This work was supported by CHDI (A-12467), the German Research Foundation (Exc 257/1 and DFG Project-ID 327654276 &#8211; SFB 1315) and intramural Research Funds of the Charit&#233;. We thank K. T&#246;r&#246;k, St. George&#39;s, University of London, and N. Helassa, University of Liverpool, for the iGluu plasmid and many helpful discussions. D. Betances and A. Sch&#246;nherr provided excellent technical assistance.

The authors have nothing to disclose.

The experiments concern a question of general interest &#8212; synapse independency and its possible loss in the course of neurodegeneration, and we describe a new approach to identify affected synapses in acute brain slices from aged (&#62;1 year) mice. Taking advantage of the improved kinetic characteristics of the recently introduced glutamate sensor iGluu the experiments illuminate the relationship between synaptic glutamate release and uptake in a way that has not been possible before.
<p class="jove_...

The relative impact of each synaptic terminal belonging to a &#34;unitary connection&#34; (i.e., the connection between 2 nerve cells) is typically assessed by its influence on the initial segment of the postsynaptic neuron1,2. Somatic and/or dendritic recordings from postsynaptic neurons represent the most common and, until now, also the most productive means to clarify information processing under a top-down or vertical perspective3,4,5. However, the presence of astrocytes with their discrete and (in rodents) non-overlapping territories may contribute a horizontal perspective that is based on local mechanisms of signal exchange, integration and synchronization at synaptic sites6,7,8,9,10.
Because it is known that astroglia play, in general, a major role in the pathogenesis of neurodegenerative disease11,12 and, in particular, a role in the maintenance and plasticity of glutamatergic synapses13,14,15,16, it is conceivable that alterations in synaptic performance evolve in accordance with the state of astrocytes in the shared target area of afferent fibers with diverse origin. To further explore the target-/astroglia-derived local regulatory mechanisms in health and disease, it is necessary to evaluate individual synapses. The present approach was worked out to estimate the range of functional glutamate release and clearance indicators and to define criteria that may be used to identify dysfunctional (or recovered) synapses in brain areas most closely related to movement initiation (i.e., first of all in the motor cortex and dorsal striatum).
The striatum lacks intrinsic glutamatergic neurons. Therefore, it is relatively easy to identify glutamatergic afferents of extrastriatal origin. The latter mostly originate in the medial thalamus and in the cerebral cortex (see17,18,19,20 for more). Corticostriatal synapses are formed by the axons of pyramidal neurons localized in cortical layers 2/3 and 5. The respective axons form bilateral intra-telencephalic (IT) connections or ipsilateral connections via a fiber system that more caudally constitutes the pyramidal tract (PT). It has further been suggested that IT- and PT-type terminals differ in their release characteristics and size21,22. In view of these data, one could also expect some differences in the handling of glutamate.
The striatum is the most affected brain area in Huntington&#39;s disease (HD)5. Human HD is a severe genetically inherited neurodegenerative disorder. The Q175 mouse model offers an opportunity to investigate the cellular basis of the hypokinetic-rigid form of HD, a state that has much in common with parkinsonism. Starting at an age of about 1 year, homozygote Q175 mice (HOM) exhibit signs of hypokinesia, as revealed by measuring the time spent without movement in an open field23. The present experiments with heterozygote Q175 mice (HET) confirmed the previous motor deficits observed in HOM and, in addition, showed that the observed motor deficits were accompanied by a reduced level of the astrocytic excitatory amino acid transporter 2 protein (EAAT2) in the immediate vicinity of corticostriatal synaptic terminals24. It has therefore been hypothesized that a deficit in astrocytic glutamate uptake could lead to dysfunction or even loss of respective synapses25,26.
Here, we describe a new approach that allows one to evaluate single synapse glutamate clearance relative to the amount of the released neurotransmitter. The new glutamate sensor iGluu was expressed in corticostriatal pyramidal neurons. It was developed by Katalin T&#246;r&#246;k27 and represents a modification of the previously introduced high-affinity but slow glutamate sensor iGluSnFR28. Both sensors are derivatives of the enhanced green fluorescent protein (EGFP). For spectral and kinetic characteristics, see Helassa et al.27. Briefly, iGluu is a low-affinity sensor with rapid de-activation kinetics and therefore particularly well suited to study glutamate clearance at glutamate-releasing synaptic terminals. The dissociation time constant of iGluu was determined in a stopped-flow device, which rendered a Tauoff value of 2.1 ms at 20 &#176;C, but 0.68 ms when extrapolated to a temperature of 34 &#176;C27. Single Schaffer collateral terminals probed at 34 &#176;C with spiral laser scanning in the CA1 region of organotypic hippocampal cultures under a 2-photon microscope exhibited a mean time constant of decay of 2.7 ms.

<table><tbody><tr><td>Stereo microsope</td><td>WPI</td><td>PZMIII</td><td>Precision Stereo Zoom Binocular Microscope</td></tr><tr><td>Stereotaxic frame</td><td>Stoelting</td><td>51500D</td><td>Digital Lab New Standard stereotaxic frame</td></tr><tr><td>High speed drill equipment</td><td>Stoelting</td><td>514439V</td><td>Foredom K1070 cromoter Kit</td></tr><tr><td>Injection system</td><td>Stoelting</td><td>53311</td><td>Quintessential Stereotaxic Injector (QSI)</td></tr><tr><td>Hamilton syringe 5 &amp;micro;l</td><td>Hamilton</td><td>87930</td><td>75RN Syr (26s/51/2)</td></tr><tr><td>Laser positioning system</td><td>Rapp OptoElectronic</td><td>UGA-40</td><td>UGA-40</td></tr><tr><td>Blue laser for iGluu excitation</td><td>Rapp OptoElectronic</td><td>DL-473-020-S</td><td>473 nm laser</td></tr><tr><td>Dichroic mirror for 473 nm</td><td>Rapp OptoElectronic</td><td>ROE TB-355-405-473</td><td>Dichroic</td></tr><tr><td>1P upright microscope</td><td>Carl Zeiss</td><td>000000-1066-600</td><td>Axioskop 2 FS Plus</td></tr><tr><td>Objective 63x/1.0</td><td>Carl Zeiss</td><td>421480-9900</td><td>W Plan-Apochromat</td></tr><tr><td>4x objective</td><td>Carl Zeiss</td><td>44-00-20</td><td>Achroplan 4x/0,10</td></tr><tr><td>Dichroic mirror for iGluu</td><td>Omega optical</td><td>XF2030</td><td></td></tr><tr><td>Emission filter for iGluu</td><td>Omega optical</td><td>XF3086</td><td></td></tr><tr><td>Dichroic mirror</td><td>Omega optical</td><td>QMAX_DI580LP</td><td></td></tr><tr><td>Emission filter for autofluorescence subtr.</td><td>Omega optical</td><td>QMAX EM600-650</td><td></td></tr><tr><td>sCMOS camera</td><td>Andor</td><td>ZYLA4.2PCL10</td><td>ZYLA 4.2MP Plus</td></tr><tr><td>Acqusition software</td><td>Andor</td><td>4.30.30034.0</td><td>Solis</td></tr><tr><td>AD/DA converter</td><td>HEKA Elektronik</td><td>895035</td><td>InstruTECH LIH8+8</td></tr><tr><td>Aquisition software</td><td>HEKA Elektronik</td><td>895153</td><td>TIDA5.25</td></tr><tr><td>Electrode positioning system</td><td>Sutter Instrument</td><td>MPC-200</td><td>Micromanipulator</td></tr><tr><td>Electrical stimulator</td><td>Charite workshops</td><td>STIM-26</td><td></td></tr><tr><td>Slicer</td><td>Leica</td><td>VT1200 S</td><td>Vibrotome</td></tr><tr><td>Brown/Flaming-type puller</td><td>Sutter Instr</td><td>SU-P1000</td><td>P-1000</td></tr><tr><td>Glass tubes for injection pipettes</td><td>WPI</td><td>1B100F3</td><td></td></tr><tr><td>Glass tubes forstimulation pipettes</td><td>WPI</td><td>R100-F3</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Abcam</td><td>ab120054</td><td>TTX</td></tr><tr><td>iGluu plasmid</td><td>Addgene</td><td>106122</td><td>pCI-syn-iGluu</td></tr><tr><td>Q175 mice</td><td>Jackson Lab</td><td>27410</td><td>Z-Q175-KI</td></tr></tbody></table>

single synapse indicators of glutamate release and uptake in acute brain slices from normal and huntington mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

Identification of two types of corticostriatal glutamatergic varicosities 
IT and PT afferents originate in layer 2/3 and 5, respectively, and exhibit differential ramification and termination patterns in the ipsilateral and contralateral (IT terminals only) striatum. Still little is known about the properties of glutamate release and clearance under repetitive activation conditions as observed during the initiation of movements, but it is well documented that the respective glutamate-releasing vari...

Watch this Scientific Journal Video about Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice at JoVE.com

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

single-synapse-indicators-glutamate-release-uptake-acute-brain-slices

Research

JoVE Journal

Neuroscience

6.1K Views.  Charité - University Medicine. Hoge resolutie beeldvorming van enkele synapsen uitdrukken van een snelle glutamaat sensor maakt een detectie van lokale mismatch tussen zender release en opname. In het geval van ziekte kan deze methode worden gebruikt om disfunctionele synapsen te identificeren. Voor autofluorescentie correctie, ten eerste, plaats een hersenschijf van de muis van belang in de opnamekamer van een one-photon microscoop.Dompel het plakje onder in zuurstofrijk, kunstmatig hersenvocht en gebruik de 20x wa...

Video: Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Biochemistry

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

<a href='https://app.jove.com/v/56153/evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices'>Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).</a>This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic architecture through their colocalization.

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Samenvatting

Meer video's verkennen