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Welcome to OXION, Universities of Oxford, Cambridge, London and MRC Harwell
Parvalbumin and Somatostatin Interneurons Contribute to the Generation of Hippocampal Gamma Oscillations.
γ-frequency oscillations (30-120 Hz) in cortical networks influence neuronal encoding and information transfer, and are disrupted in multiple brain disorders. While synaptic inhibition is important for synchronization across the γ-frequency range, the role of distinct interneuronal subtypes in slow (<60 Hz) and fast γ states remains unclear. Here, we used optogenetics to examine the involvement of parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons in γ oscillations in the mouse hippocampal CA3 ex vivo, using animals of either sex. Disrupting either PV+ or SST+ interneuron activity, via either photoinhibition or photoexcitation, led to a decrease in the power of cholinergically induced slow γ oscillations. Furthermore, photoexcitation of SST+ interneurons induced fast γ oscillations, which depended on both synaptic excitation and inhibition. Our findings support a critical role for both PV+ and SST+ interneurons in slow hippocampal γ oscillations, and further suggest that intense activation of SST+ interneurons can enable the CA3 circuit to generate fast γ oscillations.SIGNIFICANCE STATEMENT The generation of hippocampal γ oscillations depends on synchronized inhibition provided by GABAergic interneurons. Parvalbumin-expressing (PV+) interneurons are thought to play the key role in coordinating the spike timing of excitatory pyramidal neurons, but the role distinct inhibitory circuits in network synchronization remains unresolved. Here, we show, for the first time, that causal disruption of either PV+ or somatostatin-expressing (SST+) interneuron activity impairs the generation of slow γ oscillations in the ventral hippocampus ex vivo We further show that SST+ interneuron activation along with general network excitation is sufficient to generate high-frequency γ oscillations in the same preparation. These results affirm a crucial role for both PV+ and SST+ interneurons in hippocampal γ oscillation generation.
Cellular mechanisms underlying network synchrony in the medial temporal lobe
© Cambridge University Press 2009. Introduction The hippocampus lies at the apex of the hierarchical organization of cortical connectivity, receiving convergent multimodal inputs that are funneled through the adjacent entorhinal cortex (Fig. 2.1). The output of the hippocampus is relayed back through the entorhinal cortex, and thus these structures are ideally placed to both store novel associations and detect predictive errors (Lavenex and Amaral,2000; Witter et al., 2000). Indeed, while memories are likely to be stored across distributed brain regions, the learning and consolidation of explicit memories appear to depend upon the hippocampus and surrounding parahippocampal regions (Morris et al., 2003; Squire et al., 2004). However, while the anatomical substrate of such learning is becoming increasingly well defined, it remains unclear how cells act collectively within these neuronal networks to extract and store salient input correlations. Over 50 years ago, Donald Hebb postulated a simple cellular learning rule, whereby the strength of the synaptic connection between two neurons would be increased if activity in the presynaptic neuron persistently contributed to discharging the postsynaptic neuron (Hebb, 1949). It has since then been shown that such repeated pairings of synaptic events with postsynaptic action potentials (spikes), within a window of tens of milliseconds, can produce long-term changes in synaptic efficacy in many different neuronal systems, both in vitro and in vivo (Paulsen and Sejnowski, 2000; Bi and Poo, 2001).
Exploring fast hippocampal network oscillations: Combining multi-electrode recordings with optical imaging and patch-clamp techniques
© 2006 Springer Science+Business Media, Inc. All rights reserved. Cortical processing depends on orchestrated activity across distributed neuronal assemblies, and network rhythms may provide a temporal structure relative to which individual neurons within these assemblies can be coordinated. Network oscillations in the gamma-frequency range (∼30 to 100 Hz) have received particular attention because they are prominent in the awake brain, and have been implicated in cognitive processes, such as sensory binding (Singer, 1993), selective attention (Fries et al., 2001), and consciousness (Llinas et al., 1998). Gamma rhythms can be observed in the hippocampus, where they have been implicated in memory processing (Lisman and Idiart, 1995; Jensen and Lisman, 1996), and in vivo multi-electrode techniques have already uncovered some of the mechanisms underlying these hippocampal gamma oscillations in the behaving animal (Bragin et al., 1995; Csicsvari et al., 2003). Adapting such multi-electrode techniques to an in vitro model of hippocampal gamma oscillations would enable a rigorous pharmacological and physiological dissection of the cellular and synaptic mechanisms underlying these rhythms. Furthermore, as hippocampal gamma oscillations represent the coordinated activity of assemblies of neurons, such an in vitro model would provide a convenient screen for potential psychoactive drugs at the network level (Gill et al., 2002; Weiss et al., 2003). Fast network oscillations can be induced in the hippocampal slice in vitro through a variety of paradigms, including patterned afferent stimulation (Whittington et al., 1995; Traub et al., 1996; Whittington et al., 1997), local application of drugs or solutions with altered ionic composition, for example, high potassium (LeBeau et al., 2002; Towers et al., 2002), bath application of kainate (Hajos et al., 2000; Hormuzdi et al., 2001), or drugs that activate metabotropic glutamate receptors (Whittington et al., 1995; Boddeke et al., 1997; Gillies et al., 2002) or muscarinic receptors (Fisahn et al., 1998; Shimono et al., 2000). The frequency of drug-induced oscillations is temperature-dependent. At room temperature, these oscillations are often in the beta-frequency range as defined in vivo (∼10 to 30 Hz), but commonly fall in the gamma-frequency band when recorded at or above 32°C (Ecker et al., 2001; Dickinson et al., 2003), and may therefore provide models for in vivo gamma oscillations. Although planar multi-electrode arrays offer an attractive opportunity to explore the underlying mechanisms and physiological relevance of these network oscillations, the majority of previous studies have been performed in interface-style chambers. Shimono et al. (2000) succeeded in inducing fast network oscillations in hippocampal slices mounted on planar multi-electrode arrays, by lowering the fluid level to create semi-interface conditions and using an atmosphere above the chamber of humidified carbogen gas (95% O2/5% CO2). This development has facilitated the detailed study of spatiotemporal patterns of cellular and network events underlying fast hippocampal network oscillations in vitro (Shimono et al., 2000). In this chapter, we discuss how planar multi-electrode arrays can be used to study fast hippocampal network oscillations induced by activation of kainate, muscarinic, and metabotropic glutamate receptors. This includes practical guidelines for recording and analysis, as well as a discussion of current source density analysis, and emphasizes throughout how to optimize the conditions for combining multi-electrode recordings with optical imaging using voltage-sensitive dyes and patch-clamp recordings from single neurons.
Toward multi-focal spot remote focusing two-photon microscopy for high speed imaging
© 2017 SPIE. Optical sectioning techniques using two-photon excitation of fluorescent indicators are central to diverse imaging applications. The limitations of the technique are low speed and undesirable specimen agitation. In our design, high-speed axial scanning is carried out by moving a reference objective to axially displace the focal spot without introducing significant spherical aberration and any agitation of the specimen. Further, the system is configured to allow switching between single spot and multiple focal spot remote focusing to allow either high dynamic range or high speed imaging.
Cortical Up states induce the selective weakening of subthreshold synaptic inputs.
Slow-wave sleep is thought to be important for retuning cortical synapses, but the cellular mechanisms remain unresolved. During slow-wave activity, cortical neurons display synchronized transitions between depolarized Up states and hyperpolarized Down states. Here, using recordings from LIII pyramidal neurons from acute slices of mouse medial entorhinal cortex, we find that subthreshold inputs arriving during the Up state undergo synaptic weakening. This does not reflect a process of global synaptic downscaling, as it is dependent on presynaptic spiking, with network state encoded in the synaptically evoked spine Ca2+ responses. Our data indicate that the induction of synaptic weakening is under postsynaptic control, as it can be prevented by correlated postsynaptic spiking activity, and depends on postsynaptic NMDA receptors and GSK3β activity. This provides a mechanism by which slow-wave activity might bias synapses towards weakening, while preserving the synaptic connections within active neuronal assemblies.Slow oscillations between cortical Up and Down states are a defining feature of deep sleep, but their function is not well understood. Here the authors study Up/Down states in acute slices of entorhinal cortex, and find that Up states promote the weakening of subthreshold synaptic inputs, while suprathreshold inputs are preserved or strengthened.
Pathogenic potential of antibodies to the GABAB receptor.
GABAB receptor (GABABR) autoantibodies have been detected in the serum of immunotherapy-responsive patients with autoimmune encephalitis. This study aimed to investigate the effect of immunoglobulin G (IgG) from a patient with GABABR antibodies on primary neuronal cultures and acute slices of entorhinal cortex. Primary hippocampal neuronal cultures were incubated with serum immunoglobulin from patients with GABABR or AMPA receptor (AMPAR) antibodies for up to 72 h to investigate their effect on receptor surface expression. Whole-cell patch-clamp recordings from layer III pyramidal cells of the medial entorhinal cortex were used to examine the effect on neuronal activity. GABABR surface expression was unaltered by incubation with GABABR antibodies. By contrast, after 24 h application of AMPAR antibodies, AMPARs were undetectable. However, acute application of GABABR IgG decreased both the duration of network UP states and the spike rate of pyramidal cells in the entorhinal cortex. GABABR antibodies do not appear to affect GABABRs by internalization but rather reduce excitability on the medial temporal lobe networks. This unusual mechanism of action may be exploited in rational drug development strategies.
A brain-wide functional map of the serotonergic responses to acute stress and fluoxetine.
Central serotonin (5-HT) orchestrates myriad cognitive processes and lies at the core of many stress-related psychiatric illnesses. However, the basic relationship between its brain-wide axonal projections and functional dynamics is not known. Here we combine optogenetics and fMRI to produce a brain-wide 5-HT evoked functional map. We find that DRN photostimulation leads to an increase in the hemodynamic response in the DRN itself, while projection areas predominately exhibit a reduction of cerebral blood volume mirrored by suppression of cortical delta oscillations. We find that the regional distribution of post-synaptically expressed 5-HT receptors better correlates with DRN 5-HT functional connectivity than anatomical projections. Our work suggests that neuroarchitecture is not the primary determinant of function for the DRN 5-HT. With respect to two 5-HT elevating stimuli, we find that acute stress leads to circuit-wide blunting of the DRN output, while the SSRI fluoxetine noticeably enhances DRN functional connectivity. These data provide fundamental insight into the brain-wide functional dynamics of the 5-HT projection system.
IDO induces expression of a novel tryptophan transporter in mouse and human tumor cells
IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO+ tumor cells overcome the detrimental effects of local tryptophan depletion.We demonstrate that IDO+ tumor cells express a novel amino acid transporter, which accounts for ∼50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO+ tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation. Copyright © 2011 by The American Association of Immunologists, Inc.
Exosome nanotechnology: an emerging paradigm shift in drug delivery: exploitation of exosome nanovesicles for systemic in vivo delivery of RNAi heralds new horizons for drug delivery across biological barriers.
The demonstration that dendritic cell (DC)-derived exosomes can be exploited for targeted RNAi delivery to the brain after systemic injection provides the first proof-of-concept for the potential of these naturally occurring vesicles as vehicles of drug delivery. As well as being amenable to existing in vivo targeting strategies already in use for viruses and liposomes, this novel approach offers the added advantages of in vivo safety and low immunogenicity. Fulfilment of the potential of exosome delivery methods warrants a better understanding of their biology, as well as the development of novel production, characterisation, targeting and cargo-loading nanotechnologies. Ultimately, exosome-mediated drug delivery promises to overcome important challenges in the field of therapeutics, such as delivery of drugs across otherwise impermeable biological barriers, such as the blood brain barrier, and using patient-derived tissue as a source of individualised and biocompatible therapeutic drug delivery vehicles.
Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery.
Cell-derived membrane vesicles (CMVs) are endogenous carriers transporting proteins and nucleic acids between cells. They appear to play an important role in many disease processes, most notably inflammation and cancer, where their efficient functional delivery of biological cargo seems to contribute to the disease progress. CMVs encompass a variety of submicron vesicular structures that include exosomes and shedding vesicles. The lipids, proteins, mRNA and microRNA (miRNA) delivered by these vesicles change the phenotype of the receiving cells. CMVs have created excitement in the drug delivery field, because they appear to have multiple advantages over current artificial drug delivery systems. Two approaches to exploit CMVs for delivery of exogenous therapeutic cargoes in vivo are currently considered. One approach is based on engineering of natural CMVs in order to target certain cell types using CMVs loaded with therapeutic compounds. In the second approach, essential characteristics of CMVs are being used to design nano-scaled drug delivery systems. Although a number of limiting factors in the clinical translation of the exciting research findings so far exist, both approaches are promising for the development of a potentially novel generation of drug carriers based on CMVs.
Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes.
To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model.
Alzheimer's disease (AD) results in cognitive decline and altered network activity, but the mechanisms are unknown. We studied human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Electroencephalographic recordings in hAPP mice revealed spontaneous epileptiform discharges, indicating network hypersynchrony, primarily during reduced gamma oscillatory activity. Because this oscillatory rhythm is generated by inhibitory parvalbumin (PV) cells, network dysfunction in hAPP mice might arise from impaired PV cells. Supporting this hypothesis, hAPP mice and AD patients had decreased levels of the interneuron-specific and PV cell-predominant voltage-gated sodium channel subunit Nav1.1. Restoring Nav1.1 levels in hAPP mice by Nav1.1-BAC expression increased inhibitory synaptic activity and gamma oscillations and reduced hypersynchrony, memory deficits, and premature mortality. We conclude that reduced Nav1.1 levels and PV cell dysfunction critically contribute to abnormalities in oscillatory rhythms, network synchrony, and memory in hAPP mice and possibly in AD.
Flexible spike timing of layer 5 neurons during dynamic beta oscillation shifts in rat prefrontal cortex
Human brain oscillations occur in different frequency bands that have been linked to different behaviours and cognitive processes. Even within specific frequency bands such as the beta- (14-30 Hz) or gamma-band (30-100 Hz), oscillations fluctuate in frequency and amplitude. Such frequency fluctuations most probably reflect changing states of neuronal network activity, as brain oscillations arise from the correlated synchronized activity of large numbers of neurons. However, the neuronal mechanisms governing the dynamic nature of amplitude and frequency fluctuations within frequency bands remain elusive. Here we show that in acute slices of rat prefrontal cortex (PFC), carbachol-induced oscillations in the beta-band show frequency and amplitude fluctuations. Fast and slow non-harmonic frequencies are distributed differentially over superficial and deep cortical layers, with fast frequencies being present in layer 3, while layer 6 only showed slow oscillation frequencies. Layer 5 pyramidal cells and interneurons experience both fast and slow frequencies and they time their spiking with respect to the dominant frequency. Frequency and phase information is encoded and relayed in the layer 5 network through timed excitatory and inhibitory synaptic transmission. Our data indicate that frequency fluctuations in the beta-band reflect synchronized activity in different cortical subnetworks, that both influence spike timing of output layer 5 neurons. Thus, amplitude and frequency fluctuations within frequency bands may reflect activity in distinct cortical neuronal subnetworks that may process information in a parallel fashion. © 2009 The Authors. Journal compilation © 2009 The Physiological Society.
Dynamics of neuronal assemblies are modulated by anaesthetics but not analgesics.
BACKGROUND AND OBJECTIVE: Analgesics and anaesthetics have diverse synaptic actions that nonetheless have a common net inhibitory action on neuronal discharge. It is puzzling, therefore, that these two classes of compounds have fundamentally different affects, one blocking pain and the other consciousness. Indeed, beyond the isolated synapse, little is known of the larger scale mechanisms that mediate actual function, for example, transient neuronal assemblies. It was hypothesized that the two classes of drugs might have, respectively, differential effects on transient activation of these assemblies of neurons working together. METHODS: Hippocampal tissue from juvenile Wistar rats was used for in vitro optical imaging with voltage-sensitive dyes and simultaneous field potential recordings. The response to paired pulse stimulation of the hippocampus was recorded in the presence and absence of two types of analgesic (morphine and gabapentin) and two types of anaesthetic (thiopental and propofol). RESULTS: Optical imaging and electrophysiology used in parallel yield quite different results. Most consistently, the imaging technique was able to detect an enhanced period of activation following anaesthetic, but not analgesic application. This effect was not readily seen from electrophysiology field potential recordings. CONCLUSIONS: These findings suggest that, irrespective of the effects of the two drug classes at a synaptic level, the dynamics of transient neuronal assemblies are modified selectively by anaesthetics and not analgesics.
