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 fo...

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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

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Neuroscience

17.2K 次观看.  University of California, Irvine. 本文介绍了激光扫描photostimulation在表达绿色荧光蛋白在有限的抑制性神经元的人口的转基因小鼠的全细胞记录相结合的方法。该技术可以广泛的测绘和定量分析当地具体的抑制皮层神经元的突触电路。