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In Vivo Local Dye Electroporation for Ca2+ Imaging and Neuronal-Circuit Tracing Shin Nagayama, Max L. Fletcher, Wenhui Xiong, Xiaohua Lu, Shaoqun Zeng and Wei R. Chen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot083501 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Calcium Imaging (81 articles) Fluorescence (416 articles) Fluorescence, general (284 articles) Image Analysis (106 articles) Imaging for Neuroscience (274 articles) Imaging/Microscopy, general (542 articles) In Vivo Imaging (277 articles) In Vivo Imaging, general (141 articles) Mouse (302 articles) Multi-Photon Microscopy (86 articles)
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Protocol
In Vivo Local Dye Electroporation for Ca2+ Imaging and Neuronal-Circuit Tracing Shin Nagayama, Max L. Fletcher, Wenhui Xiong, Xiaohua Lu, Shaoqun Zeng, and Wei R. Chen
A major technical challenge for using optical imaging to analyze neuronal circuit functions is how to effectively load synthetic Ca2+ dyes or neural tracers into the brain. We introduce here a simple but versatile approach to label many neurons and clearly visualize their axonal and dendritic morphology. The method uses a large-tip patch pipette filled with dextran-conjugated Ca2+ dyes or fluorescent tracers. By inserting the pipette into a targeted brain area and passing microampere current pulses, dyes or tracers are electroporated into dendrites and axons near the pipette tip. The dyes are then transported to reveal the entire cell morphology, suitable for both functional Ca2+ imaging and neuronal circuit tracing. This process requires basic physiological equipment normally available in a physiological laboratory.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (1.5%) dissolved in Ringer solution Anaesthesia: urethane (1.2 g/kg body weight) or nembutal (50 mg/kg body weight) Labeling reagents (see Step 4) Dextran-conjugated Alexa Fluor 488 or 594 dyes (10,000 MW) for long-range circuit tracing Dextran-conjugated calcium indicators (dextran-conjugated Oregon Green 488 BAPTA-1 [10,000 MW] and dextran-conjugated Calcium Green-1 [3,000 MW or 10,000 MW]) Mouse to be used for in vivo dye electroporation Ringer solution, Ca2+-free Equipment
Current delivery circuit consisting of an electric stimulator connected to an isolator set at currentoutput mode (Master-8 and Iso-Flex, A.M.P.I.) Dental drill Electrode puller (Sutter P-97) Glass pipette Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083501
940
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Local Dye Electroporation
Heating pad Imaging setup In vivo two-photon imaging can be performed either with a custom-built system or with a commercially available system. We use a mode-locked laser operated at a 100-fsec pulse width, 80-MHz pulse-repeating frequency, and 810–830 nm wavelength (Tsunami and Millennia Xs, Spectra Physics).
Metal clip to ground mouse Micromanipulator Oscilloscope
METHOD
1. Anesthetize the mouse using intraperitoneal (i.p.) injection of either urethane or Nembutal. Mount the animal in a stereotaxic frame and keep it on a heating pad to maintain body temperature throughout the entire procedure. 2. Thin the skull with a dental drill and then open a small hole of 100 µm diameter (without removing the dura) for inserting a dye-loading glass pipette. A small hole is critical for reducing brain pulsation, which affects the image quality significantly. In some experiments involving a large craniotomy, 1.5% agarose dissolved in Ringer solution can be applied to reduce brain pulsation.
3. Fabricate a glass pipette using a Sutter P-97 electrode puller to make a tip size of 2.5–4 µm inner diameter. 4. Dissolve 10% (mass/volume) of dextran-conjugated Ca2+ indicators or fluorescent tracers in Ca2+-free Ringer solution. Backfill the dye-loading pipette with the dye solution. 5. Insert the pipette into a targeted brain region using a micromanipulator. 6. Carry out local electroporation by applying positive current pulses to the dye-filled glass pipette. See Figure 1A for the current delivery circuit setup; the current delivery circuit consists of an electric stimulator connected to an isolator set at current-output mode (Master-8 and Iso-Flex, A. M.P.I.). Set the current-pulse amplitude at 3–5 µA (Nagayama et al. 2007). The frequency and total number of electroporation pulses are usually 2 Hz and 1200 pulses, respectively. A pulse width of 25 msec is found to be the minimal pulse duration that can maintain loading efficiency comparable with that of longer pulses, although it can reduce background fluorescence at the electroporation site.
7. Monitor the actual waveform and amplitude of the injected current pulses on an oscilloscope by measuring the voltage drop across a 100-kΩ series resistor in the circuit. 8. Following the termination of dye electroporation, allow a suitable waiting period—usually 2 h— for the clearing of background fluorescence so that local neuronal circuits are well labeled for proper imaging. The brain, if alive and healthy, can remove excess dyes very efficiently. However, if there are concerns about temporary tissue damage caused by electroporation, a longer waiting time is recommended. One useful variant of the method is to use local electroporation of dextran-conjugated fluorescent tracers to analyze the fine organization of long-range projection circuits. In such a case, a much longer waiting period is required. We usually wait for 3 d for dextran dyes to be transported from the olfactory bulb glomerulus all the way to the piriform cortex, which covers a distance of 10 mm.
9. Carry out imaging. To reduce tissue photodamage, the laser power delivered into the brain should be
In Vivo Local Dye Electroporation for Ca2+ Imaging and Neuronal-Circuit Tracing Shin Nagayama, Max L. Fletcher, Wenhui Xiong, Xiaohua Lu, Shaoqun Zeng and Wei R. Chen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot083501 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Calcium Imaging (81 articles) Fluorescence (416 articles) Fluorescence, general (284 articles) Image Analysis (106 articles) Imaging for Neuroscience (274 articles) Imaging/Microscopy, general (542 articles) In Vivo Imaging (277 articles) In Vivo Imaging, general (141 articles) Mouse (302 articles) Multi-Photon Microscopy (86 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
In Vivo Local Dye Electroporation for Ca2+ Imaging and Neuronal-Circuit Tracing Shin Nagayama, Max L. Fletcher, Wenhui Xiong, Xiaohua Lu, Shaoqun Zeng, and Wei R. Chen
A major technical challenge for using optical imaging to analyze neuronal circuit functions is how to effectively load synthetic Ca2+ dyes or neural tracers into the brain. We introduce here a simple but versatile approach to label many neurons and clearly visualize their axonal and dendritic morphology. The method uses a large-tip patch pipette filled with dextran-conjugated Ca2+ dyes or fluorescent tracers. By inserting the pipette into a targeted brain area and passing microampere current pulses, dyes or tracers are electroporated into dendrites and axons near the pipette tip. The dyes are then transported to reveal the entire cell morphology, suitable for both functional Ca2+ imaging and neuronal circuit tracing. This process requires basic physiological equipment normally available in a physiological laboratory.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (1.5%) dissolved in Ringer solution Anaesthesia: urethane (1.2 g/kg body weight) or nembutal (50 mg/kg body weight) Labeling reagents (see Step 4) Dextran-conjugated Alexa Fluor 488 or 594 dyes (10,000 MW) for long-range circuit tracing Dextran-conjugated calcium indicators (dextran-conjugated Oregon Green 488 BAPTA-1 [10,000 MW] and dextran-conjugated Calcium Green-1 [3,000 MW or 10,000 MW]) Mouse to be used for in vivo dye electroporation Ringer solution, Ca2+-free Equipment
Current delivery circuit consisting of an electric stimulator connected to an isolator set at currentoutput mode (Master-8 and Iso-Flex, A.M.P.I.) Dental drill Electrode puller (Sutter P-97) Glass pipette Adapted from Imaging in Neuroscience (ed. Helmchen and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot083501
940
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Local Dye Electroporation
Heating pad Imaging setup In vivo two-photon imaging can be performed either with a custom-built system or with a commercially available system. We use a mode-locked laser operated at a 100-fsec pulse width, 80-MHz pulse-repeating frequency, and 810–830 nm wavelength (Tsunami and Millennia Xs, Spectra Physics).
Metal clip to ground mouse Micromanipulator Oscilloscope
METHOD
1. Anesthetize the mouse using intraperitoneal (i.p.) injection of either urethane or Nembutal. Mount the animal in a stereotaxic frame and keep it on a heating pad to maintain body temperature throughout the entire procedure. 2. Thin the skull with a dental drill and then open a small hole of 100 µm diameter (without removing the dura) for inserting a dye-loading glass pipette. A small hole is critical for reducing brain pulsation, which affects the image quality significantly. In some experiments involving a large craniotomy, 1.5% agarose dissolved in Ringer solution can be applied to reduce brain pulsation.
3. Fabricate a glass pipette using a Sutter P-97 electrode puller to make a tip size of 2.5–4 µm inner diameter. 4. Dissolve 10% (mass/volume) of dextran-conjugated Ca2+ indicators or fluorescent tracers in Ca2+-free Ringer solution. Backfill the dye-loading pipette with the dye solution. 5. Insert the pipette into a targeted brain region using a micromanipulator. 6. Carry out local electroporation by applying positive current pulses to the dye-filled glass pipette. See Figure 1A for the current delivery circuit setup; the current delivery circuit consists of an electric stimulator connected to an isolator set at current-output mode (Master-8 and Iso-Flex, A. M.P.I.). Set the current-pulse amplitude at 3–5 µA (Nagayama et al. 2007). The frequency and total number of electroporation pulses are usually 2 Hz and 1200 pulses, respectively. A pulse width of 25 msec is found to be the minimal pulse duration that can maintain loading efficiency comparable with that of longer pulses, although it can reduce background fluorescence at the electroporation site.
7. Monitor the actual waveform and amplitude of the injected current pulses on an oscilloscope by measuring the voltage drop across a 100-kΩ series resistor in the circuit. 8. Following the termination of dye electroporation, allow a suitable waiting period—usually 2 h— for the clearing of background fluorescence so that local neuronal circuits are well labeled for proper imaging. The brain, if alive and healthy, can remove excess dyes very efficiently. However, if there are concerns about temporary tissue damage caused by electroporation, a longer waiting time is recommended. One useful variant of the method is to use local electroporation of dextran-conjugated fluorescent tracers to analyze the fine organization of long-range projection circuits. In such a case, a much longer waiting period is required. We usually wait for 3 d for dextran dyes to be transported from the olfactory bulb glomerulus all the way to the piriform cortex, which covers a distance of 10 mm.
9. Carry out imaging. To reduce tissue photodamage, the laser power delivered into the brain should be
