Name: deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents
Uploaded: 2013-08-07T13:00:00
Description: Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (NS002986). AS wrote the manuscript and implemented the deconvolution analysis. JSD developed the initial version of the deconvolution analysis and commented on the text.

1. Slice Preparation
<ol>
	<li>Prepare 500 ml slicing/storage solution containing (in mM): 119 NaCl, 2.5 KCl, 0.5 CaCl2, 1.3 MgSO4&middot;7H2O, 4 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 22 glucose, 320 mOsm, pH 7.4</li>
	<li>Use a 250 ml beaker to prepare a submersion chamber for the slices, fill it with 200 ml slicing/storage solution, warm it in a water bath at 34 &deg;C and bubble it with 95% O2, 5% CO2.</li>
	<li>Keep the ...

<ol>
	<li>Ventura, R., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+relationships+between+hippocampal+synapses+and+astrocytes.">Three-dimensional relationships between hippocampal synapses and astrocytes.</a> J. Neurosci. 19, 6897-6906 (1999).</li><li>Witcher, M. R., Kirov, S. A., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+perisynaptic+astroglia+during+synaptogenesis+in+the+mature+rat+hippocampus.">Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus.</a> Glia. 55, 13-23 (2007).</li><li>Danbolt, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake.">Glutamate uptake.</a> Prog. Neurobiol. 65, 1-105 (2001).</li><li>Herman, M. A., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glutamate+concentration+in+hippocampal+slice.">Extracellular glutamate concentration in hippocampal slice.</a> J. Neurosci. 27, 9736-9741 (2007).</li><li>Arnth-Jensen, N., Jabaudon, D., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+independent+hippocampal+synapses+is+controlled+by+glutamate+uptake.">Cooperation between independent hippocampal synapses is controlled by glutamate uptake.</a> Nat. Neurosci. 5, 325-331 (2002).</li><li>Barbour, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+synapse+independence.">An evaluation of synapse independence.</a> J. Neurosci. 21, 7969-7984 (2001).</li><li>Rusakov, D. A., Kullmann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrasynaptic+glutamate+diffusion+in+the+hippocampus:+ultrastructural+constraints,+uptake,+and+receptor+activation.">Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation.</a> J. Neurosci. 18, 3158-3170 (1998).</li><li>Zerangue, N., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flux+coupling+in+a+neuronal+glutamate+transporter.">Flux coupling in a neuronal glutamate transporter.</a> Nature. 383, 634-637 (1038).</li><li>Eliasof, S., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+glutamate+transporter+is+coupled+to+an+anionic+conductance.">Retinal glial cell glutamate transporter is coupled to an anionic conductance.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 4153-4158 (1996).</li><li>Wadiche, J. I., Amara, S. G., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+fluxes+associated+with+excitatory+amino+acid+transport.">Ion fluxes associated with excitatory amino acid transport.</a> Neuron. 15, 721-728 (1995).</li><li>Wadiche, J. I., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+and+microscopic+properties+of+a+cloned+glutamate+transporter/chloride+channel.">Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel.</a> J. Neurosci. 18, 7650-7661 (1998).</li><li>Bergles, D. E., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activation+of+glutamate+transporters+in+hippocampal+astrocytes.">Synaptic activation of glutamate transporters in hippocampal astrocytes.</a> Neuron. 19, 1297-1308 (1997).</li><li>Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes:+transmitter+uptake+gets+faster+during+development.">Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.</a> J. Neurosci. 25, 2906-2916 (2005).</li><li>Scimemi, A., Tian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transporters+regulate+glutamate+clearance,+NMDA+receptor+activation,+and+synaptic+plasticity+in+the+hippocampus.">Neuronal transporters regulate glutamate clearance, NMDA receptor activation, and synaptic plasticity in the hippocampus.</a> J. Neurosci. 29, 14581-14595 (2009).</li><li>Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+enhances+glutamate+uptake+via+glutamate+transporters+in+rat+glial+cells.">Isoflurane enhances glutamate uptake via glutamate transporters in rat glial cells.</a> Neuroreport. 12, 1077-1080 (2001).</li><li>Barbour, B., Brew, H., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenic+uptake+of+glutamate+and+aspartate+into+glial+cells+isolated+from+the+salamander+(Ambystoma)+retina.">Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander (Ambystoma) retina.</a> J. Physiol. 436, 169-193 (1991).</li><li>Bergles, D. E., Tzingounis, A. V., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+coupled+and+uncoupled+currents+during+glutamate+uptake+by+GLT-1+transporters.">Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters.</a> J. Neurosci. 22, 10153-10162 (2002).</li><li>Diamond, J. S., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+glutamate+does+not+overwhelm+transporters+on+hippocampal+astrocytes+during+high-frequency+stimulation.">Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation.</a> J. Neurophysiol. 83, 2835-2843 (2000).</li><li>Benediktsson, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+regulates+glutamate+transporter+dynamics+in+developing+astrocytes.">Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.</a> Glia. 60, 175-188 (2012).</li><li>Hires, S. A., Zhu, Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+synaptic+glutamate+spillover+and+reuptake+by+linker+optimized+glutamate-sensitive+fluorescent+reporters.">Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4411-4416 (2008).</li><li>Scimemi, A., Meabon, J., Woltjer, R. L., Sullivan, J. M., Diamond, J. S., Cook, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid&#946;1-42+slows+clearance+of+synaptically-released+glutamate+by+mislocalizing+astrocytic+GLT-1.">Amyloid&#946;1-42 slows clearance of synaptically-released glutamate by mislocalizing astrocytic GLT-1.</a> J. Neurosci. 33, 5312-5318 (2013).</li></ol>

Here we describe an experimental approach to obtain electrophysiological recordings from astrocytes, an analytical protocol to isolate glutamate transporter currents in astrocytes and a mathematical method to derive the time course of glutamate clearance from astrocytic transporter currents.
The success of the analysis relies on the ability to obtain high-quality patch clamp recordings from astrocytes and on the accuracy of the fitting algorithms used to describe the transporter currents. The ...

Astrocytes are one of the most abundant cell types in the brain with star-shaped morphology and fine membrane protrusions that extend throughout the neuropil and reach neighboring synaptic contacts 1,2. The astrocytes' cell membrane is densely packed with glutamate transporter molecules 3. Under physiological conditions, glutamate transporters rapidly bind glutamate at the extracellular side of the membrane and transfer it to the cell cytoplasm. By doing so, the transporters maintain low the basal concentration of glutamate in the extracellular space 4. Glutamate transporters in fine astrocytic processes adjacent to excitatory synapses are ideally positioned to bind glutamate released during synaptic events as it diffuses away from the synaptic cleft. By doing so, the transporters also limit glutamate spillover towards peri- and extra-synaptic regions and onto neighboring synapses, reducing the spatial spread of excitatory signals in the brain 5-7.
Glutamate transport is an electrogenic process stoichiometrically coupled to the movement of 3 Na+ and 1 H+ along their electrochemical gradient and to the counter-transport of 1 K+ 8. Glutamate transport is associated with (but not stoichiometrically coupled to) an anionic conductance permeable to SCN- (thiocyanate) &gt; NO3- (nitrate) &asymp; ClO4- (perchlorate) &gt; I- &gt; Br- &gt; Cl- &gt; F-, not to CH3SO3- (methane sulfonate) and C6H11O7- (gluconate) 9-11. Both currents (stoichiometric and non-stoichiometric) can be recorded by obtaining whole-cell patch-clamp recordings from astrocytes, visually identified under Dodt illumination or infra-red differential interference contrast (IR-DIC) in acute brain slices 12. The stoichiometric component of the current associated with glutamate transport across the membrane can be isolated by using CH3SO3-, or C6H11O7- based intracellular solutions and can be evoked by flash-uncaging glutamate on astrocytes 13,14, or by activating glutamate release from neighboring synapses, either electrically 12 or with a targeted optogenetic control.
The time course of the stoichiometric component of the transporter current is shaped by the lifetime of the glutamate concentration profile at astrocytic membranes (i.e. glutamate clearance), the kinetics of glutamate transporters, the passive membrane properties of astrocytes, and during synaptic stimulations, by the synchronicity of glutamate release across the activated synapses 13. Here we describe in full detail: (1) an experimental approach to isolate the stoichiometric component of glutamate transporter currents from whole-cell patch-clamp recordings from astrocytes using acute mouse hippocampal slices as an example experimental preparation; (2) an analytical approach to derive the time course of glutamate clearance from these recordings 13,14. These methods can be used to record and analyze glutamate transporter currents from astrocytes in any region of the central nervous system.

<table border="1">
		<tbody>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>CGP52432</td>
			<td>Tocris</td>
			<td>1246</td>
			<td></td>
		 </tr>
		 <tr>
			<td>(R,S)-CPP</td>
			<td>Tocris</td>
			<td>173</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPCPX</td>
			<td>Tocris</td>
			<td>439</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LY341495 disodium salt</td>
			<td>Tocris</td>
			<td>4062</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MSOP</td>
			<td>Tocris</td>
			<td>803</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NBQX disodium salt</td>
			<td>Tocris</td>
			<td>1044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D,L-TBOA</td>
			<td>Tocis</td>
			<td>1223</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MNI-L-glutamate</td>
			<td>Tocris</td>
			<td>1490</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexa 594</td>
			<td>Life Technologies</td>
			<td>A10438</td>
			<td>Optional</td>
		 </tr>
		 <tr>
			<td>Matrix electrodes</td>
			<td>Frederick Haer Company</td>
			<td>MX21AES(JD3)</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG10165-4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual-stage glass micro-pipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Loctite 404 instant adhesive</td>
			<td>Ted Pella</td>
			<td>46551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xe lamp</td>
			<td>Rapp OptoElectronic</td>
			<td>FlashMic</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Igor Pro 6</td>
			<td>Wavemetrics</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

The success of the analytical approach described here critically depends on obtaining high-quality electrophysiological recordings of transporter currents from astrocytes in any region of the central nervous system. In acute mouse hippocampal slices, astrocytes can be readily identified under Dodt illumination or IR-DIC because of their small cell body (&Oslash; = 10 &mu;m) and prominent nucleus (Figure 1). Their distinctive star-shaped morphology can be appreciated with epifluorescence, confocal, or two...

Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

deriving-time-course-glutamate-clearance-with-deconvolution-analysis

Research

JoVE Journal

Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

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.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

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 ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...