Name: Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents (Video) | JoVE Journal
Uploaded: 2013-08-07T13:00:00.000+00:00
Duration: 9 min 42 s
Description: Learn deconvolution analysis to estimate glutamate clearance time from astrocytic membranes using electrophysiological recordings in mouse hippocampal slices. This method reveals rapid astrocytic uptake dynamics.

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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.

<ol><li>Ventura, R., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+relationships+between+hippocampal+synapses+and+astrocytes%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Plasticity+of+perisynaptic+astroglia+during+synaptogenesis+in+the+mature+rat+hippocampus%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Glutamate+uptake%5BTitle%5D)+AND+%22Prog.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Extracellular+glutamate+concentration+in+hippocampal+slice%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Cooperation+between+independent+hippocampal+synapses+is+controlled+by+glutamate+uptake%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((An+evaluation+of+synapse+independence%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Extrasynaptic+glutamate+diffusion+in+the+hippocampus%3A+ultrastructural+constraints%2C+uptake%2C+and+receptor+activation%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Flux+coupling+in+a+neuronal+glutamate+transporter%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Retinal+glial+cell+glutamate+transporter+is+coupled+to+an+anionic+conductance%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Ion+fluxes+associated+with+excitatory+amino+acid+transport%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Macroscopic+and+microscopic+properties+of+a+cloned+glutamate+transporter%2Fchloride+channel%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Synaptic+activation+of+glutamate+transporters+in+hippocampal+astrocytes%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes%3A+transmitter+uptake+gets+faster+during+development%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Neuronal+transporters+regulate+glutamate+clearance%2C+NMDA+receptor+activation%2C+and+synaptic+plasticity+in+the+hippocampus%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Isoflurane+enhances+glutamate+uptake+via+glutamate+transporters+in+rat+glial+cells%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">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/pubmed?term=((Electrogenic+uptake+of+glutamate+and+aspartate+into+glial+cells+isolated+from+the+salamander+%28Ambystoma%29+retina%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+coupled+and+uncoupled+currents+during+glutamate+uptake+by+GLT-1+transporters%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Synaptically+released+glutamate+does+not+overwhelm+transporters+on+hippocampal+astrocytes+during+high-frequency+stimulation%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Neuronal+activity+regulates+glutamate+transporter+dynamics+in+developing+astrocytes%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Optical+measurement+of+synaptic+glutamate+spillover+and+reuptake+by+linker+optimized+glutamate-sensitive+fluorescent+reporters%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Amyloid%CE%B21-42+slows+clearance+of+synaptically-released+glutamate+by+mislocalizing+astrocytic+GLT-1%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Amyloidβ1-42 slows clearance of synaptically-released glutamate by mislocalizing astrocytic GLT-1.</a> J. Neurosci. 33, 5312-5318 (2013).</li>
</ol>

The authors declare no conflict of interest.

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.

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

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 (Ø = 10 μm) and prominent nucleus (Figure 1). Their distinctive star-shaped morphology can be appreciated with epifluorescence, confocal, or two-photon la...

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 ...

The overall goal of the experiment is to estimate the time course of glutamate clearance from astrocytic membranes following its release from excitatory synapses. The first step in this process is to record synaptically activated glutamate transport occurrence from astrocytes in acute brain slices in the absence and presence of a subs saturating concentration of the broad spectrum glutamate transporter antagonist, TBOA. The second step is to analyze the recordings to isolate the synaptically activated glutamate transporter currents from all other components of the evoked response.Next, the filter time course is estimated by analyzing the transporter currents recorded in the presence of TBOA by de involving the filter from the transport occurrence recorded in controlled conditions. The time course of glutamate clearance from astrocytic membranes is obtained. The results show that in the mouse hippocampus astrocytes require only a few milliseconds to remove synaptically released glutamate from the extracellular space base.This approach provides a sensitive tool to detect changes in astrocytic uptake capacity in different brain regions during physiological and pathological conditions.

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Learn deconvolution analysis to estimate glutamate clearance time from astrocytic membranes using electrophysiological recordings in mouse hippocampal slices. This method reveals rapid astrocytic uptake dynamics.

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

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