Neurons In Slices Research Articles

Structural and Functional Plasticity in the Dorsolateral Geniculate Nucleus of Mice following Bilateral Enucleation

Within the nervous system, plasticity mechanisms attempt to stabilize network activity following disruption by injury, disease, or degeneration. Optic nerve injury and age-related diseases can induce homeostatic-like responses in adulthood. We tested this possibility in the thalamocortical (TC) neurons in the dorsolateral geniculate nucleus (dLGN) using patch-clamp electrophysiology, optogenetics, immunostaining, and single-cell dendritic analysis following loss of visual input via bilateral enucleation. We observed progressive loss of vGlut2-positive retinal terminals in the dLGN indicating degeneration post-enucleation that was coincident with changes in microglial morphology indicative of microglial activation. Consistent with the decline of vGlut2 puncta, we also observed loss of retinogeniculate (RG) synaptic function assessed using optogenetic activation of RG axons while performing whole-cell voltage clamp recordings from TC neurons in brain slices. Surprisingly, we did not detect any significant changes in the frequency of miniature post-synaptic currents (mEPSCs) or corticothalamic feedback synapses. Analysis of TC neuron dendritic structure from single-cell dye fills revealed a gradual loss of dendrites proximal to the soma, where TC neurons receive the bulk of RG inputs. Finally, analysis of action potential firing demonstrated that TC neurons have increased excitability following enucleation, firing more action potentials in response to depolarizing current injections. Our findings show that degeneration of the retinal axons/optic nerve and loss of RG synaptic inputs induces structural and functional changes in TC neurons, consistent with neuronal attempts at compensatory plasticity in the dLGN.

Characterization of orexin input to dopamine neurons of the ventral tegmental area projecting to the medial prefrontal cortex and shell of nucleus accumbens

Orexin neurons are involved in homeostatic regulatory processes, including arousal and feeding, and provide a major input from the hypothalamus to the ventral tegmental area (VTA) of the midbrain. VTA neurons are a central hub processing reward and motivation and target the medial prefrontal cortex (mPFC) and the shell part of nucleus accumbens (NAcs). We investigated whether subpopulations of dopamine (DA) neurons in the VTA projecting either to the mPFC or the medial division of shell part of nucleus accumbens (mNAcs) receive differential input from orexin neurons and whether orexin exerts differential electrophysiological effects upon these cells. VTA neurons projecting to the mPFC or the mNAcs were traced retrogradely by Cav2-Cre virus and identified by expression of yellow fluorescent protein (YFP). Immunocytochemical analysis showed that a higher proportion of all orexin-innervated DA neurons projected to the mNAcs (34.5%) than to the mPFC (5.2%). Of all sampled VTA neurons projecting either to the mPFC or mNAcs, the dopaminergic (68.3 vs. 79.6%) and orexin-innervated DA neurons (68.9 vs. 64.4%) represented the major phenotype. Whole-cell current clamp recordings were obtained from fluorescently labeled neurons in slices during baseline periods and bath application of orexin A. Orexin similarly increased the firing rate of VTA dopamine neurons projecting to mNAcs (1.99 ± 0.61 Hz to 2.53 ± 0.72 Hz) and mPFC (0.40 ± 0.22 Hz to 1.45 ± 0.56 Hz). Thus, the hypothalamic orexin system targets mNAcs and to a lesser extent mPFC-projecting dopaminergic neurons of the VTA and exerts facilitatory effects on both clusters of dopamine neurons.

Effect of Biphasic kHz Field Stimulation on CA1 Pyramidal Neurons in Slices

Electrical stimulation in the kilohertz-frequency range has been successfully used for treatment of various neurological disorders. Nevertheless, the mechanisms underlying this stimulation are poorly understood. To study the effect of kilohertz-frequency electric fields on neuronal membrane biophysics we developed a reliable experimental method to measure responses of single neurons to kilohertz field stimulation in brain slice preparations. In the submerged brain slice pyramidal neurons of the CA1 subfield were recorded in the whole-cell configuration before, during and after stimulation with a biphasic charge balanced electric field at 2kHz, 5kHz or 10 kHz. The slice was placed in a 1 mm gap between two parallel 2 mm long platinum-iridium 0.1 mm diameter wire electrodes. Whole-cell recordings lasted for an hour or longer. Typically, a few 5-10 min long sessions of kHz-field stimulation (kHz-FS) with various frequency/amplitude combinations were applied to assess the type of neuronal response and possible changes of its membrane characteristics. It was found that kHz-field stimulation at all frequencies elicited reproducible excitatory neuronal responses lasting throughout stimulation period, but not after cessation of kHz-FS. During kHz-FS the rheobase usually decreased. In addition, spontaneous firing might be initiated in some silent neurons or became more intense in previously spontaneously active neurons. Response thresholds were in the range of 0.5-2 mA and were higher at higher frequencies. Blockade of glutamatergic synaptic transmission did not alter the magnitude of responses. Inhibitory synaptic input was not changed by kilohertz field stimulation. We conclude that kHz-frequency current applied in brain tissue has an excitatory effect on pyramidal neurons during stimulation. This effect is more prominent and occurs at a lower stimulus intensity at a frequency of 2kHz as compared to 5kHz and 10kHz.

GKRP-dependent modulation of feeding behavior by tanycyte-released monocarboxylates.

Objectives: Glucokinase Regulatory Protein (GKRP) is the only known endogenous modulator of glucokinase (GK) localization and activity to date, and both proteins are localized in tanycytes, radial glia-like cells involved in metabolic and endocrine functions in the hypothalamus. However, the role of tanycytic GKRP and its impact on the regulation of feeding behavior has not been investigated. Here, we hypothesize that GKRP regulates feeding behavior by modulating tanycyte-neuron metabolic communication in the arcuate nucleus.Methods: We used primary cultures of tanycytes to evaluate the production of lactate and β-hydroxybutyrate (βHB). Similarly, we examined the electrophysiological responses to these metabolites in pro-opiomelanocortin (POMC) neurons in hypothalamic slices. To evaluate the role of GKRP in feeding behavior, we generated tanycyte-selective GKRP-overexpressing and GKRP-knock down mice (GKRPt-OE and GKRPt-KD respectively) using adenovirus-mediated transduction.Results: We demonstrated that lactate release induced by glucose uptake is favored in GKRP-KD tanycytes. Conversely, tanycytes overexpressing GKRP showed an increase in βHB efflux induced by low glucose concentration. In line with these findings, the excitability of POMC neurons was enhanced by lactate and decreased in the presence of βHB. In GKRPt-OE rats, we found an increase in post-fasting food avidity, whereas GKRPt-KD caused a significant decrease in feeding and body weight, which is reverted when MCT1 is silenced.Conclusion: Our study highlights the role of tanycytic GKRP in metabolic regulation and positions this regulator of GK as a therapeutic target for boosting satiety in patients with obesity problems.

Simultaneous Monitoring of pH and Chloride (Cl-) in Brain Slices of Transgenic Mice.

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl−]i) and hydrogen ([H+] i) the construct, called ClopHensor, which consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H+]i and [Cl−]i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H+]i and [Cl−]i under different experimental conditions including changing of external concentrations of ions (Ca2+, Cl−, K+, Na+) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl− and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl− and pH.

Medial prefrontal cortex pyramidal neurons exhibit functional defects during early stage of Alzheimer’s disease in 3xTg‐AD mice

The brain regions that show early increase of beta-amyloid (Aβ) in Alzheimer's disease (AD) overlap with the default mode network (DMN) (Palmqvist, S. et. al., 2017), which comprises, but is not limited to, the medial prefrontal cortex (mPFC), a key regulator of neurocognition (Zott, B. et al., 2018). DMN is active during mental processes that don't require attention, but it is deactivated when undertaking neurocognitive tasks (Zott, B. et al., 2018). However, such DMN deactivation is impaired in AD, often preceding formation of the Aβ plaque and onset of clinical symptoms (Chhatwal, J. P. et al., 2013; Sheline, Y. I. et al., 2010). Previous studies suggest that neuronal hyperexcitability is responsible for increased DMN vulnerability to Aβ deposition (Bero, A. W. et al., 2011; Li, X. et al., 2013). But whether and how mPFC neuronal function are altered in the early stages of AD is unknown and has not been studied.In this study, we used young 3xTg-AD mice (3-5 months of age) to mimic the early stages of AD before the formation of cortical Aβ plaques. Patch-clamp electrophysiology was applied to assess functional activity of mPFC pyramidal neurons in brain slices. Action potentials were evoked by depolarizing currents and spontaneous excitatory postsynaptic currents (sEPSCs) were assessed in Mg2+ -free bath with holding potential of -70mV. Ca2+ spikes (indicating Ca2+ influx through voltage-gated Ca2+ channels) were evoked by membrane depolarization with blockade of voltage-gated Na+ /K+ channels.We found that mPFC pyramidal neurons in young 3xTg-AD mice showed significantly increased firing, associated with depolarized resting membrane potential, decreased rheobase, increased input resistance, and reduced firing threshold. We also found that the frequency of sEPSCs was significantly increased. Meanwhile, Ca2+ spikes showed a trend of increase in their duration/area (a larger sample size is needed to confirm this finding) in mPFC neurons.Collectively, these novel findings demonstrate that the neuronal excitability and excitatory neurotransmission are aberrantly increased in the young mPFC during the early stages of AD preceding Aβ plaque formation. Whether such mPFC neuronal dysfunction could contribute to Aβ plaque accumulation is currently unknown and requires further investigation.

New monoamine antidepressant, hypidone hydrochloride (YL-0919), enhances the excitability of medial prefrontal cortex in mice via a neural disinhibition mechanism.

Hypidone hydrochloride (YL-0919) is a novel antidepressant in clinical phase II trial. Previous studies show that YL-0919 is a selective 5-HT (serotonin) reuptake inhibitor, 5-HT1A receptor partial agonist, and 5-HT6 receptor agonist, which exerts antidepressant effects in various animal models, but its effects on neural function remain unclear. Medial prefrontal cortex (mPFC), a highly evolved brain region, controls highest order cognitive functions and emotion regulation. In this study we investigated the effects of YL-0919 on the mPFC function, including the changes in neuronal activities using electrophysiological recordings. Extracellular recording (in vivo) showed that chronic administration of YL-0919 significantly increased the spontaneous discharges of mPFC neurons. In mouse mPFC slices, whole-cell recording revealed that perfusion of YL-0919 significantly increased the frequency of sEPSCs, but decreased the frequency of sIPSCs. Then we conducted whole-cell recording in mPFC slices of GAD67-GFP transgenic mice, and demonstrated that YL-0919 significantly inhibited the excitability of GABAergic neurons. In contrast, it did not alter the excitability of pyramidal neurons in mPFC slices of normal mice. Moreover, the inhibition of GABAergic neurons by YL-0919 was prevented by pre-treatment with 5-HT1A receptor antagonist WAY 100635. Finally, chronic administration of YL-0919 significantly increased the phosphorylation levels of mTOR and GSK-3β in the mPFC as compared with vehicle. Taken together, our results demonstrate that YL-0919 enhances the excitability of mPFC via a disinhibition mechanism to fulfill its rapid antidepressant neural mechanism, which was accomplished by 5-HT1A receptor-mediated inhibition of inhibitory GABAergic interneurons.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Moderate ethanol drinking is sufficient to alter Ventral Tegmental Area dopamine neurons activity via functional and structural remodeling of GABAergic transmission

Earlier studies have shown a major involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons in mediating the rewarding effects of ethanol (EtOH). Much less is known on the role of this system in mediating the transition from moderate to excessive drinking and abuse. Here we sought to explore the hypothesis that early stage drinking in rodents, resembling recreational EtOH use in humans, is sufficient to dysregulate VTA DA transmission thus increasing the propensity to use over time. To this purpose, midbrain slice recordings in mice previously exposed to an escalating (3, 6 and 12%) 18-day voluntary EtOH drinking paradigm was used. By recording from DA and γ-aminobutyric acid (GABA) VTA neurons in midbrain slices, we found that moderate EtOH drinking leads to a significant suppression of the spontaneous activity of VTA DA neurons, while increasing their response to acute EtOH application. We also found that chronic EtOH leads to the enhancement of GABA input frequency onto a subset of DA neurons. Structurally, chronic EtOH induced a significant increase in the number of GABA axonal boutons contacting DA neurons, suggesting deep rewiring of the GABA network. This scenario is consistent with a downmodulation of the reward DA system induced by moderate EtOH drinking, a neurochemical state defined as “hypodopaminergic” and previously associated with advanced stages of drug use in humans. In this context, increased sensitivity of DA neurons towards acute EtOH may represent the neurophysiological correlate of increased unitary rewarding value, possibly driving progression to addiction.

Intrinsic brainstem circuits comprised of Chx10-expressing neurons contribute to reticulospinal output in mice.

Glutamatergic reticulospinal neurons in the gigantocellular reticular nucleus (GRN) of the medullary reticular formation can function as command neurons, transmitting motor commands to spinal cord circuits to instruct movement. Recent advances in our understanding of this neuron-dense region have been facilitated by the discovery of expression of the transcriptional regulator, Chx10, in excitatory reticulospinal neurons. Here, we address the capacity of local circuitry in the GRN to contribute to reticulospinal output. We define two subpopulations of Chx10-expressing neurons in this region, based on distinct electrophysiological properties and soma size (small and large), and show that these populations correspond to local interneurons and reticulospinal neurons, respectively. Using focal release of caged glutamate combined with patch clamp recordings, we demonstrated that Chx10 neurons form microcircuits in which the Chx10 local interneurons project to and facilitate the firing of Chx10 reticulospinal neurons. We discuss the implications of these microcircuits in terms of movement selection.NEW & NOTEWORTHY Reticulospinal neurons in the medullary reticular formation integrate inputs from higher regions to effectively instruct spinal motor circuits. Using photoactivation of neurons in brainstem slices, we studied connectivity of reticular formation neurons that express the transcriptional regulator, Chx10. We show that a subpopulation of these neurons functions as local interneurons that affect descending commands. The results shed light on the internal organization and microcircuit formation of reticular formation neurons.

Physiological and Pharmacological Studies on Cervical Motor Neurons in Slices Prepared from Neonatal and Aged Mice

The effects of age on the physiological properties of cervical motor neurons were examined in slices made from an excised spinal cord graft of ICR mice from the second day after birth to age 350 days. The membrane potential of post-natal day 2 (PD2) to PD350 was about -65 mV and did not change greatly with age, although it was slightly higher at PD2. However, there were significant changes in membrane resistance, which increased with age from about 15 to 30 MΩ. The depolarization induced by the excitatory amino acid agonists, kainic acid, NMDA and AMPA, decreased with aging in spite of the increase in membrane resistance. The motor neurons of the aged mice showed delayed recovery from excitation caused by excitatory amino acid agonists. By injecting Lucifer yellow CH into motor neurons, it was observed that the dendrite trees become thin, and some of the dendrite branches were missing in older animals.

Protein Kinase C Controls the Excitability of Cortical Pyramidal Neurons by Regulating Kv2.2 Channel Activity.

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.

Roles of corticotropin-releasing factor signaling in the lateral habenula in anxiety-like and alcohol drinking behaviors in male rats

Corticotropin-releasing factor (CRF) signaling in the mesocorticolimbic system is known to modulate anxiety-like behavior and alcohol consumption, behaviors that also have been associated with the hyper-glutamatergic state of the lateral habenula (LHb) neurons in rats. However, the role of CRF signaling in the LHb on the glutamate transmission, anxiety-like behaviors and alcohol consumption is unknown. Here, we used male rats that had been consuming alcohol for three months to address this gap in the literature. First, using electrophysiological techniques, we evaluated CRF's effects on the glutamate transmission in LHb neurons in brain slices. CRF facilitated glutamate transmission. The facilitation was greater in neurons of alcohol-withdrawing rats than in those of naïve rats. The facilitation was mimicked by the activation of CRF receptor 1 (CRF1R) but attenuated by the activation of CRF receptor 2 (CRF2R). This facilitation was mediated by upregulating CRF1R-protein kinase A signaling. Conversely, protein kinase C blockade attenuated CRF's facilitation in neurons of naïve rats but promoted it in neurons of alcohol-withdrawing rats. Next, using site-direct pharmacology, we evaluated the role of CRF signaling in the LHb on anxiety-like behaviors and alcohol consumption. Intra-LHb inhibition of CRF1R or activation of CRF2R ameliorated the anxiety-like behaviors in alcohol-withdrawing rats and reduced their alcohol intake when drinking was resumed. These observations provide the first direct behavioral pharmacological and cellular evidence that CRF signaling in the LHb modulates glutamate transmission, anxiety-like behaviors and alcohol consumption, and that adaptation occurs in CRF signaling in the LHb after chronic alcohol consumption.

Reciprocal Changes in Voltage-Gated Potassium and Subthreshold Inward Currents Help Maintain Firing Dynamics of AVPV Kisspeptin Neurons during the Estrous Cycle.

Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared with diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K+ current: fast-transient slow-transient, and residual. During proestrus, the V50 of inactivation of the fast-transient current was depolarized and the amplitude of the slow-transient component was reduced compared with diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K+ currents are suppressive compared with diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling postinhibitory rebound bursting. These findings suggest modulation of voltage-gated K+ and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge.

Multimodal nonlinear-optical imaging of nucleoli.

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

Characterization of laryngeal motor neuron properties in the American bullfrog, Lithobates catesbieanus

Motor neurons represent the final output from the central respiratory network. American bullfrogs, Lithobates catesbieanus, have provided insight into development and plasticity of the breathing control system, yet cellular aspects of bullfrog motor neurons are not well-described. In this study, we characterized properties of laryngeal motor neurons that produce motor outflow to the glottal dilator, a muscle that gates airflow to the lungs of anurans. To this end, we measured several intrinsic membrane properties of labeled laryngeal motor neurons in brain slices. Using unsupervised clustering analyses, we identified two broad classes of motor neurons: those with high firing rates and strong adaptation (∼70 %), and those with lower firing rates and less adaptation (∼30 %). These results suggest that two neuronal cell types innervate the glottal dilator, roughly aligning with the composition of fast and slower twitch fibers of this muscle. In sum, these data reinforce the need to consider cell-type when assessing motor neuron function in the respiratory network.

Aquaporin 4 differentially modulates osmotic effects on vasopressin neurons in rat supraoptic nucleus.

Glial fibrillary acidic protein (GFAP) molecularly associates with aquaporin 4 (AQP4) in astrocytic plasticity. Here, we further examined how AQP4 modulates osmotic effects on vasopressin (VP) neurons in rat supraoptic nucleus (SON) through interactions with GFAP in astrocytes. Brain slices from adult male rats were kept under osmotic stimulation. Western blot, co-immunoprecipitation, immunohistochemistry and patch-clamp recordings were used for analysis of expressions and interactions between GFAP and AQP4, astrocyte-specific proteins in the SON, as well as their influence on VP neuronal activity. Data were analysed using SPSS software. Hyposmotic challenge (HOC) of acute SON slices caused an early (within 5 minutes) and transient increase in the colocalization of AQP4 with GFAP filaments. This effect was prominent at astrocytic processes surrounding VP neuron somata and was accompanied by inhibition of VP neuronal activity. Similar HOC effect was seen in the SON isolated from rats subjected to in vivo HOC, wherein a transiently increased molecular association between GFAP and AQP4 was detected using co-immunoprecipitation. The late stage rebound excitation (10minutes) of VP neurons in brain slices subjected to HOC and the associated astrocytic GFAP's 'return to normal' were both hampered by 2-(nicotinamide)-1,3,4-thiadiazole, a specific AQP4 channel blocker that itself did not influence VP neuronal activity. Moreover, this agent prevented hyperosmotic stress-evoked excitation of VP neurons and associated reduction in GFAP filaments. These findings indicate that osmotically driven increase in VP neuronal activity requires the activation of AQP4, which determines a retraction of GFAP filaments.

Scn2a severe hypomorphic mutation decreases excitatory synaptic input and causes autism-associated behaviors.

SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to autism spectrum disorders (ASDs). Most ASD-associated mutations in SCN2A are loss-of-function mutations, but studies examining how such mutations affect neuronal function and whether Scn2a mutant mice display ASD endophenotypes have been inconsistent. We generated a protein truncation variant Scn2a mouse model (Scn2aΔ1898/+) by CRISPR that eliminates the NaV1.2 channel’s distal intracellular C-terminal domain, and we analyzed the molecular and cellular consequences of this variant in a heterologous expression system, in neuronal culture, in brain slices, and in vivo. We also analyzed multiple behaviors in WT and Scn2aΔ1898/+ mice and correlated behaviors with clinical data obtained in human subjects with SCN2A variants. Expression of the NaV1.2 mutant in a heterologous expression system revealed decreased NaV1.2 channel function, and cultured pyramidal neurons isolated from Scn2aΔ1898/+ forebrain showed correspondingly reduced voltage-gated Na+ channel currents without compensation from other CNS voltage-gated Na+ channels. Na+ currents in inhibitory neurons were unaffected. Consistent with loss of voltage-gated Na+ channel currents, Scn2aΔ1898/+ pyramidal neurons displayed reduced excitability in forebrain neuronal culture and reduced excitatory synaptic input onto the pyramidal neurons in brain slices. Scn2aΔ1898/+ mice displayed several behavioral abnormalities, including abnormal social interactions that reflect behavior observed in humans with ASD and with harboring loss-of-function SCN2A variants. This model and its cellular electrophysiological characterizations provide a framework for tracing how a SCN2A loss-of-function variant leads to cellular defects that result in ASD-associated behaviors.

Gonadotropin-Releasing Hormone (GnRH) Neuron Potassium Currents and Excitability in Both Sexes Exhibit Minimal Changes upon Removal of Negative Feedback.

Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol-positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized that these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast and slow inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed among groups in most potassium current properties, input resistance, or capacitance, and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid-negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells.

Genetically and pharmacologically limiting RyR2 open time prevents neuronal hyperactivity of hippocampal CA1 neurons in brain slices of 5xFAD mice

Neuronal hyperactivity is an early, common manifestation of Alzheimer’s disease (AD), and is believed to drive AD progression. Neuronal hyperactivity in the form of baseline activity (or spontaneous Ca2+ transients) has consistently been demonstrated in mouse models of AD using two-photon in vivo Ca2+ imaging of cortical or hippocampal neurons in anesthetized animals. Notably, these AD-related spontaneous Ca2+ transients were hardly detected in acute hippocampal slices, probably due to neuronal damage during brain slicing. To better preserve neuronal activity, we employed the N-methyl-D-glucamine (NMDG) protective brain slicing protocol. We performed confocal in vitro Ca2+ imaging of hippocampal CA1 neurons in optimized hippocampal slices. Consistent with previous in vivo studies, our in vitro studies using optimized brain slices also showed that limiting the open duration of the ryanodine receptor 2 (RyR2) by the RyR2 mutation E4872Q or by the R-carvedilol enantiomer prevented and rescued neuronal hyperactivity of hippocampal CA1 neurons from 5xFAD mice. Thus, genetically and pharmacologically limiting RyR2 open time prevented and rescued AD-related neuronal hyperactivity in vitro in optimized brain slices in the absence of anesthetics’ influence. Our data also suggest that the NMDG protective brain slicing preparation offers an alternative means to study neuronal hyperactivity of various cell types in different brain regions, especially in regions that are not readily accessible to two-photon in vivo Ca2+ imaging.