inputs to the basal ganglia functionally interact to control movement. These dendritic arbourisations undergo atrophic changes in Parkinson's disease. A reduction in the dendritic complexity of SPNs is found also in animal models with severe striatal dopamine denervation. Using 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle as a model, we set out to compare morphological and electrophysiological properties of SPNs at an early versus a chronic stage of dopaminergic degeneration. Ex vivo recordings were performed in transgenic mice where SPNs forming the direct pathway (dSPNs) express a fluorescent reporter protein. At both the time points studied (5 and 28 days following 6-OHDA lesion), there was a complete loss of dopaminergic fibres through the dorsolateral striatum. A reduction in dSPN dendritic complexity and spine density was manifest at 28, but not 5days post-lesion. At the late time point, dSPN also exhibited a marked increase in intrinsic excitability (reduced rheobase current, increased input resistance, more evoked action potentials in response to depolarising currents), which was not present at 5days. The increase in neuronal excitability was accompanied by a marked reduction in inward-rectifying potassium (Kir) currents (which dampen the SPN response to depolarising stimuli). Our results show that dSPNs undergo delayed coordinate changes in dendritic morphology, intrinsic excitability and Kir conductance following dopamine denervation. These changes are predicted to interfere with the dSPN capacity to produce a normal movement-related output.

Serotonin excites medial olivocochlear neurons of the auditory efferent pathway

Medial olivocochlear (MOC) efferent neurons are located in the superior olive of the brainstem and form a sound evoked feedback loop that inhibits cochlear amplification via suppression of outer hair cell (OHC) electromotility. MOC neurons putatively receive a diverse range of synaptic input from various auditory and non-auditory brain regions. Given the proposed role of these neurons in context dependent tasks such as selective attention, we are interested in investigating the non-auditory modulation of MOC activity by the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). We used the ChAT-IRES-Cre;tdTomato mouse model to identify cholinergic MOC neurons in the brainstem. Immunohistochemical data have validated serotonergic terminals in apposition to both retrogradely labeled and genetically identified MOC neurons in mouse. During patch-clamp recordings from MOC neurons, exogenous application of serotonin increased neuron excitability by increasing action potential (AP) firing rate and decreasing both rheobase and AP threshold. Additionally, serotonin reduced the stimulation required to evoke a given AP firing rate in MOC neurons. These data will aid in our understanding of central auditory processing and how factors such as mood and attention are involved in modulating MOC responses in complex listening situations such as in the presence of background noise.

ST2-Conditioned Medium Fosters Dorsal Horn Cell Excitability and Synaptic Transmission in Cultured Mouse Spinal Cord

Conditioned medium obtained from bone marrow-derived stem cells has been proposed as a novel cell-free therapy in spinal cord injury and neuropathic pain, yet the direct effect on spinal neuron function has never been investigated. Here, we adopted spinal cord organotypic cultures (SCOCs) as an experimental model to probe the effect of ST2 murine mesenchymal stem cells-conditioned medium (ST2-CM) on dorsal horn (DH) neuron functional properties. Three days of SCOC exposure to ST2-CM increased neuronal activity measured by Fos expression, as well as spontaneous or induced firing. We showed that the increase in neuronal excitability was associated with changes in both intrinsic membrane properties and an enhanced excitatory drive. The increased excitability at the single-cell level was substantiated at the network level by detecting synchronous bursts of calcium waves across DH neurons. Altogether, SCOCs represent a viable tool to probe mesenchymal cells' effect on intact neuronal networks. Our findings indicate that ST2-CM enhances neuronal activity and synaptic wiring in the spinal dorsal horn. Our data also support the trophic role of mesenchymal cells CM in maintaining network activity in spinal circuits.Graphical

GPER1 deficiency causes sex-specific dysregulation of hippocampal plasticity and cognitive function.

Estrogens regulate synaptic properties and influence hippocampus-related learning and memory via estrogen receptors, which include the G-protein-coupled estrogen receptor 1 (GPER1). Studying mice, in which the GPER1 gene is dysfunctional (GPER1-KO), we here provide evidence for sex-specific roles of GPER1 in these processes. GPER1-KO males showed reduced anxiety in the elevated plus maze, whereas the fear response ('freezing') was specifically increased in GPER1-KO females in a contextual fear conditioning paradigm. In the Morris water maze, spatial learning and memory consolidation was impaired by GPER1 deficiency in both sexes. Notably, in the females, spatial learning deficits and the fear response were more pronounced if mice were in a stage of the estrous cycle, in which E2 serum levels are high (proestrus) or rising (diestrus). On the physiological level, excitability at Schaffer collateral synapses in CA1 increased in GPER1-deficient males and in proestrus/diestrus ('E2 high') females, concordant with an increased hippocampal expression of the AMPA-receptor subunit GluA1 in GPER1-KO males and females as compared to wildtype males. Further changes included an augmented early long-term potentiation (E-LTP) maintenance specifically in GPER1-KO females and an increased hippocampal expression of spinophilin in metestrus/estrus ('E2 low') GPER1-KO females. Our findings suggest modulatory and sex-specific functions of GPER1 in the hippocampal network, which reduce rather than increase neuronal excitability. Dysregulation of these functions may underlie sex-specific cognitive deficits or mood disorders.

Tau in cerebrospinal fluid induces neuronal hyperexcitability and alters hippocampal theta oscillations

Alzheimer’s disease (AD) and other tauopathies are characterized by the aggregation of tau into soluble and insoluble forms (including tangles and neuropil threads). In humans, a fraction of both phosphorylated and non-phosphorylated N-terminal to mid-domain tau species, are secreted into cerebrospinal fluid (CSF). Some of these CSF tau species can be measured as diagnostic and prognostic biomarkers, starting from early stages of disease. While in animal models of AD pathology, soluble tau aggregates have been shown to disrupt neuronal function, it is unclear whether the tau species present in CSF will modulate neural activity. Here, we have developed and applied a novel approach to examine the electrophysiological effects of CSF from patients with a tau-positive biomarker profile. The method involves incubation of acutely-isolated wild-type mouse hippocampal brain slices with small volumes of diluted human CSF, followed by a suite of electrophysiological recording methods to evaluate their effects on neuronal function, from single cells through to the network level. Comparison of the toxicity profiles of the same CSF samples, with and without immuno-depletion for tau, has enabled a pioneering demonstration that CSF-tau potently modulates neuronal function. We demonstrate that CSF-tau mediates an increase in neuronal excitability in single cells. We then observed, at the network level, increased input–output responses and enhanced paired-pulse facilitation as well as an increase in long-term potentiation. Finally, we show that CSF-tau modifies the generation and maintenance of hippocampal theta oscillations, which have important roles in learning and memory and are known to be altered in AD patients. Together, we describe a novel method for screening human CSF-tau to understand functional effects on neuron and network activity, which could have far-reaching benefits in understanding tau pathology, thus allowing for the development of better targeted treatments for tauopathies in the future.Graphical

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Nav1.9: A unique voltage-gated sodium channel with a role in inflammatory pain.

In inflamed tissue, sensitization of pain-sensing neurons results in exaggerated responses to mild or even innocuous stimuli. A substantial part of this increase in neuronal excitability is due to an effect on NaV1.9, an elusive member of the voltage-gated sodium (NaV) channel family that deviates from other subtypes because of its ultra-slow kinetics and hyperpolarized activation voltage. How these exceptional biophysical characteristics affect neurons at baseline and under stimulated conditions is a matter of debate, and this is likely to remain so until the major hurdles impeding its investigation can be eluded. These hurdles entail the difficulty of expression in heterologous expression systems, a lack of pharmacological tools and the general variability encountered in NaV1.9 experimentation. The goal of our research is to understand the reasons behind these complications and to establish a system that allows robust electrophysiological recordings. This work can be a starting point for future studies focusing on NaV1.9 pharmacology or its involvement in inflammatory pain pathways.

Ultrasound modulates neuronal potassium currents via ionotropic glutamate receptors

BackgroundFocused ultrasound stimulation (FUS) has the potential to provide non-invasive neuromodulation of deep brain regions with unparalleled spatial precision. However, the cellular and molecular consequences of ultrasound stimulation on neurons remains poorly understood. We previously reported that ultrasound stimulation induces increases in neuronal excitability that persist for hours following stimulation in vitro. In the present study we sought to further elucidate the molecular mechanisms by which ultrasound regulates neuronal excitability and synaptic function. ObjectivesTo determine the effect of ultrasound stimulation on voltage-gated ion channel function and synaptic plasticity. MethodsPrimary rat cortical neurons were exposed to a 40 s, 200 kHz pulsed ultrasound stimulus or sham-stimulus. Whole-cell patch clamp electrophysiology, quantitative proteomics and high-resolution confocal microscopy were employed to determine the effects of ultrasound stimulation on molecular regulators of neuronal excitability and synaptic function. ResultsWe find that ultrasound exposure elicits sustained but reversible increases in whole-cell potassium currents. In addition, we find that ultrasound exposure activates synaptic signalling cascades that result in marked increases in excitatory synaptic transmission. Finally, we demonstrate the requirement of ionotropic glutamate receptor (AMPAR/NMDAR) activation for ultrasound-induced modulation of neuronal potassium currents. ConclusionThese results suggest specific patterns of pulsed ultrasound can induce contemporaneous enhancement of both neuronal excitability and synaptic function, with implications for the application of FUS in experimental and therapeutic settings. Further study is now required to deduce the precise molecular mechanisms through which these changes occur.

PS-B05-7: HYPERTENSION IN A MODEL OF PKD IS NOT DEPENDENT ON THE SHORT-TERM ACTIONS OF TUMOUR NECROSIS FACTOR-A IN THE SFO

Objective: The development of hypertension in the Lewis polycystic kidney (LPK) disease model of kidney disease is caused, in part, by neuronal overactivity in the subfornical organ (SFO). Circulating proinflammatory cytokines, namely TNFα, are suggested to act in the central nervous system to produce an increase in neuronal excitability. As circulating cytokines are increased in kidney disease, we hypothesised that TNFα acts on the SFO to increase neuronal activity, therefore contributing to the development of hypertension in this animal model. Design and Methods: Urethane anaesthetised Lewis control (n = 23 total) and LPK (n = 18 total) animals were instrumented to record blood pressure and perform microinjections of TNFα (1–300pg/50nl), TNFα receptor 1 (TNFRI) neutralising antibody (1ng/50nl) or minocycline (0.5 μg/50nl), an inhibitor of microglial activation. Results: Exogenous TNFα microinjected into the SFO elicited a significant pressor response in the Lewis control but not the LPK animals (9 ± 2 mmHg vs -1 ± 3 mmHg, Lewis vs LPK peak change from baseline, P = 0.04). Acute inhibition of actions of local TNFα via administration of TNFRI neutralising antibody in the SFO did not reduce mean arterial blood pressure in Lewis control or LPK animals (1 ± 1mmHg vs -1 ± 1mmHg, Lewis vs LPK change from baseline, P = 0.59). Acute blockade of the actions of all proinflammatory cytokines on microglia via microinjection of minocycline in the SFO did not reduce blood pressure in Lewis control or LPK animals (-1 ± 1mmHg vs -1 ± 1mmHg, Lewis vs LPK change from baseline, P = 0.11). Prior microinjection of TNFRI neutralising antibody into the SFO abolished the pressor response observed upon microinjection of TNFα in Lewis rats (9 ± 2 mmHg vs 1 ± 1 mmHg, TNFα vs TNFα after TNFRI Ab peak change from baseline, P = 0.01), whereas prior microinjection of minocycline into the SFO only attenuated the pressor response observed upon TNFα microinjection in Lewis rats (9 ± 2 mmHg vs 3 ± 1 mmHg, TNFα vs TNFα after Minocycline peak change from baseline, P = 0.04) Conclusions: Overall, these findings demonstrate that although hypertension observed in the LPK is sustained by an increase in SFO activity, the short-term control of mean arterial blood pressure activity is not dependent on the actions of endogenous TNFα or generalised microglial activation by proinflammatory cytokines in the SFO.

Expression of cation chloride cotransporter (NKCC1/KCC2) in brain tissue of children with focal cortical dysplasia type Ⅱ

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.

Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium.

Rapid increases in cell volume reduce the size of the extracellular space (ECS) and are associated with elevated brain tissue excitability. We recently demonstrated that astrocytes, but not neurons, rapidly swell in elevated extracellular potassium (∧[K+]o) up to 26 mM. However, effects of acute astrocyte volume fluctuations on neuronal excitability in ∧[K+]o have been difficult to evaluate due to direct effects on neuronal membrane potential and generation of action potentials. Here we set out to isolate volume-specific effects occurring in ∧[K+]o on CA1 pyramidal neurons in acute hippocampal slices by manipulating cell volume while recording neuronal glutamate currents in 10.5 mM [K+]o + tetrodotoxin (TTX) to prevent neuronal firing. Elevating [K+]o to 10.5 mM induced astrocyte swelling and produced significant increases in neuronal excitability in the form of mixed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/N-methyl-D-aspartate (NMDA) receptor mEPSCs and NMDA receptor-dependent slow inward currents (SICs). Application of hyperosmolar artificial cerebrospinal fluid (ACSF) by addition of mannitol in the continued presence of 10.5 mM K+ forced shrinking of astrocytes and to a lesser extent neurons, which resisted swelling in ∧[K+]o. Cell shrinking and dilation of the ECS significantly dampened neuronal excitability in 10.5 mM K+. Subsequent removal of mannitol amplified effects on neuronal excitability and nearly doubled the volume increase in astrocytes, presumably due to continued glial uptake of K+ while mannitol was present. Slower, larger amplitude events mainly driven by NMDA receptors were abolished by mannitol-induced expansion of the ECS. Collectively, our findings suggest that cell volume regulation of the ECS in elevated [K+]o is driven predominantly by astrocytes, and that cell volume effects on neuronal excitability can be effectively isolated in elevated [K+]o conditions.

Intrinsic excitability mechanisms of neuronal ensemble formation.

Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals of both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we found that, after optogenetic or electrical stimulation, coactivated neurons increased their correlated activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Optogenetic and electrical stimulation also induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed unexpected strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reductions in spike threshold. A pharmacological agent that blocks changes in membrane resistance reverted this effect. These significant increases in excitability can explain the observed biphasic synaptic plasticity. We conclude that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an 'iceberg' model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.

Protease‐Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota is Associated with Visceral Hypersensitivity

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.

Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality.

The human brain is a complex, three‐dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human‐specific midbrain organoids. Human iPSC‐derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation‐related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC‐derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC‐derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress‐related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation.

Synaptic dysregulation and hyperexcitability induced by intracellular amyloid beta oligomers.

Intracellular amyloid beta oligomer (iAβo) accumulation and neuronal hyperexcitability are two crucial events at early stages of Alzheimer's disease (AD). However, to date, no mechanism linking iAβo with an increase in neuronal excitability has been reported. Here, the effects of human AD brain‐derived (h‐iAβo) and synthetic (iAβo) peptides on synaptic currents and action potential firing were investigated in hippocampal neurons. Starting from 500 pM, iAβo rapidly increased the frequency of synaptic currents and higher concentrations potentiated the AMPA receptor‐mediated current. Both effects were PKC‐dependent. Parallel recordings of synaptic currents and nitric oxide (NO)‐associated fluorescence showed that the increased frequency, related to pre‐synaptic release, was dependent on a NO‐mediated retrograde signaling. Moreover, increased synchronization in NO production was also observed in neurons neighboring those dialyzed with iAβo, indicating that iAβo can increase network excitability at a distance. Current‐clamp recordings suggested that iAβo increased neuronal excitability via AMPA‐driven synaptic activity without altering membrane intrinsic properties. These results strongly indicate that iAβo causes functional spreading of hyperexcitability through a synaptic‐driven mechanism and offers an important neuropathological significance to intracellular species in the initial stages of AD, which include brain hyperexcitability and seizures.