Acute Brain Slice Preparations Research Articles

Inhibition of Acute mGluR5-Dependent Depression in Hippocampal CA1 by High-Frequency Magnetic Stimulation.

High-frequency magnetic stimulation (HFMS) applied directly to the hippocampal slice preparation in vitro induces activity-dependent synaptic plasticity and metaplasticity. In addition, changes in synaptic transmission following HFMS involve the activation of N-methyl-D-aspartate and metabotropic glutamate receptors (mGluR). Here, we asked whether a short period of HFMS (5 × 10 delta-burst trains, duration of ~1 min) could alter mGluR5-mediated depression at Schaffer collateral-CA1 synapses in the acute brain slice preparation at 30 min after HFMS. To this end, we obtained field excitatory postsynaptic potential (fEPSP) slopes from Schaffer collateral-CA1 synapses after HFMS or control. First, we demonstrated that activity-dependent plasticity following HFMS depends on the slice orientation towards the magnetic coil indicating specific ion fluxes induced by magnetic fields. Second, we found that the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine reduced the field excitatory postsynaptic potential (fEPSP) slopes in control slices but rather enhanced them in HFMS-treated slices. In contrast, the compound (S)-3,5-dihydroxyphenylglycine acting at both mGluR1 and mGluR5 reduced fEPSP slopes in both control and HFMS-treated slices. Importantly, the mGluR-dependent effects were independent from the slice-to-coil orientation indicating that asynchronous glutamate release could play a role. We conclude that a short period of HFMS inhibits subsequently evoked mGluR5-dependent depression at Schaffer collateral-CA1 synapses. This could be relevant for repetitive transcranial magnetic stimulation in psychiatric disorders such as major depression.

LC-derived excitatory synaptic transmission to dorsal raphe serotonin neurons is inhibited by activation of alpha2-adrenergic receptors.

In the central nervous system, noradrenaline transmission controls the degree to which we are awake, alert, and attentive. Aberrant noradrenaline transmission is associated with pathological forms of hyper- and hypo-arousal that present in numerous neuropsychiatric disorders often associated with dysfunction in serotonin transmission. In vivo, noradrenaline regulates the release of serotonin because noradrenergic input drives the serotonin neurons to fire action potentials via activation of excitatory α1-adrenergic receptors (α1-AR). Despite the critical influence of noradrenaline on the activity of dorsal raphe serotonin neurons, the source of noradrenergic afferents has not been resolved and the presynaptic mechanisms that regulate noradrenaline-dependent synaptic transmission have not been described. Using an acute brain slice preparation from male and female mice and electrophysiological recordings from dorsal raphe serotonin neurons, we found that selective optogenetic activation of locus coeruleus terminals in the dorsal raphe was sufficient to produce an α1-AR-mediated excitatory postsynaptic current (α1-AR-EPSC). Activation of inhibitory α2-adrenergic receptors (α2-AR) with UK-14,304 eliminated the α1-AR-EPSC via presynaptic inhibition of noradrenaline release, likely via inhibition of voltage-gated calcium channels. In a subset of serotonin neurons, activation of postsynaptic α2-AR produced an outward current through activation of GIRK potassium conductance. Further, in vivo activation of α2-AR by systemic administration of clonidine reduced the expression of c-fos in the dorsal raphe serotonin neurons, indicating reduced neural activity. Thus, α2-AR are critical regulators of serotonin neuron excitability.

A piezoelectric micromachined ultrasound transducer combined with recording electrodes for acute brain preparations in vitro

BackgroundUltrasound stimulation is used to noninvasively stimulate the local and deep areas of the brain. However, the detailed cellular mechanisms of neural activation are still unclear because studies on micro-stimulation at the cellular level are lacking. New methodTo modulate neural activity at the cellular level, we developed a piezoelectric micromachined ultrasound transducer (PMUT), having circular diaphragms for application on acute brain slice preparations. To monitor neural activities, additionally, we fabricated recording microelectrodes onto the same PMUT device for closed-loop application. ResultsTo examine the PMUT-driven cellular responses of a brain slice, intracellular calcium signals in individual cells were measured using two calcium indicators. We successfully observed the intracellular responses triggered by the ultrasound of our novel PMUT. In addition, we performed recordings of local field potentials in a brain slice, demonstrating its usefulness as a simultaneous recording interface. Comparison with existing method(s)Conventional ultrasound stimulators are open-loop systems that risk inducing excessive neural activity because of the absence of neural activity monitoring. In contrast, our PMUT is packaged in a single device with both stimulation and sensor interface for neuromodulation. Further, there are no published reports on in vitro microdevices that can be used for ultrasound stimulation in rodent cortical slices that are several hundred micrometers thick, which maintain the cortical laminar structure and intrinsic neural networks. ConclusionsOur findings suggest that this novel PMUT device has the potential for being a powerful tool for in vitro brain slice applications and effective closed loop ultrasound stimulation.

SAT593 Mechanism Of Kisspeptin Neuron Synchronization In Acute Brain Slices From Female Mice

Abstract Disclosure: P.G. Morris: None. A.E. Herbison: None. Arcuate kisspeptin (ARNKISS) neurons, which co-express glutamate, neurokinin B and dynorphin, intermittently synchronize their activity to drive pulsatile hormone secretion. However, the mechanisms underlying such network behaviour remains unclear in females. Using an acute Kiss1-Cre GCaMP6 brain slice preparation which maintains spontaneous synchronized ARNKISS neuron burst firing, we were able to both characterise this behaviour and reveal the neurotransmitter receptors contributing to it. Results in diestrous female mice revealed that the order individual neurons took part in synchronizations was only mildly consistent, with a mean Spearman correlation coefficient of 0.43. Spontaneous ARNKISS neuron synchronizations were found to be highly dependent on glutamate transmission via AMPA receptors, as well as Neurokinin B transmission. Inhibition of NMDA receptors did not alter rates of synchronization, and similarly inhibition of kappa opioid receptors had no effect on rate of synchronization or duration of spontaneous events. Combined with previously published data in male mice, these data underscore the importance of AMPA receptors in synchronizing the ARNKISS neuron network, but additionally suggest a sex difference in the relative contribution of neurokinin B to synchronizations. Presentation: Saturday, June 17, 2023

Mechanism of kisspeptin neuron synchronization for pulsatile hormone secretion in male mice

The mechanism by which arcuate nucleus kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to generate pulsatile hormone secretion remains unknown. An acute brain slice preparation maintaining synchronized ARNKISS neuron burst firing was used alongside invivo GCaMP GRIN lens microendoscope and fiber photometry imaging coupled with intra-ARN microinfusion. Studies in intact and gonadectomized male mice revealed that ARNKISS neuron synchronizations result from near-random emergent network activity within the population and that this was critically dependent on local glutamate-AMPA signaling. Whereas neurokinin B operated to potentiate glutamate-generated synchronizations, dynorphin-kappa opioid tone within the network served as a gate for synchronization initiation. These observations force a departure from the existing "KNDy hypothesis" for ARNKISS neuron synchronization. A "glutamate two-transition" mechanism is proposed to underlie synchronizations in this key hypothalamic central pattern generator driving mammalian fertility.

S-ketamine enhances thalamocortical and corticocortical synaptic transmission in acute murine brain slices via increased AMPA-receptor-mediated pathways.

Despite ongoing research efforts and routine clinical use, the neuronal mechanisms underlying the anesthesia-induced loss of consciousness are still under debate. Unlike most anesthetics, ketamine increases thalamic and cortical activity. Ketamine is considered to act via a NMDA-receptor antagonism-mediated reduction of inhibition, i.e., disinhibition. Intact interactions between the thalamus and cortex constitute a prerequisite for the maintenance of consciousness and are thus a promising target for anesthetics to induce loss of consciousness. In this study, we aim to characterize the influence of s-ketamine on the thalamocortical network using acute brain-slice preparation. We performed whole-cell patch-clamp recordings from pyramidal neurons in cortical lamina IV and thalamocortical relay neurons in acute brain slices from CB57BL/6N mice. Excitatory postsynaptic potentials (EPSPs) were obtained via electrical stimulation of the cortex with a bipolar electrode that was positioned to lamina II/III (electrically induced EPSPs, eEPSPs) or via optogenetic activation of thalamocortical relay neurons (optogenetically induced EPSPs, oEPSPs). Intrinsic neuronal properties (like resting membrane potential, membrane threshold for action potential generation, input resistance, and tonic action potential frequency), as well as NMDA-receptor-dependent and independent spontaneous GABAA-receptor-mediated inhibitory postsynaptic currents (sIPSCs) were evaluated. Wilcoxon signed-rank test (level of significance < 0.05) served as a statistical test and Cohen's U3_1 was used to determine the actual effect size. Within 20 min, s-ketamine (5 μM) significantly increased both intracortical eEPSPs as well as thalamocortical oEPSPs. NMDA-receptor-mediated intracortical eEPSPs were significantly reduced. Intrinsic neuronal properties of cortical pyramidal neurons from lamina IV and thalamocortical relay neurons in the ventrobasal thalamic complex were not substantially affected. Neither a significant effect on NMDA-receptor-dependent GABAA sIPSCs (thought to underly a disinhibitory effect) nor a reduction of NMDA-receptor independent GABAA sIPSCs was observed. Both thalamocortical and intracortical AMPA-receptor-mediated EPSPs were significantly increased.In conclusion, our findings show no evidence for a NMDA-receptor antagonism-based disinhibition, but rather suggest an enhanced thalamocortical and intracortical synaptic transmission, which appears to be driven via increased AMPA-receptor-mediated transmission.

Spatiotemporal Control of Noradrenaline-Dependent Synaptic Transmission in Mouse Dorsal Raphe Serotonin Neurons.

Activity of dorsal raphe neurons is controlled by noradrenaline afferents. In this brain region, noradrenaline activates Gαq-coupled α1-adrenergic receptors (α1-AR), causing action potential (AP) firing and serotonin release. In vitro, electrical stimulation elicits vesicular noradrenaline release and subsequent activation of α1-AR to produce an EPSC (α1-AR-EPSC). The duration of the α1-AR-EPSC (∼27 s) is much longer than that of most other synaptic currents, but the factors that govern the spatiotemporal dynamics of α1-AR are poorly understood. Using an acute brain slice preparation from adult male and female mice and electrophysiological recordings from dorsal raphe neurons, we found that the time course of the α1-AR-EPSC was slow, but highly consistent within individual serotonin neurons. The amount of noradrenaline released influenced the amplitude of the α1-AR-EPSC without altering the time constant of decay suggesting that once released, extracellular noradrenaline was cleared efficiently. Reuptake of noradrenaline via noradrenaline transporters was a primary means of terminating the α1-AR-EPSC, with little evidence for extrasynaptic diffusion of noradrenaline unless transporter-dependent reuptake was impaired. Taken together, the results demonstrate that despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is temporally and spatially controlled and noradrenaline transporters are critical regulators of serotonin neuron excitability. Given the functionally distinct types of neurons intermingled in the dorsal raphe nucleus and the unique roles of these neural circuits in physiological responses, transporters may preserve independence of each synapse to encode a long-lasting but discrete signal.SIGNIFICANCE STATEMENT The dorsal raphe nucleus is the predominant source of serotonin in the brain and is controlled by another monoamine, noradrenaline. In this brain region, noradrenaline activates G-protein-coupled α1-adrenergic receptors (α1-AR) causing action potential (AP) firing and serotonin release. Despite high interest in pharmacotherapies to enhance serotonin signaling, the factors that govern noradrenaline α1-AR signaling have received little attention. Here, we show using mouse brain slices that the time course of α1-AR signaling is slow, persisting for tens of seconds. Despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is controlled temporally and spatially by efficient noradrenaline transporter-dependent clearance of extracellular noradrenaline. Thus, noradrenaline transporters are critical regulators of serotonin neuron excitability.

Elevated expression of endogenous glial cell line-derived neurotrophic factor impairs spatial memory performance and raises inhibitory tone in the hippocampus.

Parvalbumin-positive interneurons (PV+) are a key component of inhibitory networks in the brain and are known to modulate memory and learning by shaping network activity. The mechanisms of PV+ neuron generation and maintenance are not fully understood, yet current evidence suggests that signalling via the glial cell line-derived neurotrophic factor (GDNF) receptor GFRα1 positively modulates the migration and differentiation of PV+ interneurons in the cortex. Whether GDNF also regulates PV+ cells in the hippocampus is currently unknown. In this study, we utilized a Gdnf "hypermorph" mouse model where GDNF is overexpressed from the native gene locus, providing greatly increased spatial and temporal specificity of protein expression over established models of ectopic expression. Gdnfwt/hyper mice demonstrated impairments in long-term memory performance in the Morris water maze test and an increase in inhibitory tone in the hippocampus measured electrophysiologically in acute brain slice preparations. Increased PV+ cell number was confirmed immunohistochemically in the hippocampus and in discrete cortical areas and an increase in epileptic seizure threshold was observed in vivo. The data consolidate prior evidence for the actions of GDNF as a regulator of PV+ cell development in the cortex and demonstrate functional effects upon network excitability via modulation of functional GABAergic signalling and under epileptic challenge.

Multiple sources of internal calcium stores mediate ethanol-induced presynaptic inhibitory GABA release in the central nucleus of the amygdala in mice.

Ethanol can enhance GABA release in various brain regions via presynaptic mechanisms. However, the presynaptic action of ethanol on inhibitory GABA release is still not well understood. Since calcium is required for neurotransmitter release from presynaptic terminals, the purpose of this study was to investigate the role of both internal and external calcium signaling in ethanol-induced enhancement of GABA release within the central amygdala nucleus (CeA) in acute brain slice preparations. Whole-cell patch clamp electrophysiology was used to record miniature GABAA receptor-mediated inhibitory postsynaptic currents (mIPSCs) from CeA neurons. Ethanol-enhanced mIPSCs were recorded in the presence of antagonists that regulate internal and external calcium-mediated processes. Bath-applied ethanol dose-dependently increased the mean frequency of mIPSCs without altering mIPSC amplitude. Ethanol-induced increases in mIPSC frequency were antagonized by dantrolene, 2-APB, and the endoplasmic reticulum calcium pump (SERCA) antagonists thapsigargin and cyclopiazonic acid (CPA). Blocking calcium release from mitochondria or via exocytosis with ruthenium red also attenuated mIPSCs while frequency was not altered in the presence of a non-selective calcium channel blocker cadmium. The L-type calcium blocker nifedipine, but not its analogue nimodipine, blocked ethanol-induced enhancement in CeA neurons. These results demonstrate ethanol-induced presynaptic release of GABA is mediated by internal calcium stores and by disrupting neurotransmitter exocytosis within the CeA, a critical brain area involved in drugs of abuse and alcohol addiction.

Biased signaling by endogenous opioid peptides

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (μ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([35S]GTPγS binding and β-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of β-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction.

Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions.

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions.

Assessing Neuron-Astrocyte Spatial Interactions Using the Neuron-Astrocyte Proximity Assay.

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.

A novel method using ambient glutamate for the electrophysiological quantification of extrasynaptic NMDA receptor function in acute brain slices.

We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83nM resulting in a block of more than 82% of ES-NMDARs during a 5min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.

Intravenous lipid emulsion modifies synaptic transmission in hippocampal CA1 pyramidal neurons after bupivacaine-induced central nervous system toxicity.

Local anesthetics can cause severe toxicity when absorbed systemically. Rapid intravenous administration of lipid emulsion (LE) is the standard of care for severe local anesthetic systemic toxicity which can cause cardiovascular and central nervous system (CNS) injury. The biological mechanism by which LE alleviates CNS toxicity remains unknown and understudied. Previous research has suggested that local anesthetics cause an imbalance of excitatory and inhibitory transmission in the brain. Therefore, this study aimed to observe the effect of LE on glutamate‐ and GABA‐induced currents in CA1 pyramidal neurons after bupivacaine‐induced CNS toxicity. We further characterized post‐synaptic modifications in these cells to try to elucidate the mechanism by which LE mediates bupivacaine‐induced CNS toxicity. Sprague–Dawley rats received intravenous bupivacaine (1 mg kg−1 min−1) in either normal saline or LE (or LE without bupivacaine) for 5 min. An acute brain slice preparation and a combination of whole‐cell patch clamp techniques and whole‐cell recordings were used to characterize action potential properties, miniature excitatory, and inhibitory post‐synaptic currents, and post‐synaptic modifications of excitatory and inhibitory transmission in CA1 hippocampal pyramidal neurons. The expression level of GABAA receptors were assessed with western blotting, whereas H&E and TUNEL staining were used to assess cytoarchitecture and apoptosis levels respectively. Bupivacaine treatment significantly increased the number of observed action potentials, whereas significantly decreasing rheobase, the first interspike interval (ISI), and hyperpolarization‐activated cation currents (Ih) in CA1 pyramidal neurons. LE treatment significantly reduced the frequency of miniature inhibitory post‐synaptic currents and enhanced GABA‐induced paired pulse ratio with 50 ms interval stimulation in bupivacaine‐treated rats. Regulation of GABAA levels is a promising mechanism by which LE may ameliorate CNS toxicity after systemic absorption of bupivacaine.

PDTM-21. MULTI-MODAL PROFILING OF PEDIATRIC HIGH-GRADE GLIOMA SINGLE CELLS USING PATCH-SEQ

Abstract Pediatric and adult high-grade gliomas are characterized by extensive intra-tumoral transcriptional heterogeneity. When measured by single cell RNA sequencing, gliomas reveal themselves as continuums of stemness and differentiation programs with important implications for therapy, but to date this transcriptional information has not been directly linked to physiological behaviors of cells. Recent work from our group establishes the electrical integration of glioma cells into neural circuitry. One subpopulation of glioma cells participates in glutamatergic synaptic communication with neurons, and a distinct subpopulation of cells sense and respond to extracellular potassium flux of neuronal networks by an entirely distinct mechanism. Our data support a model in which both modes of electrical communication are critical to glioma growth, but current associations between the electrophysiological properties of a cell, its transcriptional profile and developmental state are correlational in nature. Patch-seq is needed to clarify the relationship between transcriptional profiles of quiescent/cycling stem-like cells and the observed electrophysiological behaviors. Put more simply, patch-seq will clarify where the synaptically-connected glioma cells exist along a developmental hierarchy. METHODS Here, we adapt a recently described technique called patch-seq to record the electrophysiological profiles of individual pediatric high-grade glioma cells by whole cell patch-clamp and subsequently isolate their mRNA for single cell sequencing by smart-seq2 and analysis using Seurat. In this way, we couple electrophysiological and transcriptomic profiles to unambiguously assign functional identities to cells with transcriptional profiles along a developmental hierarchy. RESULTS We report the successful adaptation of patch-seq for use with patient-derived diffuse intrinsic pontine glioma (DIPG) xenografts in acute brain slice preparations, enabling evaluation of single glioma cells integrated in intact neural circuitry. CONCLUSIONS Data synthesizing the electrophysiological and transcriptomic profiles of single glioma cells in the context of the developmental hierarchy will be presented.

Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

Electroencephalogram monitoring during propofol (PRO) anesthesia typically features low-frequency oscillations, which may be involved with thalamic reticular nucleus (TRN) modulation. TRN receives noradrenergic inputs from the locus coeruleus (LC). We hypothesized that specific noradrenergic connections in the TRN may contribute to the emergence from PRO anesthesia. Intranuclei norepinephrine (NE) injections (n = 10) and designer receptors exclusively activated by designer drugs (DREADDs) (n = 10) were used to investigate the role of noradrenergic inputs from the LC to the TRN during PRO anesthesia. Whole-cell recording in acute brain slice preparations was used to identify the type of adrenoceptor that regulates noradrenergic innervation in the TRN. An intracerebral injection of NE into the TRN delays arousal in mice recovering from PRO anesthesia (means ± sd; 486.6 ± 57.32 s for the NE injection group vs. 422.4 ± 48.19 s for the control group; P = 0.0143) and increases the cortical-δ (0.1-4 Hz, 25.4 ± 2.9 for the NE injection group vs. 21.0 ± 1.7 for the control group; P = 0.0094) oscillation. An intra-TRN injection of NE also decreased the EC50 of PRO-induced unconsciousness (57.05 ± 1.78 mg/kg for the NE injection group vs. 72.44 ± 3.23 mg/kg for the control group; P = 0.0096). Moreover, the activation of LC-noradrenergic nerve terminals in the TRN using DREADDs increased the recovery time [466.1 ± 44.57 s for the clozapine N-oxide (CNO) injection group vs. 426.1 ± 38.75 s for the control group; P = 0.0033], decreased the EC50 of PRO-induced unconsciousness (64.77 ± 3.40 mg/kg for the CNO injection group vs. 74.00 ± 2.08 mg/kg for the control group; P = 0.0081), and increased the cortical-δ oscillation during PRO anesthesia (23.29 ± 2.58 for the CNO injection group vs. 19.56 ± 1.9 for the control group; P = 0.0213). In addition, whole-cell recording revealed that NE augmented the inhibitory postsynaptic currents in the TRN neurons via the α1-adrenoceptor. Our data indicated that enhanced NE signaling at the noradrenergic terminals of the LC-TRN projection delays arousal from general anesthesia, which is likely mediated by the α1-adrenoceptor activation. Our findings open a door for further understanding of the functions of various LC targets in both anesthesia and arousal.-Zhang, Y., Fu, B., Liu, C., Yu, S., Luo, T., Zhang, L., Zhou, W., Yu, T. Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

Investigating KYNA production and kynurenergic manipulation on acute mouse brain slice preparations

Manipulation of kynurenic acid (KYNA) level through kynurenine aminotransferase-2 (KAT-2) inhibition with the aim of therapy in neuro-psychiatric diseses has been the subject of extensive recent research. Although mouse models are of particular importance, neither the basic mechanism of KYNA production and release nor the relevance of KAT-2 in the mouse brain has yet been clarified.Using acute mouse brain slice preparations, we investigated the basal and L-kynurenine (L-KYN) induced KYNA production and distribution between the extracellular and intracellular compartments. Furthermore, we evaluated the effect of specific KAT-2 inhibition with the irreversible inhibitor PF-04859989. To ascertain that the observed KYNA release is not a simple consequence of general cell degradation, we examined the structural and functional integrity of the brain tissue with biochemical, histological and electrophysiological tools.We did not find relevant change in the viability of the brain tissue after several hours incubation time. HPLC measurements proved that mouse brain slices intensively produce and liberate KYNA to the extracellular compartment, while only a small proportion retained in the tissue both in the basal and L-KYN supplemented state. Finally, specific KAT-2 inhibition significantly reduced the extracellular KYNA content.Taken together, these results provide important data about KYNA production and release, and in vitro evidence for the first time of the function of KAT-2 in the adult mouse brain. Our study extends investigations of KAT-2 manipulation to mice in a bid to fully understand the function; the final, future aim is to assign therapeutical kynurenergic manipulation strategies to humans.

Serotonin regulates dynamics of cerebellar granule cell activity by modulating tonic inhibition.

Understanding how afferent information is integrated by cortical structures requires identifying the factors shaping excitation and inhibition within their input layers. The input layer of the cerebellar cortex integrates diverse sensorimotor information to enable learned associations that refine the dynamics of movement. Specifically, mossy fiber afferents relay sensorimotor input into the cerebellum to excite granule cells, whose activity is regulated by inhibitory Golgi cells. To test how this integration can be modulated, we have used an acute brain slice preparation from young adult rats and found that encoding of mossy fiber input in the cerebellar granule cell layer can be regulated by serotonin (5-hydroxytryptamine, 5-HT) via a specific action on Golgi cells. We find that 5-HT depolarizes Golgi cells, likely by activating 5-HT2A receptors, but does not directly act on either granule cells or mossy fibers. As a result of Golgi cell depolarization, 5-HT significantly increases tonic inhibition onto both granule cells and Golgi cells. 5-HT-mediated Golgi cell depolarization is not sufficient, however, to alter the probability or timing of mossy fiber-evoked feed-forward inhibition onto granule cells. Together, increased granule cell tonic inhibition paired with normal feed-forward inhibition acts to reduce granule cell spike probability without altering spike timing. Hence, these data provide a circuit mechanism by which 5-HT can reduce granule cell activity without altering temporal representations of mossy fiber input. Such changes in network integration could enable flexible, state-specific suppression of cerebellar sensorimotor input that should not be learned or enable reversal learning for unwanted associations. NEW & NOTEWORTHY Serotonin (5-hydroxytryptamine, 5-HT) regulates synaptic integration at the input stage of cerebellar processing by increasing tonic inhibition of granule cells. This circuit mechanism reduces the probability of granule cell spiking without altering spike timing, thus suppressing cerebellar input without altering its temporal representation in the granule cell layer.

Docking and pharmacodynamic studies on hGAT1 inhibition activity in the presence of selected neuronal and astrocytic inhibitors. Part I

Inhibition of 4-aminobutanoic acid (GABA) uptake is a strategy for enhancing GABA transmission. The utility of this approach is demonstrated by the successful development of such agents for the treatment of epilepsy and pain. Existing reports on acute brain slice preparations indicate the intersecting of complementary channels and receptors sets between astrocytes and neurons cells. Thorough analysis of astroglial cells by means of molecular and functional studies demonstrated their active modulatory role in intercellular communication. The chemical interactions between sixteen GABA analogues and isoform of hGAT1 is outlined in the light of molecular docking results. In the in vivo part antinociceptive properties of racemic nipecotic acid, its R and S enantiomers and isonipecotic acid, each administered intraperitoneally at 3 fixed doses (10, 30 and 100 mg/kg), were assessed in a thermally-induced acute pain model i.e. the mouse hot plate test. Docking analyses provided complex binding energies, specific h-bond components, and h-bond properties, such as energies, distances and angles. In vivo tests revealed statistically significant antinociceptive properties of isonipecotic acid (10 and 30 mg/kg), R-nipecotic acid (30 and 100 mg/kg) and S-nipecotic acid (100 mg/kg) in mice. The docking data endorse the hypothesis of correlation between the strength of their chemical interactions with hGAT1 and analgesic action of studied compounds.

Long-Term Plasticity at the Mitral and Tufted Cell to Granule Cell Synapse of the Olfactory Bulb Investigated with a Custom Multielectrode in Acute Brain Slice Preparations.

Single extracellular stimulation electrodes are a widespread means to locally activate synaptic inputs in acute brain slices. Here we describe the fabrication and application of a multielectrode stimulator that was developed for conditions under which independent stimulation of several nearby sites is desirable. For the construction of the multielectrode we have developed a method by which electrode wires can be spaced at minimal distances of 100μm. This configuration increases the efficiency of stimulation paradigms, such as the comparison of proximal induced and control inputs for studies of synaptic plasticity.In our case the multielectrode was used for acute olfactory bulb slices to independently excite individual nearby glomeruli; the technique allowed us to demonstrate homosynaptic bidirectional long-term plasticity at the mitral/tufted cell to granule cell synapse. We also describe the determinants for successful recordings of long-term plasticity at this synapse, with mechanical and electrophysiological recording stability being tantamount. Finally, we briefly discuss data analysis procedures.