Biphasic Npas4 expression promotes inhibitory plasticity and suppression of fear memory consolidation in mice.

Long-term memories are believed to be encoded by unique transcriptional signatures in the brain. The expression of immediate early genes (IEG) promotes structural and molecular changes required for memory consolidation. Recent evidence has shown that the brain is equipped with mechanisms that not only promote, but actively constrict memory formation. However, it remains unknown whether IEG expression may play a role in memory suppression. Here we uncovered a novel function of the IEG neuronal PAS domain protein 4 (Npas4), as an inducible memory suppressor gene of highly salient aversive experiences. Using a contextual fear conditioning paradigm, we found that low stimulus salience leads to monophasic Npas4 expression, while highly salient learning induces a biphasic expression of Npas4 in the hippocampus. The later phase requires N-methyl-D-aspartate (NMDA) receptor activity and is independent of dopaminergic neurotransmission. Our in vivo pharmacological and genetic manipulation experiments suggested that the later phase of Npas4 expression restricts the consolidation of a fear memory and promote behavioral flexibility, by facilitating fear extinction and the contextual specificity of fear responses. Moreover, immunofluorescence and electrophysiological analysis revealed a concomitant increase in synaptic input from cholecystokinin (CCK)-expressing interneurons. Our results demonstrate how salient experiences evoke unique temporal patterns of IEG expression that fine-tune memory consolidation. Moreover, our study provides evidence for inducible gene expression associated with memory suppression as a possible mechanism to balance the consolidation of highly salient memories, and thereby to evade the formation of maladaptive behavior.

Hydrogen peroxide is not generated intracellularly in human neural spheroids during ischemia-reperfusion.

Reactive oxygen species (ROS) are considered a primary source of damage during ischemic stroke. However, the precise timing of ROS production (during hypoxia or reperfusion) remains unclear. Cellular 3D spheroids are often proposed as an optimal alternative to both 2D cell cultures and animal models in modeling disease conditions. Here we report live imaging of hydrogen peroxide dynamics during the acute phase of hypoxia and reperfusion in human iPSC-derived neural spheroids, stably expressing fluorescent biosensor HyPer7. Contrary to previous reports, we did not observe a hydrogen peroxide production burst neither during hypoxia nor in course of reperfusion. Our data suggest either lack of oxidative stress during ischemia-reperfusion in spheroids or existence of different mechanisms of oxidative damage.

Acute stress modulates hippocampal to entorhinal cortex communication.

Feed-forward inhibition is vital in the transfer and processing of synaptic information within the hippocampal-entorhinal loop by controlling the strength and direction of excitation flow between different neuronal populations and individual neurons. While the cellular targets in the hippocampus that receive excitatory inputs from the entorhinal cortex have been well studied, and the role of feedforward inhibitory neurons has been attributed to neurogliafom cells, the cortical interneurons providing feed-forward control over receiving layer V in the entorhinal cortex remain unknown. We used sharp-wave ripple oscillations as a natural excitatory stimulus of the entorhinal cortex, driven by the hippocampus, to study the function of synaptic interactions between neurons in the deep layers of the entorhinal cortex. We discovered that CB1R-expressing interneurons in the deep layers of the entorhinal cortex constitute the major relay station that translates hippocampal excitation into efficient inhibition of cortical pyramidal cells. The impact of inhibition provided by these interneurons is under strong endocannabinoid control and can be drastically reduced either by enhanced activity of postsynaptic targets or by stress-induced elevation of cannabinoids.

Distinct effects of AMPAR subunit depletion on spatial memory.

Pharmacological studies established a role for AMPARs in the mammalian forebrain in spatial memory performance. Here we generated global GluA1/3 double knockout mice (Gria1/3-/-) and conditional knockouts lacking GluA1 and GluA3 AMPAR subunits specifically from principal cells across the forebrain (Gria1/3ΔFb). In both models, loss of GluA1 and GluA3 resulted in reduced hippocampal GluA2 and increased levels of the NMDAR subunit GluN2A. Electrically-evoked AMPAR-mediated EPSPs were greatly diminished, and there was an absence of tetanus-induced LTP. Gria1/3-/- mice showed premature mortality. Gria1/3ΔFb mice were viable, and their memory performance could be analyzed. In the Morris water maze (MWM), Gria1/3ΔFb mice showed profound long-term memory deficits, in marked contrast to the normal MWM learning previously seen in single Gria1-/- and Gria3-/- knockout mice. Our results suggest a redundancy of function within the pool of available ionotropic glutamate receptors for long-term spatial memory performance.

Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity.

Oxidative stress, a state of disrupted redox signaling, reactive oxygen species (ROS) overproduction, and oxidative cell damage, accompanies numerous brain pathologies, including aging-related dementia and Alzheimer's disease, the most common neurodegenerative disorder of the elderly population. However, a causative role of neuronal oxidative stress in the development of aging-related cognitive decline and neurodegeneration remains elusive because of the lack of approaches for modeling isolated oxidative injury in the brain. Here, we present a chemogenetic approach based on the yeast flavoprotein d-amino acid oxidase (DAAO) for the generation of intraneuronal hydrogen peroxide (H2O2). To validate this chemogenetic tool, DAAO and HyPer7, an ultrasensitive genetically encoded H2O2 biosensor, were targeted to neurons. Changes in the fluorescence of HyPer7 upon treatment of neurons expressing DAAO with d-norvaline (D-Nva), a DAAO substrate, confirmed chemogenetically induced production of intraneuornal H2O2. Then, using the verified chemogenetic tool, we emulated isolated intraneuronal oxidative stress in acute brain slices and, using electrophysiological recordings, revealed that it does not alter basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals but reduces long-term potentiation (LTP). Moreover, treating neurons expressing DAAO with D-Nva via the patch pipette also decreases LTP. This observation indicates that isolated oxidative stress affects synaptic plasticity at single cell level. Our results broaden the toolset for studying normal redox regulation in the brain and elucidating the role of oxidative stress to the pathogenesis of cognitive aging and the early stages of aging-related neurodegenerative diseases. The proposed approach is useful for identification of early markers of neuronal oxidative stress and may be used in screens of potential antioxidants effective against neuronal oxidative injury.

Dendritic axon origin enables information gating by perisomatic inhibition in pyramidal neurons.

Information processing in neuronal networks involves the recruitment of selected neurons into coordinated spatiotemporal activity patterns. This sparse activation results from widespread synaptic inhibition in conjunction with neuron-specific synaptic excitation. We report the selective recruitment of hippocampal pyramidal cells into patterned network activity. During ripple oscillations in awake mice, spiking is much more likely in cells in which the axon originates from a basal dendrite rather than from the soma. High-resolution recordings in vitro and computer modeling indicate that these spikes are elicited by synaptic input to the axon-carrying dendrite and thus escape perisomatic inhibition. Pyramidal cells with somatic axon origin can be activated during ripple oscillations by blocking their somatic inhibition. The recruitment of neurons into active ensembles is thus determined by axonal morphological features.

On the accuracy of cell-attached current-clamp recordings from cortical neurons.

Cell-attached current-clamp (CA/CC) recordings have been proposed to measure resting membrane potential and synaptic/agonist responses in neurons without disrupting the cell membrane, thus avoiding the intracellular dialysis that occurs in conventional whole-cell recordings (WC). However, the accuracy of CA/CC recordings in neurons has not been directly assessed. Here, we used concomitant CA and WC current clamp recordings from cortical neurons in brain slices. Resting membrane potential values and slow voltage shifts showed variability and were typically attenuated during CA/CC recordings by ~10-20% relative to WC values. Fast signals were slowed down and their amplitude was greatly reduced: synaptic potentials by nearly 2-fold, and action potentials by nearly 10-fold in CA/CC mode compared to WC. The polarity of GABAergic postsynaptic responses in CA/CC mode matched the responses in WC, and depolarising GABAergic potentials were predominantly observed during CA/CC recordings of intact neonatal CA3 hippocampal pyramidal neurons. Similarly, CA/CC recordings reliably detected neuronal depolarization and excitation during network-induced giant depolarizing potentials in the neonatal CA3 hippocampus, and revealed variable changes, from depolarization to hyperpolarization, in CA1 pyramidal cells during sharp wave ripples in the adult hippocampus. Thus, CA/CC recordings are suitable for assessing membrane potential but signal distortion, probably caused by leakage via the seal contact and RC filtering should be considered.

Transient Oxygen-Glucose Deprivation Causes Region- and Cell Type-Dependent Functional Deficits in the Mouse Hippocampus In Vitro.

Neurons are highly vulnerable to conditions of hypoxia-ischemia (HI) such as stroke or transient ischemic attacks. Recovery of cognitive and behavioral functions requires re-emergence of coordinated network activity, which, in turn, relies on the well-orchestrated interaction of pyramidal cells (PYRs) and interneurons. We therefore modelled HI in the mouse hippocampus, a particularly vulnerable region showing marked loss of PYR and fast-spiking interneurons (FSIs) after hypoxic-ischemic insults. Transient oxygen-glucose deprivation (OGD) in ex vivo hippocampal slices led to a rapid loss of neuronal activity and spontaneous network oscillations (sharp wave-ripple complexes; SPW-Rs), and to the occurrence of a spreading depolarization. Following reperfusion, both SPW-R and neuronal spiking resumed, but FSI activity remained strongly reduced compared with PYR. Whole-cell recordings in CA1 PYR revealed, however, a similar reduction of both EPSCs and IPSCs, leaving inhibition-excitation (I/E) balance unaltered. At the network level, SPW-R incidence was strongly reduced and the remaining network events showed region-specific changes including reduced ripple energy in CA3 and increased ripple frequency in CA1. Together, our data show that transient hippocampal energy depletion results in severe functional alterations at the cellular and network level. While I/E balance is maintained, synaptic activity, interneuron spiking and coordinated network patterns remain reduced. Such alterations may be network-level correlates of cognitive and functional deficits after cerebral HI.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Processing of Hippocampal Network Activity in the Receiver Network of the Medial Entorhinal Cortex Layer V.

The interplay between hippocampus and medial entorhinal cortex (mEC) is of key importance for forming spatial representations. Within the hippocampal-entorhinal loop, the hippocampus receives context-specific signals from layers II/III of the mEC and feeds memory-associated activity back into layer V (LV). The processing of this output signal within the mEC, however, is largely unknown. We characterized the activation of the receiving mEC network by evoked and naturally occurring output patterns in mouse hippocampal-entorhinal cortex slices. Both types of glutamatergic neurons (mEC LVa and LVb) as well as fast-spiking inhibitory interneurons receive direct excitatory input from the intermediate/ventral hippocampus. Connections between the two types of excitatory neurons are sparse, and local processing of hippocampal output signals within mEC LV is asymmetric, favoring excitation of far projecting LVa neurons over locally projecting LVb neurons. These findings suggest a new role for mEC LV as a bifurcation gate for feedforward (telencephalic) and feedback (entorhinal-hippocampal) signal propagation.SIGNIFICANCE STATEMENT Patterned network activity in hippocampal networks plays a key role in the formation and consolidation of spatial memories. It is, however, largely unclear how information is transferred to the neocortex for long-term engrams. Here, we elucidate the propagation of network activity from the hippocampus to the medial entorhinal cortex. We show that patterned output from the hippocampus reaches both major cell types of deep entorhinal layers. These cells are, however, only weakly connected, giving rise to two parallel streams of activity for local and remote signal propagation, respectively. The relative weight of both pathways is regulated by local inhibitory interneurons. Our data reveal important insights into the hippocampal-neocortical dialogue, which is of key importance for memory consolidation in the mammalian brain.

Lactate Attenuates Synaptic Transmission and Affects Brain Rhythms Featuring High Energy Expenditure.

Lactate shuttled from blood, astrocytes, and/or oligodendrocytes may serve as the major glucose alternative in brain energy metabolism. However, its effectiveness in fueling neuronal information processing underlying complex cortex functions like perception and memory is unclear. We show that sole lactate disturbs electrical gamma and theta-gamma oscillations in hippocampal networks by either attenuation or neural bursts. Bursting is suppressed by elevating the glucose fraction in substrate supply. By contrast, lactate does not affect electrical sharp wave-ripple activity featuring lower energy use. Lactate increases the oxygen consumption during the network states, reflecting enhanced oxidative ATP synthesis in mitochondria. Finally, lactate attenuates synaptic transmission in excitatory pyramidal cells and fast-spiking, inhibitory interneurons by reduced neurotransmitter release from presynaptic terminals, whereas action potential generation in the axon is regular. In conclusion, sole lactate is less effective and potentially harmful during gamma-band rhythms by omitting obligatory ATP delivery through fast glycolysis at the synapse.

Overexpression of Calretinin Enhances Short-Term Synaptic Depression.

Analysis of the effects of various proteins on short-term synaptic plasticity is a difficult task, which may require the use of knockout animals. Here, we propose an alternative experimental approach for studying the roles of desired proteins in synaptic plasticity. We packed the Ca2+-binding protein calretinin and the fluorescent protein Venus into AAV and injected the concentrated viral suspension into the neocortex of newborn rats. The infected layer 2/3 pyramidal cells were identified in rat cortical slices using Venus fluorescence. Analysis of short-term synaptic plasticity using paired patch clamp recordings between layer 2/3 pyramidal cells (presynaptic cell) and fast-spiking (FS) interneurons (post-synaptic cell) showed that calretinin expression in the pyramidal cells did not change the failure rate in this synapse but did decrease synaptic delay. Analysis of the parameters of short-term synaptic plasticity showed that the amplitude of the first EPSP in the train was not affected by calretinin, however, calretinin strongly enhanced short-term depression. In addition, we found that the effect of calretinin depended on the presynaptic firing frequency: an increase in frequency resulted in enhancement of synaptic depression.

The Ever-Growing Puzzle of Asynchronous Release.

Invasion of an action potential (AP) to presynaptic terminals triggers calcium dependent vesicle fusion in a relatively short time window, about a millisecond, after the onset of the AP. This allows fast and precise information transfer from neuron to neuron by means of synaptic transmission and phasic mediator release. However, at some synapses a single AP or a short burst of APs can generate delayed or asynchronous synaptic release lasting for tens or hundreds of milliseconds. Understanding the mechanisms underlying asynchronous release (AR) is important, since AR can better recruit extrasynaptic metabotropic receptors and maintain a high level of neurotransmitter in the extracellular space for a substantially longer period of time after presynaptic activity. Over the last decade substantial work has been done to identify the presynaptic calcium sensor that may be involved in AR. Several models have been suggested which may explain the long lasting presynaptic calcium elevation a prerequisite for prolonged delayed release. However, the presynaptic mechanisms underlying asynchronous vesicle release are still not well understood. In this review article, we provide an overview of the current state of knowledge on the molecular components involved in delayed vesicle fusion and in the maintenance of sufficient calcium concentration to trigger AR. In addition, we discuss possible alternative models that may explain intraterminal calcium dynamics underlying AR.

The Role of Polyamine-Dependent Facilitation of Calcium Permeable AMPARs in Short-Term Synaptic Enhancement.

Depending on subunit composition AMPA receptor channels can be subdivided into two groups: GluA2-containing calcium impermeable AMPARs, and GluA2-lacking calcium permeable, AMPARs. These two groups differ in a number of biophysical properties and, most likely, in their functional role at glutamatergic synapses. GluA2-lacking channels have received a lot of attention over the last two decades mainly due to high calcium permeability, which was suggested to play a significant role in the induction of long-term synaptic plasticity in healthy tissue and neuronal death under neuropathological conditions. However, calcium permeable AMPARs possess another property that can contribute substantially to frequency dependent dynamics of synaptic efficacy. In the closed state calcium permeable AMPARs are blocked by endogenous polyamines, however, repetitive activation leads to progressive relief from the block and to the facilitation of ion flux through these channels. Polyamine-dependent facilitation of AMPARs can contribute to short-term plasticity at synapses that have high initial release probability and express calcium permeable AMPARs. During synaptic transmission activity-dependent relief from polyamine block of postsynaptic calcium-permeable AMPARs either counteracts presynaptic short-term depression in a frequency-dependent manner or, under specific stimulation conditions, induces facilitation of a synaptic response. Taking into account the fact that expression of calcium permeable AMPARs is developmentally regulated, depends on network activity and increases in diseased brain states, polyamine-dependent facilitation of calcium permeable AMPARs is an important, entirely postsynaptic mechanism of synaptic gain regulation.

Functional Analysis of Recombinant Channels in Host Cells Using a Fast Agonist Application System.

A reduced recombinant system provides a unique opportunity to study the biophysical properties of NMDAR channels with known subunit compositions, by using a point mutation approach to analyze the structural determinants of receptor function (Wollmuth and Sobolevsky, Trends Neurosci 27:321-328, 2004). However, in addition to the well-developed repertoire of molecular biological techniques, these types of studies also require electrophysiological methods that allow a wide range of receptor activation protocols that can adequately assess desensitization, inactivation, ion permeability, and other properties of the channels. Currently, one of the most well-developed techniques suitable for addressing these issues is use of the fast agonist application system for rapid activation of ligand gated ion-channels (Colquhoun et al., J Physiol 458:261-287, 1992; Jonas and Sakmann, J Physiol 455:143-171, 1992).

Fast interaction between AMPA and NMDA receptors by intracellular calcium.

Suppression of NMDA receptor (NMDAR)-mediated currents by intracellular Ca2+ has been described as a negative feedback loop in NMDAR modulation. In the time scale of tenths of milliseconds the depth of the suppression does not depend on the Ca2+ source. It may be caused by Ca2+ influx through voltage-gated calcium channels, NMDAR channels or release from intracellular stores. However, NMDARs are often co-expressed in synapses with Ca2+-permeable AMPA receptors (AMPARs). Due to significant differences in activation kinetics between these two types of glutamate receptors (GluRs), Ca2+ entry through AMPARs precedes full activation of NMDARs, and therefore, might have an impact on the amplitude of NMDAR-mediated currents. The study of Ca2+-mediated crosstalk between AMPAR and NMDAR in native synapses is challenging due to high NMDAR Ca2+ permeability. Therefore, recombinant Ca2+-permeable AMPAR and Ca2+-impermeable NMDAR mutant channels were co-expressed in HEK 293 cells to examine their interaction. An AMPAR-mediated increase in intracellular Ca2+ concentration ([Ca2+]i) reversibly reduced the size of NMDAR-mediated whole-cell currents. The time course of the NMDAR channel inactivation and recovery from inactivation followed the time course of the [Ca2+]i transient. When brief (1ms) pulses of glutamate were applied to outside-out patches, the degree of NMDAR inactivation increased with the increase in charge carried by the currents through co-activated AMPARs. However, AMPAR-mediated NMDAR inactivation was abolished in the presence of intracellular fast Ca2+ buffer BAPTA or in Ca2+-free extracellular solution. We conclude that Ca2+ entering through AMPARs inactivates co-localized NMDARs in the time range of excitatory postsynaptic currents.

Causal Interrogation of Neuronal Networks and Behavior through Virally Transduced Ivermectin Receptors.

The causal interrogation of neuronal networks involved in specific behaviors requires the spatially and temporally controlled modulation of neuronal activity. For long-term manipulation of neuronal activity, chemogenetic tools provide a reasonable alternative to short-term optogenetic approaches. Here we show that virus mediated gene transfer of the ivermectin (IVM) activated glycine receptor mutant GlyRα1 (AG) can be used for the selective and reversible silencing of specific neuronal networks in mice. In the striatum, dorsal hippocampus, and olfactory bulb, GlyRα1 (AG) promoted IVM dependent effects in representative behavioral assays. Moreover, GlyRα1 (AG) mediated silencing had a strong and reversible impact on neuronal ensemble activity and c-Fos activation in the olfactory bulb. Together our results demonstrate that long-term, reversible and re-inducible neuronal silencing via GlyRα1 (AG) is a promising tool for the interrogation of network mechanisms underlying the control of behavior and memory formation.

GABABR-Dependent Long-Term Depression at Hippocampal Synapses between CB1-Positive Interneurons and CA1 Pyramidal Cells.

Activity induced long lasting modifications of synaptic efficacy have been extensively studied in excitatory synapses, however, long term plasticity is also a property of inhibitory synapses. Inhibitory neurons in the hippocampal CA1 region can be subdivided according to the compartment they target on the pyramidal cell. Some interneurons preferentially innervate the perisomatic area and axon hillock of the pyramidal cells while others preferentially target dendritic branches and spines. Another characteristic feature allowing functional classification of interneurons is cell type specific expression of different neurochemical markers and receptors. In the hippocampal CA1 region, nearly 90% of the interneurons expressing cannabinoid type 1 receptors (CB1R) also express cholecystokinin (CCK). Therefore, the functional presence of CB1 receptors can be used for identification of the inhibitory input from CCK positive (CCK+) interneurons to CA1 pyramidal cells. The goal of this study was to explore the nature of long term plasticity at the synapses between interneurons expressing CB1Rs (putative CCK+) and pyramidal neurons in the CA1 region of the hippocampus in vitro. We found that theta burst stimulation triggered robust long-term depression (LTD) at this synapse. The locus of LTD induction was postsynaptic and required activation of GABAB receptors. We also showed that LTD at this synaptic connection involves GABABR-dependent suppression of adenylyl cyclase and consequent reduction of PKA activity. In this respect, CB1+ to pyramidal cell synapses differ from the majority of the other hippocampal inhibitory connections where theta burst stimulation results in long-term potentiation.

The Relative Contribution of NMDARs to Excitatory Postsynaptic Currents is Controlled by Ca(2+)-Induced Inactivation.

NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca(2+). At the same time, they are themselves inhibited by the elevation of intracellular Ca(2+) concentration. It is unclear however, whether the Ca(2+) entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca(2+) buffers. Loading of pyramidal cells with exogenous Ca(2+) buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca(2+) influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg(2+) concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca(2+) buffer capacity of postsynaptic neurons.

Developmental Changes in Electrophysiological Properties and a Transition from Electrical to Chemical Coupling between Excitatory Layer 4 Neurons in the Rat Barrel Cortex.

During development, sensory systems switch from an immature to an adult mode of function along with the emergence of the active cortical states. Here, we used patch-clamp recordings from neocortical slices in vitro to characterize the developmental changes in the basic electrophysiological properties of excitatory L4 neurons and their connectivity before and after the developmental switch, which occurs in the rat barrel cortex in vivo at postnatal day P8. Prior to the switch, L4 neurons had higher resting membrane potentials, higher input resistance, lower membrane capacity, as well as action potentials (APs) with smaller amplitudes, longer durations and higher AP thresholds compared to the neurons after the switch. A sustained firing pattern also emerged around the switch. Dual patch-clamp recordings from L4 neurons revealed that recurrent connections between L4 excitatory cells do not exist before and develop rapidly across the switch. In contrast, electrical coupling between these neurons waned around the switch. We suggest that maturation of electrophysiological features, particularly acquisition of a sustained firing pattern, and a transition from the immature electrical to mature chemical synaptic coupling between excitatory L4 neurons, contributes to the developmental switch in the cortical mode of function.