Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures.

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.

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

KEY POINTS: 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 ∼77 nM 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. ABSTRACT: 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 72-83 nM resulting in a block of more than 82% of ES-NMDARs during a 5 min 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.

Human in vitro systems for examining synaptic function and plasticity in the brain.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Adenosine A2A receptor antagonism reverses inflammation-induced impairment of microglial process extension in a model of Parkinson's disease.

Microglia, the immune cells of the central nervous system, constantly survey the parenchyma in the healthy brain to maintain homeostasis. When a disturbance, such as cell death, results in ATP release in vivo, microglial processes respond by utilizing P2Y12 purinergic receptors to trigger extension toward the site of damage. Processes ultimately surround the injury site, preventing the spread of harmful cellular constituents and assisting with tissue repair. In contrast to the healthy brain, many neurodegenerative diseases, including Parkinson's disease, are characterized by the presence of neuroinflammation. Yet, the ability of microglia to respond to tissue damage under pro-inflammatory conditions has not been well studied. To assess the ability of microglia to respond to tissue injury and localized cell death in the context of Parkinson's disease, we performed confocal imaging of acute brain slices from mice with microglia-specific green fluorescent protein expression. Microglia in coronal slices containing the substantia nigra extend processes toward a mechanical injury in a P2Y12 receptor-dependent manner. However, microglia in mice treated for 5days with 20mg/kg/day 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) show significantly reduced process displacement toward the injury compared to microglia in control animals. Pre-treatment of slices from MPTP-injected mice with the A2A receptor-selective antagonist preladenant restores the ability of activated microglia to respond to tissue damage. These data support the hypothesis that chronic inflammation impedes microglial motility in response to further injury, such as cell death, and suggest that some aspects of the neuroprotection observed with adenosine A2A receptor antagonists may involve direct or indirect actions at microglia.

Optogenetic Interrogation of Electrophysiological Dendritic Properties and Their Effect on Pacemaking Neurons from Acute Rodent Brain Slices.

Understanding dendritic excitability is essential for a complete and precise characterization of neurons' input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology. Key features • A method for studying the effects of electrotonic and nonlinear dendritic properties on the sub- and suprathreshold responses of pacemaking neurons. • Combines somatic patch clamp or perforated patch recordings with optogenetic activation in acute brain slices to investigate dendritic linear transformation without patching the dendrite. • Oscillatory illumination at various frequencies estimates spectral properties of the dendrite using subthreshold voltage-clamp recordings and studies entrainment of pacemakers in current clamp recordings. • This protocol uses Poisson white noise illumination to estimate dendritic phase response curves and peri-stimulus time histograms.

Assaying activity-dependent arteriole and capillary responses in brain slices.

Significance: Neurovascular coupling (NVC) is the process that increases cerebral blood flow in response to neuronal activity. NVC is orchestrated by signaling between neurons, glia, and vascular cells. Elucidating the mechanisms underlying NVC at different vascular segments and in different brain regions is imperative for understanding of brain function and mechanisms of dysfunction. Aim: Our goal is to describe a protocol for concurrently monitoring stimulation-evoked neuronal activity and resultant vascular responses in acute brain slices. Approach: We describe a step-by-step protocol that allows the study of endogenous NVC mechanisms engaged by neuronal activity in a controlled, reduced preparation. Results: This ex vivo NVC assay allows researchers to disentangle the mechanisms regulating the contractile responses of different vascular segments in response to neuronal firing independent of flow and pressure mediated effects from connected vessels. It also enables easy pharmacological manipulations in a simplified, reduced system and can be combined with Ca 2 + imaging or broader electrophysiology techniques to obtain multimodal data during NVC. Conclusions: The ex vivo NVC assay will facilitate investigations of cellular and molecular mechanisms that give rise to NVC and should serve as a valuable complement to in vivo imaging methods.

Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions.

BACKGROUND: Acute brain slices represent a powerful tool for analysis of brain function in physiology and pathology. Commercial systems and custom-build solutions with carbogen (95% O2/5% CO2) aeration, but they are expensive, have a high working volume requiring large amount of substances, and only limited options for treatment in parallel are possible. NEW METHOD: We developed a novel cost-effective incubation system using materials available in every laboratory, allowing parallel incubation of several treatment conditions, thus also reducing the number of experimental animals. Our system incubation parameters were optimized for cortical neuron observation. RESULTS: We tested several different options using 6, 12 or 24 standard culture well plates, combining them with cell strainer baskets inside. The system was placed in a pre-warmed incubator at 37 °C. Carbogen was injected through a 22 gauge needle, positioned between the basket and the wall of the well. Best results were achieved in a 6-well plate. In 12 and 24-well plates bubbles accumulated beneath the basket, displacing it upwards, making it unsuitable for our purposes. The gas oxygenized the medium without mechanically disturbing the slices, protected within the strainer basket, but still allowing optimal diffusion through the 100 µm pores. In a 6-well plate, six simultaneous treatments were possible in parallel. LDH/Cytotoxicity tests showed an acute toxicity of less than 7%. The system lost about 2.5% per hour of the fluid through evaporation, which was replenished every 2 h. Up to 6 h after treatment, however, this evaporation was excellently tolerated by the neurons even without fluid replenishment, most probably due to the anti-swelling effect of the mildly hypertonic medium. We performed two staining procedures, working excellently with this experimental setup, namely - a modified DiI staining and a slice silver impregnation method, both confirming the intact neuronal morphology. Preserved CA3 calcium influx and removal response following KCl depolarization confirmed the normal physiology of the pyramidal neurons 6 h after exposure in the system. COMPARISON TO EXISTING METHODS: The proposed system is much cheaper than the commercial solutions, can be constructed in any lab, allows up to 6 different treatments in parallel, which none of the existing systems allows. Antibiotic presence in the incubation medium and adequate evaporation control is required if longer incubation (> 6 h) is needed. Lower incubation volumes (3-6 ml) allow sparing expensive reagents. Our procedure was optimized for cortical neurons, further fine tuning to meet other specific requirements is possible. CONCLUSIONS: The system we propose allows filling the gap for budget solutions for short to mid-term incubation of acute brain slices.

Single or Double Patch-Clamp Recordings In Ex Vivo Slice Preparation: Functional Connectivity, Synapse Dynamics, and Optogenetics.

Patch-clamp recordings are the method of choice to define cell-type specific electrophysiological properties of single neurons and the synaptic connectivity between pairs of connected neurons in brain slices. In combination with optogenetic tools, patch-clamp recordings allow for the investigation of long-range afferent connectivity from identified distant brain areas. Here we describe the necessary equipment to carry out patch clamp recordings, surgical methods for dissection and preparation of horizontal brain slices containing the hippocampus, and a step-by-step guide for establishing patch clamp recordings in the whole-cell configuration. We provide protocols for single neuron stimulation via the patch pipette and for photostimulation experiments that activate axon terminals expressing light sensitive ion channels.

Minimizing shrinkage of acute brain slices using metal spacers during histological embedding.

The morphological structure of neurons provides the basis for their functions and is a major focus of contemporary neuroscience studies. Intracellular staining of single cells in acute slices is a well-established approach, offering high-resolution information on neuronal morphology, complementing their physiology. Despite major technical advances, however, a common histological artifact often precludes precise morphological analysis: shrinkage of the sampled tissue after embedding for microscopy. Here, we describe a new approach using a metal spacer, sandwiched between two coverslips to reduce shrinkage of whole-mount slice preparations during embedding with aqueous mounting medium under a coverslip. This approach additionally allows imaging the slices from both sides to obtain better quality images of deeper structures. We demonstrate that the use of this spacer system can efficiently and stably reduce the shrinkage of slices, whereas conventional embedding methods without spacer or with agar spacer cause severe, progressive shrinkage after embedding. We further show that the shrinkage of slices is not uniform and artifacts in morphology and anatomical parameters produced cannot be compensated using linear correction algorithms. Our study, thus, emphasizes the importance of preventing the deformation of slice preparations and offers an effective means for reducing shrinkage and associated artifacts during embedding.

The Synaptic Vesicle Priming Protein CAPS-1 Shapes the Adaptation of Sensory Evoked Responses in Mouse Visual Cortex.

Short-term plasticity gates information transfer across neuronal synapses and is thought to be involved in fundamental brain processes, such as cortical gain control and sensory adaptation. Neurons employ synaptic vesicle priming proteins of the CAPS and Munc13 families to shape short-term plasticity in vitro, but the relevance of this phenomenon for information processing in the intact brain is unknown. By combining sensory stimulation with in vivo patch-clamp recordings in anesthetized mice, we show that genetic deletion of CAPS-1 in thalamic neurons results in more rapid adaptation of sensory-evoked subthreshold responses in layer 4 neurons of the primary visual cortex. Optogenetic experiments in acute brain slices further reveal that the enhanced adaptation is caused by more pronounced short-term synaptic depression. Our data indicate that neurons engage CAPS-family priming proteins to shape short-term plasticity for optimal sensory information transfer between thalamic and cortical neurons in the intact brain in vivo.

Intrinsic and Synaptic Dynamics Contribute to Adaptation in the Core of the Avian Central Nucleus of the Inferior Colliculus.

The reduction of neuronal responses to repeated stimulus presentation occurs in many sensory neurons, also in the inferior colliculus of birds. The cellular mechanisms that cause response adaptation are not well described. Adaptation must be explicable by changes in the activity of input neurons, short-term synaptic plasticity of the incoming connections, excitability changes of the neuron under consideration or influences of inhibitory or modulatory network connections. Using whole-cell recordings in acute brain slices of the embryonic chicken brain we wanted to understand the intrinsic and synaptic contributions to adaptation in the core of the central nucleus of the inferior colliculus (ICCc). We described two neuron types in the chicken ICCc based on their action potential firing patterns: Phasic/onset neurons showed strong intrinsic adaptation but recovered more rapidly. Tonic/sustained firing neurons had weaker adaptation but often had additional slow components of recovery from adaptation. Morphological analysis suggested two neuron classes, but no physiological parameter aligned with this classification. Chicken ICCc neurons received mostly mixed AMPA- and NMDA-type glutamatergic synaptic inputs. In the majority of ICCc neurons the input synapses underwent short-term depression. With a simulation of the putative population output activity of the chicken ICCc we showed that the different adaptation profiles of the neuron classes could shift the emphasize of stimulus encoding from transients at long intervals to ongoing parts at short intervals. Thus, we report here that description of biophysical and synaptic properties can help to explain adaptive phenomena in central auditory neurons.

Analysis of lipid raft molecules in the living brain slices.

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.

Two-Photon STED Microscopy for Nanoscale Imaging of Neural Morphology In Vivo.

The advent of super-resolution microscopy offers to bridge the gap between electron and light microscopy. It has opened up the possibility of visualizing cellular structures and dynamic signaling events on the "mesoscale" well below the classic diffraction barrier of light microscopy (10-200 nm), while essentially retaining the advantages of fluorescence microscopy concerning multicolor labeling, detection sensitivity, signal contrast, live-cell imaging, and temporal resolution.From among the new super-resolution techniques, STED microscopy stands out as a laser-scanning imaging modality, which enables nanoscale volume-metric imaging of cellular morphology. In combination with two-photon (2P) excitation, STED microscopy facilitates the visualization of the highly complex and dynamic morphology of neurons and glia cells deep inside living brain slices and in the intact brain in vivo.Here, we present an overview of the principles and implementation of 2P-STED microscopy in vivo, providing the neurobiological context and motivation for this technique, and illustrating its capacity by showing images of dendritic spines and microglial processes obtained from living brain tissue.

Stretch induced hyperexcitability of mice callosal pathway.

Memory and learning are thought to result from changes in synaptic strength. Previous studies on synaptic physiology in brain slices have traditionally been focused on biochemical processes. Here, we demonstrate with experiments on mouse brain slices that central nervous system plasticity is also sensitive to mechanical stretch. This is important, given the host of clinical conditions involving changes in mechanical tension on the brain, and the normal role that mechanical tension plays in brain development. A novel platform is developed to investigate neural responses to mechanical stretching. Flavoprotein autofluoresence (FA) imaging was employed for measuring neural activity. We observed that synaptic excitability substantially increases after a small (2.5%) stretch was held for 10 min and released. The increase is accumulative, i.e., multiple stretch cycles further increase the excitability. We also developed analytical tools to quantify the spatial spread and response strength. Results show that the spatial spread is less stable in slices undergoing the stretch-unstretch cycle. FA amplitude and activation rate decrease as excitability increases in stretch cases but not in electrically enhanced cases. These results collectively demonstrate that a small stretch in physiological range can modulate neural activities significantly, suggesting that mechanical events can be employed as a novel tool for the modulation of neural plasticity.

Quantitative comparison of novel GCaMP-type genetically encoded Ca(2+) indicators in mammalian neurons.

New variants of GCaMP-type genetically encoded Ca(2+) indicators (GECIs) have been continuously developed and heavily used in many areas of biology including neuroscience. The latest subfamily called "GECOs" were developed with in vitro high-throughput screening, and shown to have novel spectral properties and/or improved fluorescent responses over their ancestor GCaMP3. The most critical parameter in evaluating performance in neurons, however, remains uncharacterized: the relationship between the GECI responses and the number of action potentials (APs). Here we analyzed the GECI responses to APs in cortical pyramidal cells of mouse acute brain slices. Unexpectedly, we found that none of the GECOs exhibited any improved performance over GCaMP3. Our results imply that careful validation is required for the accurate prediction of the actual performance of GECIs in mammalian neurons. We propose that appropriate guidelines for evaluating their efficacy should be established for the benefit of research community, given the rapidly expanding use of GECIs in neuroscience.

Quantitative comparison of genetically encoded Ca indicators in cortical pyramidal cells and cerebellar Purkinje cells.

Genetically encoded Ca(2+) indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 ± 2°C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20 APs evoked at 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1-10 CSs at 10 Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide useful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano15 is suitable for detecting CSs.