Summary

Сопоставление ингибирующих нейронов схем с Фотостимуляция лазерного сканирования

Published: October 06, 2011
doi:

Summary

Эта статья представляет подход объединения лазерного сканирования фотостимуляции с целой записи ячейки в трансгенных мышей, экспрессирующих GFP в ограниченном ингибирующее популяций нейронов. Методика позволяет для широкого отображения и количественного анализа местных синаптических схемы конкретных ингибирующих нейронов коры.

Abstract

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.

Protocol

1. Подготовка среза мозга Трансгенные мыши, глубоко анестезировали фенобарбиталом натрия (> 100 мг / кг, внутрибрюшинно) и быстро обезглавлены, и их мозг извлекается в замороженном и кислородом резки решение. GFP очки, которые используются для визуального экрана, если мозг мыши …

Discussion

Фотостимуляция на основе методов картирования были эффективно применены для анализа корковых цепей. Лазерное сканирование фотостимуляции в сочетании с все записи ячейки обеспечивает высокое разрешение отображения ламинарного распределения пресинаптических источники входного сиг…

Declarações

The authors have nothing to disclose.

Acknowledgements

Мы благодарим Tran Huynh, Эндрю Сан-Антонио, Джерри Линь за их техническую помощь. Эта работа финансировалась Национальным институтом здравоохранения грантов DA023700 и DA023700-04S1 в XX

Materials

Name of the reagent Company Catalogue number Comments
transgenic mouse lines Jackson lab or other sources Please refer to Xu and Callaway (2009)
GFP goggles BLS Ltd., Hungary
vibratome Leica Systems VT1200S
MNI caged glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate) Tocris Bioscience, Ellisville, MO Cat No. 1490
biocytin B4261
electrode puller Sutter Instrument, Novato, CA P-97
glass tubes for making electrodes BF150-86-10
Multiclamp 700B amplifier Molecular Devices, Sunnyvale, CA Multiclamp 700B
digital CCD camera Q-imaging, Austin, TX Retiga 2000
Research microscope Olympus, Tokyo, Japan BW51X
UV laser unit DPSS Lasers, Santa Clara, CA model 3501
Other equipment for Laser scanning phostimulation Please refer to Xu et al. (2010)

Solutions:

  • 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).
  • Recording ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose)
  • 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).

Referências

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Citar este artigo
Ikrar, T., Olivas, N. D., Shi, Y., Xu, X. Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation. J. Vis. Exp. (56), e3109, doi:10.3791/3109 (2011).

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