We thank Tran Huynh, Andrew San Antonio, Jerry Lin for their technical assistance. This work was funded by the National Institutes of Health grants DA023700 and DA023700-04S1 to X.X.

1. Brain slice preparation
<ol>
 <li>Transgenic mice are deeply anesthetized with pentobarbital sodium (&gt;100 mg/kg, ip) and rapidly decapitated, and their brains extracted into a frozen and oxygenated cutting solution.</li>
 <li>GFP goggles are used to visually screen if the mouse brain indeed expresses GFP.</li>
 <li>Primary somatosensory cortical sections of 400 &mu;m thick are cut with a vibratome in sucrose-containing artificial cerebrospinal fluid (ACSF). Slices are first incubated in sucrose-containing ACSF for 30 min to 1 h at 32&deg;C, and after the initial incubation period, transferred to recording ACSF at room temperature. Throughout the incubation and recording, the slices are continuously bubbled with 95% O2-5% CO2.</li>
</ol>
2. Equipment startup
The overall system implementation is illustrated in Figure 1.
<ol>
 <li>The laser cooling system and power supplier is turned on, getting the laser ready.</li>
 <li>All hardware related to photostimulation control (including the scanning mirror system, an optical modulator, an electronic shutter and a photodiode input amplifier) is turned on.</li>
 <li>The electrophysiological equipment (including a Multiclamp 700B amplifier and micromanipulators) is turned on. Electrophysiological recordings, photostimulation, and imaging of the slice preparations are done in a slice perfusion chamber mounted on the motorized stage of the microscope.</li>
 <li>The imaging camera (Retiga 2000, Q-imaging, Austin, TX) is turned on. Slices are visualized with the upright microscope (BW51X, Olympus) with infrared differential interference contrast (DIC) and epi- fluorescent optics through the imaging system.</li>
 <li>The Matlab-based EPHUS software and Q-capture camera software are started up. A custom-modified version of Ephus software (Ephus, available at <a href="https://openwiki.janelia.org/">https://openwiki.janelia.org/</a>) is used to control photostimulation protocols and acquire photostimulation data.</li>
 <li>After the system is on, one should check to see if the laser photostimulation system is at a working state. As the 4x microscope objective is used for delivering the UV flashes for glutamate uncaging, a piece of white paper can be put under to observe the laser scanning pattern while the system runs.</li>
</ol>
3. Setting up slice perfusion
<ol>
 <li>A pressurized perfusion system is turned on to feed the recording ACSF into the slice chamber. Care is taken to ensure a constant fluid level of 2.0 mm above the slice in the chamber.</li>
 <li>An aliquot of the stock solution of MNI-caged-glutamate is added to 25 ml of circulating ACSF for a concentration of 0.2 mM caged glutamate. Please note that after 5-6 h of experimentation, the bath solution and MNI-glutamate will be refreshed.</li>
 <li>A brain slice is moved into the recording chamber. One can run the imaging system to check the slice quality and anatomically locate the primary somatosensory area. Then the slice is anchored with a custom-made stringed platinum ring. Be careful not to put the anchor over the brain region intended for recordings.</li>
</ol>
4. Whole cell patch-clamping recording
<ol>
 <li>Glass electrodes (4-6 M&Omega; resistance) are pulled and filled with an internal solution containing 0.1% biocytin for cell labeling and morphological identification.</li>
 <li>To perform patch recording, cells are visualized at the 60x objective. Inhibitory cell types are first identified and selected based on visualization of GFP expression under a DIC/fluorescent Olympus microscope; recordings are subsequently performed under visual control aided by infrared DIC video monitoring. Please note that a few times of switching between the DIC and fluorescent modes is necessary to confirm the GFP cell location while approaching the electrode to the target cell.</li>
 <li>Conventional patch-clamping techniques are applied. The electrode is filled with positive pressure, moved close to the cell surface to establish a discernible dimple upon the targeted cell membrane; and then negative pressure is quickly applied to form a gigaseal and break in while monitoring the current injection responses on the video monitor.</li>
 <li>Upon breaking in, the images of the cell at 60x under the DIC and fluorescent modes are taken for the on-line verification. Before collection of photostimulation data, hyperpolarizing and depolarizing current pulses are injected to examine each cell's basic electrophysiological properties.</li>
 <li>Once stable whole cell recordings are achieved with good access resistance (usually &lt;20 M&Omega;), the microscope objective is switched from 60x to 4x for laser scanning photostimulation. The slice image at 4x is acquired and will be used for guiding and registering photostimulation sites.</li>
</ol>
5. Laser scanning photostimulation
<ol>
 <li>The photostimulation and data acquisition parameters are set by activating the configuration switch module of Ephus. Normally, 1 ms laser (20 mW) with a stimulus interval of 1 second is used for photostimulation mapping. Data traces of 1 second sampled at 10 kHz are acquired.</li>
 <li>The 4x slice image is loaded into the mapper module of Ephus. The location of cell soma is defined, and photostimulation sites of a 16x16 pattern (80 &mu;m x 80 &mu;m spacing) are set up around the cell location, covering all the cortical layers.</li>
 <li>The excitation profile of the recorded neuron is mapped by examining the spiking locations in response to photostimulation at the current clamp mode. Our user-friendly software with the on-line display features facilitates the mapping experiments.</li>
 <li>Local excitatory circuit connections are mapped by detection of excitatory postsynaptic currents (EPSC) from the recorded cell while laser scanning at different locations. The cell is held at -70 mV in voltage clamp mode to detect inward excitatory currents. The EPSC maps are repeated 2-3 times with the photostimulation pattern rotated or flipped.</li>
 <li>Optional: Local inhibitory circuit connections can also mapped by detection of inhibitory postsynaptic currents (IPSC) from the recorded cell held at close to 0 mV in voltage clamp mode to detect outward inhibitory currents. Note that it is best to use the electrode internal solution with potassium replaced with cesium for IPSC mapping.</li>
 <li>After all physiological assays are completed, the electrode is gently removed from the recorded cell. The brain slice is taken out and fixed in 4% paraformaldehyde overnight. The recorded cells is stained against biocytin. Cell morphology is examined with confocal or epi- fluorescent microscopy, which also confirms that each recorded cell is indeed the GFP-expressing interneuron originally targeted.</li>
</ol>
6. Photostimulation data analysis
Our newly developed methodology and software implementation11 is applied to the detection and measurement of photostimulation-evoked synaptic events in the raw data maps. As exemplified in Figure 2, color-coded maps are constructed to illustrate the pattern of synaptic input to the recorded neuron.
7. Representative Results:
Example results of electrophysiological recording and photostimulation mapping are shown in Figure 2. Targeted recordings of specific inhibitory cell types are made possible by using transgenic mice expressing GFP in known inhibitory cell types. Given the diversity of inhibitory interneurons, analyses of GFP expression, intrinsic electrophysiology and morphological characteristics (Figure 2A) are combined to arrive at the final cell type classification. Figure 2B-C illustrate that laser scanning photostimulation allows for extensive mapping of local cortical circuit connections to single inhibitory neurons. The raw data map is shown in Figure 2C. The direct uncaging responses are excluded from data analysis (Figure 2C). The quantitative input maps are color-coded, shown in Figure 2D-F. The example fast-spiking inhibitory interneurons received strong excitatory synaptic input (EPSCs) from layer 4 and deeper layers. By relating the cell's synaptic input organization to defined cortical pathways, we are able to infer its possible role (e.g., feedback and feedforward inhibition) in cortical information processing.
<img alt="Figure 1" src="/files/ftp_upload/3109/3109fig1.jpg" /> 
Figure 1. General system instrumentation for laser scanning photostimulation. Our overall system consists of photostimulation control, video imaging, and electrophysiological recording systems. We adopted the design of the LSPS system described previously7,17. A laser unit is used to generate 355 nm UV laser for glutamate uncaging. The laser beam is directed through the optical path of our system. Short durations of laser flashes (e.g., 1- 3 ms) are controlled by using an electro-optical modulator (i.e., pockels cell) and a mechanical shutter. Laser beam power is modulated by a neutral density gradient wheel and monitored by diverting a small fraction of the laser beam with a glass coverslip to a photodiode. The laser scanning system includes an X-Y pair of scan mirrors, the scan lens, the tube lens, and the objective lens. The mirrors deliver the laser beam through a scan lens; then the beam enters the microscope via a dichroic mirror and is focused by a custom-made UV-transmitting tube lens. The beam underfills the back aperture of the microscope objective to provide a more columnar (as opposed to conical) illuminating beam, keeping the mapping as two-dimensional as possible by reducing the axial resolution. Under the 4x objective, the laser beam forms uncaging spots, each approximating a Gaussian profile with a width of 153 &mu;m laterally at the focal plane (see the insert). Various laser stimulation positions can be achieved through galvanometers-driven XY scanning mirrors, as the mirrors and the back aperture of the objective are in conjugate planes, translating mirror positions into different scanning locations at the objective lens focal plane. During uncaging, a variable number of patterned sites that covers the whole field are stimulated sequentially in a nonraster, nonrandom sequence to avoid revisiting the vicinity of recently stimulated sites. Please refer to Xu et al. (2010) for more detailed information.
<img alt="Figure 2" src="/files/ftp_upload/3109/3109fig2.jpg" /> 
Figure 2. Mapping local excitatory circuit connections to a GFP-expressing fast-spiking interneuron in layer 4 of mouse primary sensory cortex. A1 and A2 show the DIC and GFP fluorescent images of the targeted cell under the 60x objective in the living brain slice, while A3 shows the cell morphology revealed by biocytin staining in the post-recording, fixed slice. A4 shows the firing patterns of the recorded GFP cell, characteristic of a fast-spiking inhibitory interneuron, in response to intrasomatic current injections of different strengths. B show the slice image superimposed with the 16 x16 photostimulation sites (*). The cell location is indicated by the small red circle. C shows an array of photostimulation-evoked response traces from the stimulation locations shown in B, while the cell was held at -70 mV to detect inward excitatory currents. Different forms of photostimulation responses are illustrated by the traces at locations of 1 and 2, which are expanded and separately shown in the insert. The trace 1 is an example of the direct responses (indicated by the red arrowheads) to glutamate uncaging on the cell body and promximal dendrites. Other traces in 2 are typical examples of excitatory synaptic input responses (blue). Direct responses and synaptic inputs can be distinguished by their amplitudes and response latencies. D, E and F are the color-coded maps (16x16 sites) of average EPSC amplitude, the EPSC numbers, and the first detected EPSC latency per site, respectively, for the raw data map shown in C. The average input amplitude from each stimulation site is the mean amplitude of EPSCs in the analysis window, with the baseline spontaneous response subtracted from the photostimulation response of the same site. The number of EPSCs and the arrival time or latency of the first detected EPSC per site are also measured and plotted. Please refer to (Shi et al., 2010) for more details.

<ol>
	<li>Ascoli, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Petilla+terminology:+nomenclature+of+features+of+GABAergic+interneurons+of+the+cerebral+cortex.">Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.</a> Nature. 9, 557-568 (2008).</li><li>Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nature. 5, 793-807 (2004).</li><li>Xu, X., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+specificity+of+functional+input+to+distinct+types+of+inhibitory+cortical+neurons.">Laminar specificity of functional input to distinct types of inhibitory cortical neurons.</a> J Neurosci. 29, 70-85 (2009).</li><li>Xu, X., Roby, K. D., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+characterization+of+inhibitory+mouse+cortical+neurons:+three+chemically+distinct+classes+of+inhibitory+cells.">Immunochemical characterization of inhibitory mouse cortical neurons: three chemically distinct classes of inhibitory cells.</a> J Comp Neurol. 518, 389-404 (2010).</li><li>Dantzker, J. L., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+sources+of+synaptic+input+to+cortical+inhibitory+interneurons+and+pyramidal+neurons.">Laminar sources of synaptic input to cortical inhibitory interneurons and pyramidal neurons.</a> Nat Neurosci. 3, 701-707 (2000).</li><li>Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine-scale+specificity+of+cortical+networks+depends+on+inhibitory+cell+type+and+connectivity.">Fine-scale specificity of cortical networks depends on inhibitory cell type and connectivity.</a> Nat Neurosci. 8, 1552-1559 (2005).</li><li>Shepherd, G. M., Pologruto, T. A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit+analysis+of+experience-dependent+plasticity+in+the+developing+rat+barrel+cortex.">Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex.</a> Neuron. 38, 277-289 (2003).</li><li>Weiler, N., Wood, L., Yu, J., Solla, S. A., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+laminar+organization+of+the+excitatory+network+in+motor+cortex.">Top-down laminar organization of the excitatory network in motor cortex.</a> Nat Neurosci. 11, 360-366 (2008).</li><li>Xu, X., Olivas, N. D., Levi, R., Ikrar, T., Nenadic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+precision+and+fast+functional+mapping+of+cortical+circuitry+through+a+combination+of+voltage+sensitive+dye+imaging+and+laser+scanning+photostimulation.">High precision and fast functional mapping of cortical circuitry through a combination of voltage sensitive dye imaging and laser scanning photostimulation.</a> J Neurophysiol. 103, 2301-2312 (2010).</li><li>Xu, X., Roby, K. D., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cortical+inhibitory+neuron+type+that+coexpresses+somatostatin+and+calretinin.">Mouse cortical inhibitory neuron type that coexpresses somatostatin and calretinin.</a> J Comp Neurol. 499, 144-160 (2006).</li><li>Shi, Y., Nenadic, Z., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+of+matched+filtering+for+synaptic+event+detection+and+extraction.">Novel use of matched filtering for synaptic event detection and extraction.</a> PLoS ONE. 5, e15517-e15517 (2010).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nat Neurosci. 8, 1263-1268 (2005).</li><li>Kuhlman, S. J., Huang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+labeling+and+functional+manipulation+of+specific+neuron+types+in+mouse+brain+by+Cre-activated+viral+gene+expression.">High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.</a> PLoS ONE. 3, e2005-e2005 (2008).</li><li>Petreanu, L., Huber, D., Sobczyk, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2-assisted+circuit+mapping+of+long-range+callosal+projections.">Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.</a> Nat Neurosci. 10, 663-668 (2007).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457, 1142-1145 (2009).</li><li>Cardin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Driving+fast-spiking+cells+induces+gamma+rhythm+and+controls+sensory+responses.">Driving fast-spiking cells induces gamma rhythm and controls sensory responses.</a> Nature. 459, 663-667 (2009).</li><li>Shepherd, G. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+and+columnar+organization+of+ascending+excitatory+projections+to+layer+2/3+pyramidal+neurons+in+rat+barrel+cortex.">Laminar and columnar organization of ascending excitatory projections to layer 2/3 pyramidal neurons in rat barrel cortex.</a> J Neurosci. 25, 5670-5679 (2005).</li></ol>

No conflicts of interest declared.

Photostimulation-based mapping techniques have been effectively applied for analyzing cortical circuits. Laser scanning photostimulation combined with whole cell recording allows high resolution mapping of laminar distributions of presynaptic input sources to single neurons, because the simultaneous recording from a postsynaptic neuron with photostimulation of clusters of presynaptic neurons at many different locations provides quantitative measures of spatial distribution of excitatory or inhibitory inputs. This tech...

<table border="1"> <tbody> <tr> <td>Name of the reagent</td> <td>Company</td> <td>Catalogue number</td> <td>Comments</td> </tr> <tr> <td>transgenic mouse lines</td> <td>Jackson lab or other sources</td> <td></td> <td>Please refer to Xu and Callaway (2009)</td> </tr> <tr> <td>GFP goggles</td> <td>BLS Ltd., Hungary</td> <td></td> <td></td> </tr> <tr> <td>vibratome</td> <td>Leica Systems</td> <td>VT1200S</td> <td></td> </tr> <tr> <td>MNI caged glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate)</td> <td>Tocris Bioscience, Ellisville, MO</td> <td>Cat No. 1490</td> <td></td> </tr> <tr> <td>biocytin</td> <td></td> <td>B4261</td> <td></td> </tr> <tr> <td>electrode puller</td> <td>Sutter Instrument, Novato, CA</td> <td>P-97</td> <td></td> </tr> <tr> <td>glass tubes for making electrodes</td> <td></td> <td>BF150-86-10</td> <td></td> </tr> <tr> <td>Multiclamp 700B amplifier</td> <td>Molecular Devices, Sunnyvale, CA</td> <td>Multiclamp 700B</td> <td></td> </tr> <tr> <td>digital CCD camera</td> <td>Q-imaging, Austin, TX</td> <td>Retiga 2000</td> <td></td> </tr> <tr> <td>Research microscope</td> <td>Olympus, Tokyo, Japan</td> <td>BW51X</td> <td></td> </tr> <tr> <td>UV laser unit</td> <td>DPSS Lasers, Santa Clara, CA</td> <td>model 3501</td> <td></td> </tr> <tr> <td>Other equipment for Laser scanning phostimulation</td> <td></td> <td></td> <td>Please refer to Xu et al. (2010)</td> </tr> </tbody> </table> Solutions: <ul> <li>Sucrose-containing artificial cerebrospinal fluid (ACSF) for slice cutting (in mM: 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH2PO4, 4 MgCl2, 0.5 CaCl2, and 24 NaHCO3).</li> <li>Recording ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose)</li> <li>Electrode internal solution (in mM: 126 K-gluconate, 4 KCl, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na, and 10 phosphocreatine; pH 7.2, 300 mOsm).</li> </ul>

mapping inhibitory neuronal circuits by laser scanning photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

Watch this Scientific Journal Video about Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation at JoVE.com

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

mapping-inhibitory-neuronal-circuits-laser-scanning

Research

JoVE Journal

Neuroscience

17.2K ビュー.  University of California, Irvine. 本稿では、限られた抑制性ニューロン集団でGFPを発現するトランスジェニックマウスにおける細胞全体の録音でレーザ走査光刺激を組み合わせたアプローチを紹介します。手法は特異的な阻害皮質ニューロンの局所シナプス回路の広範なマッピングと定量分析が可能になります。

ビデオ: Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

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 ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.