对小鼠脑切片中突触连接进行光遗传学图谱绘制来确定已鉴定的神经元群体的功能连接体

Susana Mingote; Nao Chuhma; Stephen Rayport

doi:10.21769/BioProtoc.2090

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Peer-reviewed

Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations

对小鼠脑切片中突触连接进行光遗传学图谱绘制来确定已鉴定的神经元群体的功能连接体

SM Susana Mingote

NC Nao Chuhma

SR Stephen Rayport email

发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2090 浏览次数: 10542

评审: Geoff LauPascal Fossat Ehsan Kheradpezhouh

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Dec 2015

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Abstract

Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DAT^IREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.

Keywords: Channelrhodopsin (通道视紫红质)

Dopamine (多巴胺)

Cotransmission (共同传递)

Patch-clamp (膜片钳)

Immunofluorescence (免疫荧光)

Adeno associated virus (腺相关病毒)

Background

To establish the function of specific neural circuits it is necessary to determine the anatomical connectome, the mapping of anatomical connections, and its functional connectome, the mapping of the strength, incidence and neurotransmitter nature of connections. The use of viral transsynaptic tracing techniques that are monosynaptically restricted, allows for the description of complex anatomical connections of neural circuits, including the dopamine system (Callaway and Luo, 2015; Faget et al., 2016). The functional connectivity of these circuits has been harder to determine due to the intermingling of axons that make selective electrical stimulation impossible. With the advent of optogenetics it became possible to stimulate genetically defined populations of cells selectively. This allowed for the identification of new connections made by striatal medium spiny neurons (Chuhma et al., 2011), ventral midbrain glutamate neurons (Hnasko et al., 2012; Root et al., 2014) and dopamine/glutamate neurons (Mingote et al., 2015). Moreover, optogenetics used in a systematic and comprehensive manner to map the incidence and strength of connections of specific neuronal populations to defined postsynaptic target regions, determines the functional connectome of defined neuronal populations (Chuhma et al., 2011; Mingote et al., 2015). In this protocol, we describe how to determine the functional connectome of any genetically defined neuronal population. As an example, we focus on the excitatory functional connectome of dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015).

Part I: Inducing channelrhodopsin expression in dopamine neurons by viral transfection

Materials and Reagents

Glass PCR micropipettes with 1 μl marks (Drummond Scientific, catalog number: 5-000-1001-X10 )
Kimwipe
Syringe
Q-tip
Surgical blades No.11 (Thomas Scientific, catalog number: 3883B59 )
Needle
Mice (THE JACKSON LABORATORY, Strain 006660: DAT^IREScre )
Note: This knock-in mouse expresses cre recombinase under the transcriptional control of the endogenous dopamine transporter (DAT) promoter. To minimize the interference with the DAT promoter function, cre recombinase expression is driven from the 3’ untranslated region via an internal ribosomal entry sequence (IRES).
AAV5-EF1α-DIO-hChR2(H134R)-EYFP (titer: 8x10e-12 virions/ml) (Addgene, catalog number: 20298 ) is used to drive cre-dependent expression of ChR2-EYFP. The adeno-associated virus (AAV) can be obtained under a MTA from Dr. Karl Deisseroth from the vector core at the University of North Carolina; this AAV is serotype 5 and replication-incompetent.
Paraffin
10% bleach solution
Carprofen (Rimadyl, Zoetis)
Ketamine HCl (KetaVed, Vedco)
Xylazine (Akorn, AnaSed^®)
Puralube VET ointment, sterile ocular lubricant (Dechra)
Lidocaine HCl (40 mg/ml) for local anesthesia (Boehringer Ingelheim)
Vetbond, n-butyl cyanoacrylate adhesive (3M)
70% ethanol solution
Betadine
Neosporin
Saline

Equipment

Pipette puller (Sutter Instruments, model: P97 )
Nalgene^TM 280 polyurethane tubing (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 8030-0060 )
Nalgene^TM 180 clear plastic PVC tubing (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 8000-0004 )
Custom valve controller that delivers momentary pulses of 24 V with 5 watts power (modified pulse controller obtained from General Valve Corporation, model: 9-82-902 )
Note: This product has been discontinued. As an alternative, use UltraMicroPumP III (World Precision Instruments, model: UMP3 ) with a TAXIC900 stereotaxic frame, which allows for a precise control of the rate of delivery of the virus.
Air pressure regulator with gauge (General Cryogenics & Specialty Gas Co.)
Hair clipper (e.g., Wahl Clipper, model: 09916-4301 )
Heating pad, water-circulating
Mouse stereotaxic apparatus (Stoeling, catalog number: 51730D )
Solenoid valve (e.g., Parker Hannifin, part number: 001-0028-900 )
Scalpel handle (Thomas Scientific, catalog number: 3883H10 )
Camera (e.g., Microscope, model: 5MP , catalog number: AM7115MZT)
Video monitor (e.g., Acer)
Drill (Black & Decker, model: RTX )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Mingote, S., Chuhma, N. and Rayport, S. (2017). Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations. Bio-protocol 7(1): e2090. DOI: 10.21769/BioProtoc.2090.
Mingote, S., Chuhma, N., Kusnoor, S. V., Field, B., Deutch, A. Y. and Rayport, S. (2015). Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J Neurosci 35(49): 16259-16271.