Acute Slices Research Articles

Dual Role of the P2X7 Receptor in Dendritic Outgrowth during Physiological and Pathological Brain Development.

At high levels, extracellular ATP operates as a "danger" molecule under pathologic conditions through purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Its endogenous activation is associated with neurodevelopmental disorders; however, its function during early embryonic stages remains largely unclear. Our objective was to determine the role of P2X7R in the regulation of neuronal outgrowth. For this purpose, we performed Sholl analysis of dendritic branches on primary hippocampal neurons and in acute hippocampal slices from WT mice and mice with genetic deficiency or pharmacological blockade of P2X7R. Because abnormal dendritic branching is a hallmark of certain neurodevelopmental disorders, such as schizophrenia, a model of maternal immune activation (MIA)-induced schizophrenia, was used for further morphologic investigations. Subsequently, we studied MIA-induced behavioral deficits in young adult mice females and males. Genetic deficiency or pharmacological blockade of P2X7R led to branching deficits under physiological conditions. Moreover, pathologic activation of the receptor led to deficits in dendritic outgrowth on primary neurons from WT mice but not those from P2X7R KO mice exposed to MIA. Likewise, only MIA-exposed WT mice displayed schizophrenia-like behavioral and cognitive deficits. Therefore, we conclude that P2X7R has different roles in the development of hippocampal dendritic arborization under physiological and pathologic conditions.SIGNIFICANCE STATEMENT Our main finding is a novel role for P2X7R in neuronal branching in the early stages of development under physiological conditions. We show how a decrease in the expression of P2X7R during brain development causes the receptor to play pathologic roles in adulthood. Moreover, we studied a neurodevelopmental model of schizophrenia and found that, at higher ATP concentrations, endogenous activation of P2X7R is necessary and sufficient for the development of positive and cognitive symptoms.

Angiotensin II increases respiratory rhythmic activity in the preBötzinger complex without inducing astroglial calcium signaling.

Angiotensin II (Ang II) is the primary modulator of the renin-angiotensin system and has been widely studied for its effect on the cardiovascular system. While a few studies have also indicated an involvement of Ang II in the regulation of breathing, very little is known in this regard and its effect on brainstem respiratory regions such as the preBötzinger complex (preBötC), the kernel for inspiratory rhythm generation, has not been investigated yet. This study reports that Ang II temporarily increases phrenic nerve activity in the working heart-brainstem preparation, indicating higher central respiratory drive. Previous studies have shown that the carotid body is involved in mediating this effect and we revealed that the preBötC also plays a part, using acute slices of the brainstem. It appears that Ang II is increasing the respiratory drive in an AT1R-dependent manner by optimizing the interaction of inhibitory and excitatory neurons of the preBötC. Thus, Ang II-mediated effects on the preBötC are potentially involved in dysregulating breathing in patients with acute lung injury.

Abstract WP240: TRPM2 Channel Deletion In Neurons Reduces Injury Following Ischemic Stroke And Improves Post-stroke Cognitive Function

Background: Emerging evidence implicates post-stroke cognitive impairment as a major contributor to long-term disability. Therefore, optimal therapeutic targets reduce acute ischemic injury and enhance post-stroke brain function. Strong data demonstrates that a novel TRPM2 channel antagonist (tat-M2NX) provides neuroprotection and improves synaptic function, thereby reducing post-stroke cognitive impairment (PSCI). Hypothesis: Knockout of neuron-specific TRPM2 channel expression reduces infarct volume and enhances functional recovery following MCAO Methods: Transient MCAO (60 min) was performed on adult (8-10 week) male and female TRPM2 neuron-specific KO (TRPM2fl/fl, CaMKII Cre) and TRPM2 floxed controls (TRPM2fl/fl)Hemispheric infarct volume analyzed from MRI (T2) images 3 days post-injury by a blinded investigator. Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 7 days after recovery from MCAO to analyze synaptic placticity (LTP). Results: We observed that neuronal-specific TRPM2 channel knockout reduces acute ischemic injury (infarct volume) in male animals, while having minimal effect on female mice. Consistent with the hypothesis that neuronal TRPM2 channels contribute to ischemia-induced synaptic dysfunction, recordings obtained in brain slices from neuronal TRPM2 channel KO mice (TRPM2fl/fl-CaMKII CRE) mice 7 days after recovery from 60 min MCAo exhibited intact hippocampal plasticity compared to control TRPM2fl/fl mice not expressing CRE. Male sham control mice exhibit robust LTP of 163±10.4% (n=3) compared to 115±5.6% (n=4) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 175±9.8% (n=2). Similarly, female mice had control LTP of 170±6.8% (n=4) compared to 125±6.8% (n=3) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 208±7.8% (n=5, p<0.05 ANOVA compared to TRPM2fl/fl MCAO). Conclusion: Our data highlight that TRPM2 channels expressed in neurons contribute to both acute injury following transient ischemic stroke and subacute/chronic functional recovery.

Transient astrocytic accumulation of fluorescein during spreading depolarizations

Spreading depolarizations (SDs) occur frequently in acute cerebral injuries. They are characterized by a breakdown of transmembrane ion gradients resulting in a reduced extracellular sodium ([Na+]o) and increased extracellular potassium concentration ([K+]o). Elevated [K+]o induces astrocytic swelling, another feature of SD; however, the solutes that drive astrocytic swelling remain incompletely understood. We incidentally found astrocytic accumulation of fluorescein (Fluo) – a low molecular weight anionic dye – during SDs induced by elevated [K+]o. Herein, we aimed to explore the properties of astrocytic Fluo accumulation during SDs, electrical stimulation, [K+]o and glutamate elevation and elucidate underlying mechanisms and its relation to swelling. Experiments were performed in acute neocortical slices from adult male C57Bl6 mice and transgenic mice expressing tdTomato in parvalbumin (PV)-positive neurons. We labeled astrocytes with sulforhodamine-101 (SR-101), measured Fluo kinetics using 2-photon laser scanning microscopy and recorded local field potentials (LFP) to detect SDs. Elevations of [K+]o lead to an increase of the astrocytic Fluo intensity in parallel with astrocytic swelling. Pharmacological inhibitors of sodium‑potassium ATPase (Na/K-ATPase), secondary-active transporters and channels were used to address the underlying mechanisms. Fluo accumulation as well as swelling were only prevented by inhibition of the sodium‑potassium ATPase. Application of glutamate or hypoosmolar solution induced astrocytic swelling independent of Fluo accumulation and glutamate opposed Fluo accumulation when co-administered with high [K+]o. Astrocytes accumulated Fluo and swelled during electrical stimulation and even more during SDs. Taken together, Fluo imaging can be used as a tool to visualize yet unidentified anion fluxes during [K+]o- but not glutamate- or hypoosmolarity induced astrocytic swelling. Fluo imaging may thereby help to elucidate mechanisms of astrocytic swelling and associated fluid movements between brain compartments during physiological and pathological conditions, e.g. SDs.

Presenilins regulate synaptic plasticity in the perforant pathways of the hippocampus

Mutations in the Presenilin genes (PSEN1 and PSEN2) are the major cause of familial Alzheimer’s disease (AD), highlighting the importance of Presenilin (PS) in AD pathogenesis. Previous studies of PS function in the hippocampus demonstrated that loss of PS results in the impairment of short- and long-term synaptic plasticity and neurotransmitter release at hippocampal Schaffer collateral (SC) and mossy fiber (MF) synapses. Cortical input to the hippocampus through the lateral perforant pathway (LPP) and the medial perforant pathway (MPP) is critical for normal cognitive functions and is particularly vulnerable during aging and early stages of AD. Whether PS regulates synaptic function in the perforant pathways, however, remained unknown. In the current study, we investigate PS function in the LPP and MPP by performing whole-cell and field-potential electrophysiological recordings using acute hippocampal slices from postnatal forebrain-restricted excitatory neuron-specific PS conditional double knockout (cDKO) mice. We found that paired-pulse ratio (PPR) is reduced in the LPP and MPP of PS cDKO mice. Moreover, synaptic frequency facilitation or depression in the LPP or MPP, respectively, is impaired in PS cDKO mice. Notably, depletion of intracellular Ca2+ stores by inhibition of sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) minics and occludes the effects of PS inactivation, as evidenced by decreases of the evoked excitatory postsynaptic currents (EPSCs) amplitude in the LPP and MPP of control neurons but no effect on the EPSC amplitude in PS cDKO neurons, suggesting that impaired intracellular calcium homeostasis in the absence of PS may contribute to the observed deficits in synaptic transmission. While spontaneous synaptic events, such as both the frequency and the amplitude of spontaneous or miniature EPSCs, are similar between PS cDKO and control neurons, long-term potentiation (LTP) is impaired in the LPP and MPP of PS cDKO mice, accompanied with reduction of evoked NMDA receptor-mediated responses. These findings show the importance of PS in the regulation of synaptic plasticity and intracellular calcium homeostasis in the hippocampal perforant pathways.

Bi-directional modulation of hyperpolarization-activated cation currents (Ih) by ethanol in rat hippocampal CA3 pyramidal neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60–80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity.

13C Isotope Labeling and Mass Spectrometric Isotope Enrichment Analysis in Acute Brain Slices.

Feeding of stable 13C-labeled compounds coupled to mass spectrometric analysis has enabled the characterization of dynamic metabolite partitioning in various experimental conditions. This information is particularly relevant for the study and functional understanding of brain metabolic heterogeneity. We here describe a protocol for the analysis of metabolic enrichment analysis upon feeding of murine acute cerebellar slices with 13C-labeled substrates.

The positive allosteric modulator of NMDA receptors, GNE-9278, blocks the ethanol-induced decrease of excitability in developing retrosplenial cortex neurons from mice.

Binge-like exposure to ethanol during the brain growth spurt triggers apoptotic neurodegeneration in multiple brain regions, including the retrosplenial cortex, a brain region that is part of the hippocampal-diencephalic-cingulate memory network. This is mediated, in part, by reduced Ca2+ influx through N-methyl-d-aspartate (NMDA) receptors followed by a decrease in the activation of pro-survival genes. Here, we tested whether a positive allosteric modulator of NMDA receptors could counteract the inhibitory effect of ethanol on developing retrosplenial cortex pyramidal neurons. We used patch-clamp electrophysiological techniques in acute slices from postnatal day 6-8mice to test the effect of the positive allosteric modulator GNE-9278 on ethanol-induced inhibition of NMDA receptor function. GNE-9278 dose-dependently increased the amplitude, decay time, and total charge of NMDA excitatory postsynaptic currents. At a concentration of 5μmol L-1 , GNE-9278 significantly reduced the 90 mmol L-1 ethanol-induced inhibition of NMDA excitatory postsynaptic current amplitude, decay time, and total charge. Current-clamp experiments showed that 5μmol L-1 GNE-9278 ameliorated the 90 mmol L-1 ethanol-induced inhibition of synaptically-evoked action potential firing and compound excitatory postsynaptic potential amplitude. These findings indicate that positive allosteric modulators mitigate ethanol-induced hypofunction of NMDA receptors in developing cerebral cortex neurons, an effect that could ameliorate its pro-apoptotic effects during the late stages of fetal development.

Microglial Pten safeguards postnatal integrity of the cortex and sociability.

Microglial abnormalities may contribute to neurodevelopmental disorders. PTEN is implicated as a susceptibility gene for autism spectrum disorders and its germline ablation in mice causes behavioral abnormalities. Here we find postnatal PTEN deletion in microglia causes deficits in sociability and novel object recognition test. Mutant mice harbor markedly more activated microglia that manifest enhanced phagocytosis. Interestingly, two-week postponement of microglia PTEN ablation leads to no social interaction defects, even though mutant microglia remain abnormal in adult animals. Disturbed neurodevelopment caused by early PTEN deletion in microglia is characterized by insufficient VGLUT1 protein in synaptosomes, likely a consequence of enhanced removal by microglia. In correlation, in vitro acute slice recordings demonstrate weakened synaptic inputs to layer 5 pyramidal neurons in the developing cortex. Therefore, microglial PTEN safeguards integrity of neural substrates underlying sociability in a developmentally determined manner.

Long-Term Synaptic Plasticity Tunes the Gain of Information Channels through the Cerebellum Granular Layer.

A central hypothesis on brain functioning is that long-term potentiation (LTP) and depression (LTD) regulate the signals transfer function by modifying the efficacy of synaptic transmission. In the cerebellum, granule cells have been shown to control the gain of signals transmitted through the mossy fiber pathway by exploiting synaptic inhibition in the glomeruli. However, the way LTP and LTD control signal transformation at the single-cell level in the space, time and frequency domains remains unclear. Here, the impact of LTP and LTD on incoming activity patterns was analyzed by combining patch-clamp recordings in acute cerebellar slices and mathematical modeling. LTP reduced the delay, increased the gain and broadened the frequency bandwidth of mossy fiber burst transmission, while LTD caused opposite changes. These properties, by exploiting NMDA subthreshold integration, emerged from microscopic changes in spike generation in individual granule cells such that LTP anticipated the emission of spikes and increased their number and precision, while LTD sorted the opposite effects. Thus, akin with the expansion recoding process theoretically attributed to the cerebellum granular layer, LTP and LTD could implement selective filtering lines channeling information toward the molecular and Purkinje cell layers for further processing.

Dynamic regulation of excitatory and inhibitory synaptic transmission by growth hormone in the developing mouse brain.

Normal sensory and cognitive function of the brain relies on its intricate and complex neural network. Synaptogenesis and synaptic plasticity are critical to neural circuit formation and maintenance, which are regulated by coordinated intracellular and extracellular signaling. Growth hormone (GH) is the most abundant anterior pituitary hormone. Its deficiencies could alter brain development and impair learning and memory, while GH replacement therapy in human patients and animal models has been shown to ameliorate cognitive deficits caused by GH deficiency. However, the underlying mechanism remains largely unknown. In this study, we investigated the neuromodulatory function of GH in young (pre-weaning) mice at two developmental time points and in two different brain regions. Neonatal mice were subcutaneously injected with recombinant human growth hormone (rhGH) on postnatal day (P) 14 or 21. Excitatory and inhibitory synaptic transmission was measured using whole-cell recordings in acute cortical slices 2 h after the injection. We showed that injection of rhGH (2 mg/kg) in P14 mice significantly increased the frequency of mEPSCs, but not that of mIPSCs, in both hippocampal CA1 pyramidal neurons and L2/3 pyramidal neurons of the barrel field of the primary somatosensory cortex (S1BF). Injection of rhGH (2 mg/kg) in P21 mice significantly increased the frequency of mEPSCs and mIPSCs in both brain regions. Perfusion of rhGH (1 μM) onto acute brain slices in P14 mice had similar effects. Consistent with the electrophysiological results, the dendritic spine density of CA1 pyramidal neurons and S1BF L2/3 pyramidal neurons increased following in vivo injection of rhGH. Furthermore, NMDA receptors and postsynaptic calcium-dependent signaling contributed to rhGH-dependent regulation of both excitatory and inhibitory synaptic transmission. Together, these results demonstrate that regulation of excitatory and inhibitory synaptic transmission by rhGH occurs in a developmentally dynamic manner, and have important implication for identifying GH treatment strategies without disturbing excitation/inhibition balance.

Rapid presynaptic maturation in naturally regenerating axons of the adult mouse olfactory nerve.

Successful neuronal regeneration requires the reestablishment of synaptic connectivity. This process requires the reconstitution of presynaptic neurotransmitter release, which we investigate here in a model of entirely natural regeneration. After toxin-induced injury, olfactory sensory neurons in the adult mouse olfactory epithelium can regenerate fully, sending axons via the olfactory nerve to reestablish synaptic contact with postsynaptic partners in the olfactory bulb. Using electrophysiological recordings in acute slices, we find that, after initial recontact, functional connectivity in this system is rapidly established. Reconnecting presynaptic terminals have almost mature functional properties, including high release probability and strong capacity for presynaptic inhibition. Release probability then matures quickly, rendering reestablished terminals functionally indistinguishable from controls just 1week after initial contact. These data show that successful synaptic regeneration in the adult mammalian brain is almost a "plug-and-play" process, with presynaptic terminals undergoing a rapid phase of functional maturation as they reintegrate into target networks.

Calcineurin activation and scaffolding in Aß‐induced synaptic dysfunction

AbstractBackgroundCalcineurin (CaN) is a protein phosphatase that is essential for synaptic long‐term depression (LTD) through its regulation of AMPA‐type glutamate receptors (AMPAR). AMPAR regulation by CaN is critically mediated by the postsynaptic scaffold protein A‐kinase anchoring protein 150 (AKAP150), which localizes a pool of CaN to the postsynaptic membrane. Our lab has generated knockin mice carrying a mutant version of AKAP150 lacking its CaN‐binding site, the PxIxIT motif; AKAP150ΔPIX (PIX). LTD does not occur in these mice, underscoring the importance of the synaptic pool of CaN for normal synaptic function and plasticity. Pharmacological inhibition of calcineurin prevents diverse manifestations of Aβ‐mediated synaptotoxicity, including long‐term potentiation (LTP) blockade, synapse loss, and impaired learning and memory in AD mouse models. However, the importance of synaptically‐localized calcineurin in Aβ synaptotoxicity is unknown.MethodTo test our novel hypothesis that Aβ synaptotoxicity requires spatial positioning of CaN at the synapse via AKAP150 anchoring, we used our innovative AKAP150ΔPIX (PIX) knock‐in mouse model. Primary hippocampal neuronal cultures and acute hippocampal slices from WT and PIX mice were treated with various doses of Aβ oligomers and assayed for Aβ’s effect on dendritic spine density, quantal calcium transients through NMDA receptors (both in culture), and LTP through extracellular field recordings (in slices). Additionally, utilizing the 5xFAD AD mouse model, we generated PIX/5xFAD mice to test long‐term behavioral (via radial arm water maze and contextual fear conditioning tasks) and electrophysiological effects (through field recordings) of the PIX mutation on the 5xFAD phenotype in 6‐month‐old animals.ResultThe PIX mutation protects against acute Aβ‐mediated synaptic loss, decreased Ca2+ entry through NMDA receptors, and LTP deficits. Long‐term, the PIX mutation rescues contextual fear conditioning memory, but not radial arm water maze performance or chronic LTP deficits of 5xFAD mice.ConclusionSynaptically‐localized calcineurin is necessary for early Aβ‐mediated synaptic deficits. Long‐term Aβ exposure engages mechanisms beyond its AKAP‐anchored calcineurin pathway.

Targeting the Nav1.6:GSK3β Protein:Protein Interaction Complex to Mitigate Hippocampal Hyperexcitability in Early‐Stage Alzheimer’s Disease

AbstractBackgroundNav1.6 is the most densely expressed sodium channel isoform in the adult brain and is a primary determinant of action potential initiation and propagation due to its subcellular localization at the axon initial segment (AIS). Importantly, it has been shown that the firing frequency and Na+ currents of Nav 1.6 channels in hippocampal neurons are specifically increased by Aβ exposure in a concentration‐dependent manner. Nav1.6 is regulated through its interactions with key auxiliary proteins, notably Glycogen Synthase Kinase 3‐β (GSK3β), which has increased expression in the hippocampus of AD patients as well as peripheral circulating lymphocytes in both MCI and AD patients. We have shown that GSK3β regulates Nav1.6 channel activity through phosphorylation of and direct binding to the Nav1.6’s intracellular C‐terminal domain (CTD). Furthermore, genetic silencing of GSK3Ββ significantly decreased both Na+ currents and intrinsic firing compared to controls specifically through Nav1.6, while GSK3Ββ over‐expression or pharmacological disinhibition produced opposing phenotypes. Multiple lines of evidence indicate that hippocampal hyperactivity is present in patients with MCI and early‐stage AD and it is observed that reduction of hippocampal hyperactivity in MCI patients improves cognition and memory performance. We hypothesize that small‐molecule modulation of the Nav1.6:GSK3β PPI interface can counteract hippocampal hyperexcitability and could be used to treat early phases of AD, preventing the onset of cognitive decline.MethodHEK293 cells co‐transfected with WT or mutant Nav1.6 and GSK3β were utilized to interrogate the Nav1.6:GSK3β PPI and screen a series of 50 validated hit compounds using split‐luciferase complementation (LCA). Whole‐cell patch‐clamp electrophysiology was performed in heterologous cells and acute slice preparations to validate GSK3β‐mediated regulation of Nav1.6 activity and evaluate lead compound efficacy.ResultMutagenesis screening revealed putative interaction sites of the Nav1.6:GSK3β PPI. Counter‐screening of 50 validated hits revealed 5 compounds that inhibit Nav1.6:GSK3β complex assembly at low micromolar concentrations (Figure 1). Compound 5671063, identified from the counter‐screen, exhibits functional modulation of Nav1.6 activity in heterologous cells (Figure 2).ConclusionThe Nav1.6:GSK3β complex is a critical mediator of neuronal activity and represents a novel target for treatment of pathological hippocampal hyperexcitability associated with cognitive decline.

Effects of early life stress and subsequent re-exposure to stress on neuronal activity in the lateral habenula

Early life stress can result in depression in humans and depressive-like behaviour in rodents. In various animal models of depression, the lateral habenula (LHb) has been shown to become hyperactive immediately after early life stress. However, whether these pathological changes persist into adulthood is less well understood. Hence, we utilised the maternal separation (MS) model of depression to study how early life stress alters LHb physiology and depressive behaviour in adult mice. We find that only a weak depressive phenotype persists into adulthood which surprisingly is underpinned by LHb hypoactivity in acute slices, accompanied by alterations in both excitatory and inhibitory signalling. However, while we find the LHb to be less active at rest, we report that the neurons reside in a sensitised state where they are more responsive to re-exposure to stress in adulthood in the form of acute restraint, thus priming them to respond to aversive events with an increase in neuronal activity mediated by changes in glutamatergic transmission. These findings thus suggest that in addition to LHb hyperactivity, hypoactivity likely also promotes an adverse phenotype. Re-exposure to stress results in the reappearance of LHb hyperactivity offering a possible mechanism to explain how depression relapses occur following previous depressive episodes.

Action potential firing rhythms in the suprachiasmatic nucleus of the diurnal grass rat, Arvicanthis niloticus

In mammals, daily physiological activities are regulated by a central circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, an increasing number of studies have used diurnal grass rats to analyze neuronal mechanisms regulating diurnal behavior. However, spontaneous action potential firing rhythms in SCN neurons have not been demonstrated clearly in diurnal grass rats. Therefore, the present study examined extracellular single-unit recordings from SCN neurons in acute hypothalamic slices of Arvicanthis niloticus (Nile grass rats). The results of this study found that circadian firing rhythms with the highest frequency occurred at dusk (6.4 Hz at zeitgeber time (ZT)10–12), while the secondary peak occurred at dawn (5.6 Hz at ZT0-2), and the lowest frequency took place in the middle of the night (3.6 Hz at ZT14-16). Locomotor activity recordings from a separate group of animals demonstrated that the Nile grass rats of the laboratory colony used in this study displayed diurnal behaviors that coincided with large crepuscular peaks under 12:12 h light–dark cycles and bimodal rhythms under constant dim red light. Thus, a positive correlation between SCN firing frequencies and locomotor activity levels was observed in the Nile grass rats. Previously, behavioral coupling of action potential firings in SCN neurons has been suggested by in vivo recordings while the present study demonstrates that the sustenance of bimodal firing rhythms in grass rat SCN neurons can last at least one day in vitro.

Enhanced recruitment of glutamate receptors underlies excitotoxicity of mitral cells in acute hyperammonemia.

Hepatic encephalopathy (HE)-a major complication of liver disease-has been found to increase the risk of olfactory dysfunction, which may be attributed to elevated levels of ammonia/ammonium in the blood and cerebrospinal fluid. However, the cellular mechanisms underlying hyperammonemia-induced olfactory dysfunction remain unclear. By performing patch-clamp recordings of mitral cells (MCs) in the mouse olfactory bulb (OB), we found that 3 mM ammonium (NH4 +) increased the spontaneous firing frequency and attenuated the amplitude, but synaptic blockers could prevent the changes, suggesting the important role of glutamate receptors in NH4 +-induced hyperexcitability of MCs. We also found NH4 + reduced the currents of voltage-gated K+ channel (Kv), which may lead to the attenuation of spontaneous firing amplitude by NH4 +. Further studies demonstrated NH4 + enhanced the amplitude and integral area of long-lasting spontaneous excitatory post-synaptic currents (sEPSCs) in acute OB slices. This enhancement of excitatory neurotransmission in MCs occurred independently of pre-synaptic glutamate release and re-uptake, and was prevented by the exocytosis inhibitor TAT-NSF700. In addition, an NH4 +-induced increasement in expression of NR1 and GluR1 was detected on cytoplasmic membrane, indicating that increased trafficking of glutamate receptors on membrane surface in MCs is the core mechanism. Moreover, NH4 +-induced enhanced activity of glutamate receptors in acute OB slices caused cell death, which was prevented by antagonizing glutamate receptors or chelating intracellular calcium levels. Our study demonstrates that the enhancement of the activity and recruitment of glutamate receptor directly induces neuronal excitotoxicity, and contributes to the vulnerability of OB to acute hyperammonemia, thus providing a potential pathological mechanism of olfactory defects in patients with hyperammonemia and HE.

GABAA receptor function is enhanced by Interleukin-10 in human epileptogenic gangliogliomas and its effect is counteracted by Interleukin-1β

Gangliogliomas (GGs) are low-grade brain tumours that cause intractable focal epilepsy in children and adults. In GG, as in epileptogenic focal malformations (i.e., tuberous sclerosis complex, TSC), there is evidence of sustained neuroinflammation with involvement of the pro-inflammatory cytokine IL-1β. On the other hand, anti-inflammatory mediators are less studied but bear relevance for understanding seizure mechanisms. Therefore, we investigated the effect of the key anti-inflammatory cytokine IL-10 on GABAergic neurotransmission in GG. We assessed the IL-10 dependent signaling by transcriptomic analysis, immunohistochemistry and performed voltage-clamp recordings on Xenopus oocytes microtransplanted with cell membranes from brain specimens, to overcome the limited availability of acute GG slices. We report that IL-10-related mRNAs were up-regulated in GG and slightly in TSC. Moreover, we found IL-10 receptors are expressed by neurons and astroglia. Furthermore, GABA currents were potentiated significantly by IL-10 in GG. This effect was time and dose-dependent and inhibited by blockade of IL-10 signaling. Notably, in the same tissue, IL-1β reduced GABA current amplitude and prevented the IL-10 effect. These results suggest that in epileptogenic tissue, pro-inflammatory mechanisms of hyperexcitability prevail over key anti-inflammatory pathways enhancing GABAergic inhibition. Hence, boosting the effects of specific anti-inflammatory molecules could resolve inflammation and reduce intractable seizures.

WNK3 kinase maintains neuronal excitability by reducing inwardly rectifying K+ conductance in layer V pyramidal neurons of mouse medial prefrontal cortex.

The with-no-lysine (WNK) family of serine-threonine kinases and its downstream kinases of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) may regulate intracellular Cl− homeostasis through phosphorylation of cation-Cl− co-transporters. WNK3 is expressed in fetal and postnatal brains, and its expression level increases during development. Its roles in neurons, however, remain uncertain. Using WNK3 knockout (KO) mice, we investigated the role of WNK3 in the regulation of the intracellular Cl− concentration ([Cl−]i) and the excitability of layer V pyramidal neurons in the medial prefrontal cortex (mPFC). Gramicidin-perforated patch-clamp recordings in neurons from acute slice preparation at the postnatal day 21 indicated a significantly depolarized reversal potential for GABAA receptor-mediated currents by 6 mV, corresponding to the higher [Cl−]i level by ~4 mM in KO mice than in wild-type littermates. However, phosphorylation levels of SPAK and OSR1 and those of neuronal Na+-K+-2Cl− co-transporter NKCC1 and K+-Cl− co-transporter KCC2 did not significantly differ between KO and wild-type mice. Meanwhile, the resting membrane potential of neurons was more hyperpolarized by 7 mV, and the minimum stimulus current necessary for firing induction was increased in KO mice. These were due to an increased inwardly rectifying K+ (IRK) conductance, mediated by classical inwardly rectifying (Kir) channels, in KO neurons. The introduction of an active form of WNK3 into the recording neurons reversed these changes. The potential role of KCC2 function in the observed changes of KO neurons was investigated by applying a selective KCC2 activator, CLP290. This reversed the enhanced IRK conductance in KO neurons, indicating that both WNK3 and KCC2 are intimately linked in the regulation of resting K+ conductance. Evaluation of synaptic properties revealed that the frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced, whereas that of inhibitory currents (mIPSCs) was slightly increased in KO neurons. Together, the impact of these developmental changes on the membrane and synaptic properties was manifested as behavioral deficits in pre-pulse inhibition, a measure of sensorimotor gating involving multiple brain regions including the mPFC, in KO mice. Thus, the basal function of WNK3 would be the maintenance and/or development of both intrinsic and synaptic excitabilities.

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells.

Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.