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In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber Helmuth Adelsberger, Christine Grienberger, Albrecht Stroh and Arthur Konnerth Cold Spring Harb Protoc; doi: 10.1101/pdb.prot084145 Email Alerting Service Subject Categories
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Protocol
In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber Helmuth Adelsberger, Christine Grienberger, Albrecht Stroh, and Arthur Konnerth
We describe here an approach for the fluorometric monitoring of population activity in neurons in live mice combined with the activation of optogenetic actuators in vivo. In this protocol, a thin multimode fiber, which is used for both delivering excitation light and collecting emitted fluorescence signals, is inserted into the skull of a mouse. When combined with multicell bolus loading of Ca2+ indicators, this optical fiber and its associated fluorescence detection system can be used for the in vivo recording of brain Ca2+ signals from a local cluster of coactive neurons. The fiber can also be used for the optogenetic stimulation of light-activated ion channels, such as channelrhodopsin-2, allowing the monitoring of local calcium signals evoked by optogenetic stimulation.
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.
Reagents
Anesthetics appropriate for the species under investigation (see, e.g., Flecknell 2000) For mice, we typically use inhalation anesthesia with 0.8%–1.5% isoflurane in pure O2 or intraperitoneal injection of ketamine/xylazine (0.1/0.01 mg/g body weight). Xylocaine is used for local anesthesia.
Cyanoacrylate glue Eye protection cream (Isopto-Max, Alcon Pharma) Mice for analysis For combined calcium recordings and optogenetic activation, use animals expressing optogenetic actuators (e.g., channelrhodopsin-2 [ChR2]) in the brain area of interest.
Reagents and equipment for multicell bolus loading of the brain tissue with calcium-sensitive dye Oregon Green-488 BAPTA-1 (OGB-1) AM See In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
Equipment
Biomonitoring system for monitoring body temperature, heartbeat, and breathing rates (e.g., AD Instruments) Diamond Wedge scribe (Thorlabs, Inc.) Fiber-stripping tool (Thorlabs, Inc.) 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.prot084145
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In Vivo Recording Through an Optical Fiber
Imaging setup A diagram of the recording and stimulation setup is shown in Figure 1A. The light for excitation of the fluorescent calcium-sensitive dye, such as OGB-1, and activation of the ChR2 is delivered by a 10-mW solid-state laser (Sapphire, Coherent, Inc.) with a wavelength of 488 nm. The rapid control of the intensity of the laser beam is achieved by an acousto-optic modulator (AOM; 3080–125, Crystal Technology). After deflection by a dichroic mirror (Fig. 1A), the beam is focused by means of a collimator into one end of a multimode fiber (Thorlabs, Inc.) with a diameter of ≤200 µm. The emitted fluorescence light is guided back through the same fiber and focused on an avalanche photodiode (APD; S5343, Hamamatsu Photonics) with an aperture of 1 mm. The recorded fluorescence signals are digitized with a sampling frequency of 2000 Hz using a multifunction I/O device USB-6221 (National Instruments) and custom-written LabView-based software. The fluorescence signals were low-pass filtered offline with frequencies of 5–20 Hz.
Materials for the fixation of the optical fiber in the skull Syntac Adhesive Syntac Primer Tetric dental cement (Ivoclar Vivadent) Total Etch (Heraeus Kulzer) UV lamp Optical fiber Typically, multimode fibers with a diameter of 200 µm and a numerical aperture of 0.48 are most commonly used. However, thinner (100-μm) or thicker (500-μm) fibers may be implanted by using the same protocol.
Stereotaxic device with ear bars B
APD
A Focusing lens
Em. filter Shutter Shutter Beam splitter Laser AOM Collimator Optical fiber
C
D Optical fiber Metal tube
Dental cement Skull Cortex
Illuminated volume
Stained volume
200 µm
FIGURE 1. (A) Scheme of the optical fiber recording setup. AOM, acousto-optic modulator; APD, avalanche photodiode; Em, emission. (B) Image showing the tip of an optical fiber (diameter 200 µm) glued into a metal tube. (C ) Illustration of the fiber tip inserted into the brain region with neurons labeled by the multicell bolus loading technique with calcium-sensitive dye (green). The blue cone indicates the volume in which the dye in the loaded neurons is excited by the laser light. (D) The tip of an optical fiber was placed perpendicular to an acute cortical slice in which the neurons were stained with Oregon Green-488 BAPTA (OGB-1) AM. The image was acquired using a charge-coupled device (CCD) camera and shows the neuron-containing fiber-illuminated cortical region.
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Surgical equipment (dental drill, fine scissors, fine forceps, needles) Syringe needle (see Step 12) Warming plate (to keep the animal’s body temperature constant) METHOD Anesthesia
1. Anesthetize the animal either by injection of ketamine/xylazine intraperitoneally or by inhalation of isoflurane in pure O2. When using isoflurane, we recommend the placement of the animal for 10 min in a closed box containing 2% isoflurane before its fixation in the stereotaxic apparatus. The depth of anesthesia needed for surgery is indicated by the loss of the reflexes (e.g., eyelid reflex, pinch withdrawal). At this stage, inhalation of anesthesia with isoflurane concentrations of 0.8%–1.2% is sufficient to keep the mouse at respiration rates of 80–130/min.
Preparation of the Skull
2. Place the anesthetized mouse onto a warming plate (37˚C) and fix the head in a stereotaxic frame with ear bars. 3. Apply sensors for the control of the body temperature and breathing rates. 4. Apply eye protection cream onto the eyes to protect them from drying. 5. Inject 100 µL 2% xylocaine solution subcutaneously at the site of brain surgery. 6. Ten minutes after xylocaine injection, gently remove the skin and muscles above the desired brain region with fine scissors. 7. Dry the skull with warm air. 8. Perform the following steps to ensure stable fixation of the optic fiber: i. Apply a thin film of Total Etch for precisely 15 sec onto the exposed region. ii. Rinse the skull with water and dry with air. iii. Apply Syntac Primer for 15 sec and dry with air again. iv. Apply Syntac Adhesive for 10 sec followed by air drying. 9. Use the dental drill to thin the skull at the site of recording. Create a small opening with a diameter of 0.5 mm by gently using fine forceps and needles. 10. Perform multicell bolus loading of the brain region of choice. See In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
Implantation and Fixation of the Optical Fiber
11. Remove the coating from the end of the fiber with a standard fiber-stripping tool. 12. Prepare a thin metal tube with the appropriate inner diameter and a length of 10 mm from a syringe needle. 13. Pull the metal tube over the fiber and fix it with cyanoacrylate glue. 14. Trim the protruding short end of the optic fiber (1 cm length) with a Diamond Wedge scribe to a length corresponding to the depth of insertion into the brain (Fig. 1B). For example, trim to 200 µm when recording from a layer two-thirds into the mouse cortex. 15. Place the optic fiber transiently into a manipulator and use the site covered with the metal tube for fixation. 16. Thirty minutes after multicell bolus loading of the calcium-sensitive dye (e.g., OGB-1), insert the tip of the optic fiber gently into the brain through the skull opening. The optimal depth corresponds to the site with the highest fluorescence intensity. See Troubleshooting.
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In Vivo Recording Through an Optical Fiber
17. Apply Tetric dental cement around the metal tube onto the skull (Fig. 1C) and harden it with UV light for 20 sec. The fiber can now be released from the micromanipulator. 18. For the recording of calcium signals, increase the laser beam to an intensity such that it is just below the level in which bleaching is detected (typical range is 0.01–0.1 mW at the tip of the optical fiber). See Troubleshooting.
Calcium Recording and ChR2 Activation
19. For combined calcium recordings and optogenetic activation, apply the procedures described in the preceding steps to animals expressing optogenetic actuators (e.g., ChR2) in the brain area of interest (see Fig. 2). ChR2 stimulation can be performed in combination with fluorometric calcium recordings and also without calcium recordings.
20. For combined calcium recording–ChR2 stimulation experiments, increase the excitation light intensity 20–80 times for the period of stimulation. Because of the saturation of the light detected by APD, calcium recordings are not possible during ChR2 stimulation.
TROUBLESHOOTING Problem (Step 16): There is little or no fluorescence after implanting the optical fiber. Solution: Aside from a failure in dye injection (see In Vivo Two-Photon Calcium Imaging Using
Multicell Bolus Loading [Garaschuk and Konnerth 2010]), this problem is mainly caused by A
B
Optical fiber
1% 1 sec
C
D
E
0.5 sec
Layer 5 neurons expressing ChR2
2.3 mW pulse power
% Response
1%
100 90 80 70 60 50 40 30 20 10 0
2
3
5
20
50
300
Pulse duration (msec) 0.03 mW baseline power
FIGURE 2. (A) (Left) Photograph of a mouse brain indicating the site of posthoc sectioning at the level of the visual cortex. Scale bar, 2 mm. (Right) Section through the primary visual cortex indicating the OGB-1 AM-stained region (green in false color) and the position of the optical fiber. (B) Calcium transient detected in the primary visual cortex evoked by a 50-msec light flash applied to the contralateral eye in an anesthetized adult mouse. (C ) Schematic showing the tip of an optic fiber implanted into a stained cortical region above neurons expressing ChR2 (red). (D) Calcium transient evoked by optogenetic stimulation of ChR2-expressing layer 5 neurons. The optical fiber was implanted at a depth of 500 µm in the visual cortex of an adult transgenic mouse. Light pulses of 488 nm wavelength and 50 msec duration, with a power of 2.3 mW, were used for stimulation. (E) Graph depicting, for constant excitation light intensity, the relationship between the duration of the stimulus pulse and the response probability. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084145
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H. Adelsberger et al.
placement of the fiber tip outside of the stained area, especially when recording from deep brain regions. To avoid this problem, take care to insert the staining pipette and the optical fiber exactly at the same position and with the same angle, by optimizing the use of the stereotaxic device. Another reason for low fluorescence levels is bleeding into the tissue after damage of a blood vessel, either with the staining pipette or with the optical fiber. Blood vessel rupture may be partially avoided by lowering the pipette and the fiber very slowly. Problem (Step 18): The traces are too noisy. Solution: This problem is often caused by heartbeat-mediated pulsation of a large blood vessel located
close to the tip of the optical fiber. Minor changes in the depths of the fiber tip may lead to major improvements. Problem (Step 18): There are movement artifacts. Solution: Artifacts can occur in anesthetized animals by strong breathing excursions or, in awake
animals by fast movements. Proper fixation of the fiber in the metal tube and on the skull helps to minimize movement artifacts. DISCUSSION
In many instances (e.g., when recording oscillatory wave activity from simultaneously active cells), useful information can be extracted from monitoring the activity of large neuronal populations. We therefore developed the fluorescence detection system described here (see also Adelsberger et al. 2005). When combined with multicell bolus loading of calcium indicators (Stosiek et al. 2003; In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading [Garaschuk and Konnerth 2010]), it allows the in vivo recording of brain Ca2+ signals from a local cluster of coactive neurons. Such recordings may be performed in any brain region in both anesthetized and nonanesthetized animals. Importantly, the recordings can be readily combined with the stimulation of light-activated ion channels, such as ChR2 (Boyden et al. 2005), allowing the monitoring of the local calcium signals evoked by optogenetic stimulation. The system we describe here includes a thin multimode fiber that is inserted to the skull of the mouse. This optical fiber is used for the detection of fluorescence signals from all stained cells and the surrounding neuropile within the volume illuminated by the excitation light (Fig. 1D). For twophoton imaging recordings, the calcium signals detected by the optical fiber predominantly represent the action potential activity in the stained region (Stosiek et al. 2003; Kerr et al. 2005). From combined two-photon imaging and optical fiber experiments, we estimate that about 20 coactive neurons located near the tip are sufficient for producing a detectable calcium signal. Note that a variant of this approach has been used for the detection of calcium signals from the upper dendrites of layer 5 cortical neurons (Murayama et al. 2007; see also Fiber-Optic Calcium Monitoring of Dendritic Activity In Vivo [Murayama and Larkum 2012]). The original application of this approach was concerned with the monitoring of spontaneous calcium waves in the cortex of awake newborn mice (Adelsberger et al. 2005). An example of a population calcium signal evoked by sensory stimulation in the adult mouse is shown in Figure 2. The optical fiber was implanted in the primary visual cortex (Fig. 2A). Stimulation of the contralateral eye with a white light flash (Fig. 2B) evoked a large calcium transient lasting for 2–3 sec. The transient occurred with a delay of 80 msec, which corresponds closely to the range of the delays observed with electrical recordings (Porciatti et al. 1999). For the illustration of calcium recordings combined with optogenetic stimulation, we used mice expressing ChR2 under the control of the Thy1 promoter in cortical layer 5 neurons (Arenkiel et al. 2007). Figure 2C shows the experimental arrangement of an optical fiber placed in the stained cortical region after multicell bolus loading. The calcium signals were monitored by using low light levels (0.03 mW) from the 488-nm excitation laser. For ChR2 stimulation, the excitation light intensity was increased for 50 msec to a level of 2.3 mW. The resulting calcium transients had time courses and 1078
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In Vivo Recording Through an Optical Fiber
amplitudes that were comparable with those evoked by sensory stimulation. This experimental configuration was extremely robust and allowed many trials of repeated stimulations and recordings. For example, in Fig. 2E we illustrate the result of such a long-lasting experiment, showing that for constant laser intensity, the response probability was proportional to the duration of the stimulus.
ACKNOWLEDGMENTS
This work was supported by the Bundesministerium für Bildung und Forschung (NGFN-2) and the Deutsche Forschungsgemeinschaft (International Graduate School 1373). REFERENCES Adelsberger H, Garaschuk O, Konnerth A. 2005. Cortical calcium waves in resting newborn mice. Nat Neurosci 8: 988–990. Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, Augustine GJ, Ehlers MD, Feng G. 2007. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54: 205–218. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. 2005. Millisecondtimescale, genetically targeted optical control of neural activity. Nat Neurosci 8: 1263–1268. Flecknell P. 2000. Laboratory animal anesthesia. Academic, New York. Garaschuk O, Konnerth A. 2010. In vivo two-photon calcium imaging using multicell bolus loading. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5482.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084145
Kerr JN, Greenberg D, Helmchen F. 2005. Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci 102: 14063–14068. Murayama M, Larkum M. 2012. Fiber-optic calcium monitoring of dendritic activity in vivo. Cold Spring Harb Protoc doi: 10.1101/pdb .prot067835. Murayama M, Perez-Garci E, Lüscher H-R, Larkum ME. 2007. Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J Neurophysiol 98: 1791–1805. Porciatti V, Pizzorusso T, Maffei L. 1999. The visual physiology of the wild type mouse determined with pattern VEPs. Vision Res 39: 3071–3081. Stosiek C, Garaschuk O, Holthoff K, Konnerth A. 2003. In vivo two-photon calcium imaging using multi-cell bolus loading (MCBL). Proc Natl Acad Sci 100: 7319–7324.
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In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber Helmuth Adelsberger, Christine Grienberger, Albrecht Stroh and Arthur Konnerth Cold Spring Harb Protoc; doi: 10.1101/pdb.prot084145 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 (83 articles) Fluorescence (419 articles) Fluorescence, general (287 articles) Imaging for Neuroscience (275 articles) Imaging/Microscopy, general (543 articles) In Vivo Imaging (280 articles) In Vivo Imaging, general (144 articles) Mouse (307 articles)
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Protocol
In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber Helmuth Adelsberger, Christine Grienberger, Albrecht Stroh, and Arthur Konnerth
We describe here an approach for the fluorometric monitoring of population activity in neurons in live mice combined with the activation of optogenetic actuators in vivo. In this protocol, a thin multimode fiber, which is used for both delivering excitation light and collecting emitted fluorescence signals, is inserted into the skull of a mouse. When combined with multicell bolus loading of Ca2+ indicators, this optical fiber and its associated fluorescence detection system can be used for the in vivo recording of brain Ca2+ signals from a local cluster of coactive neurons. The fiber can also be used for the optogenetic stimulation of light-activated ion channels, such as channelrhodopsin-2, allowing the monitoring of local calcium signals evoked by optogenetic stimulation.
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.
Reagents
Anesthetics appropriate for the species under investigation (see, e.g., Flecknell 2000) For mice, we typically use inhalation anesthesia with 0.8%–1.5% isoflurane in pure O2 or intraperitoneal injection of ketamine/xylazine (0.1/0.01 mg/g body weight). Xylocaine is used for local anesthesia.
Cyanoacrylate glue Eye protection cream (Isopto-Max, Alcon Pharma) Mice for analysis For combined calcium recordings and optogenetic activation, use animals expressing optogenetic actuators (e.g., channelrhodopsin-2 [ChR2]) in the brain area of interest.
Reagents and equipment for multicell bolus loading of the brain tissue with calcium-sensitive dye Oregon Green-488 BAPTA-1 (OGB-1) AM See In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
Equipment
Biomonitoring system for monitoring body temperature, heartbeat, and breathing rates (e.g., AD Instruments) Diamond Wedge scribe (Thorlabs, Inc.) Fiber-stripping tool (Thorlabs, Inc.) 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.prot084145
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In Vivo Recording Through an Optical Fiber
Imaging setup A diagram of the recording and stimulation setup is shown in Figure 1A. The light for excitation of the fluorescent calcium-sensitive dye, such as OGB-1, and activation of the ChR2 is delivered by a 10-mW solid-state laser (Sapphire, Coherent, Inc.) with a wavelength of 488 nm. The rapid control of the intensity of the laser beam is achieved by an acousto-optic modulator (AOM; 3080–125, Crystal Technology). After deflection by a dichroic mirror (Fig. 1A), the beam is focused by means of a collimator into one end of a multimode fiber (Thorlabs, Inc.) with a diameter of ≤200 µm. The emitted fluorescence light is guided back through the same fiber and focused on an avalanche photodiode (APD; S5343, Hamamatsu Photonics) with an aperture of 1 mm. The recorded fluorescence signals are digitized with a sampling frequency of 2000 Hz using a multifunction I/O device USB-6221 (National Instruments) and custom-written LabView-based software. The fluorescence signals were low-pass filtered offline with frequencies of 5–20 Hz.
Materials for the fixation of the optical fiber in the skull Syntac Adhesive Syntac Primer Tetric dental cement (Ivoclar Vivadent) Total Etch (Heraeus Kulzer) UV lamp Optical fiber Typically, multimode fibers with a diameter of 200 µm and a numerical aperture of 0.48 are most commonly used. However, thinner (100-μm) or thicker (500-μm) fibers may be implanted by using the same protocol.
Stereotaxic device with ear bars B
APD
A Focusing lens
Em. filter Shutter Shutter Beam splitter Laser AOM Collimator Optical fiber
C
D Optical fiber Metal tube
Dental cement Skull Cortex
Illuminated volume
Stained volume
200 µm
FIGURE 1. (A) Scheme of the optical fiber recording setup. AOM, acousto-optic modulator; APD, avalanche photodiode; Em, emission. (B) Image showing the tip of an optical fiber (diameter 200 µm) glued into a metal tube. (C ) Illustration of the fiber tip inserted into the brain region with neurons labeled by the multicell bolus loading technique with calcium-sensitive dye (green). The blue cone indicates the volume in which the dye in the loaded neurons is excited by the laser light. (D) The tip of an optical fiber was placed perpendicular to an acute cortical slice in which the neurons were stained with Oregon Green-488 BAPTA (OGB-1) AM. The image was acquired using a charge-coupled device (CCD) camera and shows the neuron-containing fiber-illuminated cortical region.
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H. Adelsberger et al.
Surgical equipment (dental drill, fine scissors, fine forceps, needles) Syringe needle (see Step 12) Warming plate (to keep the animal’s body temperature constant) METHOD Anesthesia
1. Anesthetize the animal either by injection of ketamine/xylazine intraperitoneally or by inhalation of isoflurane in pure O2. When using isoflurane, we recommend the placement of the animal for 10 min in a closed box containing 2% isoflurane before its fixation in the stereotaxic apparatus. The depth of anesthesia needed for surgery is indicated by the loss of the reflexes (e.g., eyelid reflex, pinch withdrawal). At this stage, inhalation of anesthesia with isoflurane concentrations of 0.8%–1.2% is sufficient to keep the mouse at respiration rates of 80–130/min.
Preparation of the Skull
2. Place the anesthetized mouse onto a warming plate (37˚C) and fix the head in a stereotaxic frame with ear bars. 3. Apply sensors for the control of the body temperature and breathing rates. 4. Apply eye protection cream onto the eyes to protect them from drying. 5. Inject 100 µL 2% xylocaine solution subcutaneously at the site of brain surgery. 6. Ten minutes after xylocaine injection, gently remove the skin and muscles above the desired brain region with fine scissors. 7. Dry the skull with warm air. 8. Perform the following steps to ensure stable fixation of the optic fiber: i. Apply a thin film of Total Etch for precisely 15 sec onto the exposed region. ii. Rinse the skull with water and dry with air. iii. Apply Syntac Primer for 15 sec and dry with air again. iv. Apply Syntac Adhesive for 10 sec followed by air drying. 9. Use the dental drill to thin the skull at the site of recording. Create a small opening with a diameter of 0.5 mm by gently using fine forceps and needles. 10. Perform multicell bolus loading of the brain region of choice. See In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading (Garaschuk and Konnerth 2010).
Implantation and Fixation of the Optical Fiber
11. Remove the coating from the end of the fiber with a standard fiber-stripping tool. 12. Prepare a thin metal tube with the appropriate inner diameter and a length of 10 mm from a syringe needle. 13. Pull the metal tube over the fiber and fix it with cyanoacrylate glue. 14. Trim the protruding short end of the optic fiber (1 cm length) with a Diamond Wedge scribe to a length corresponding to the depth of insertion into the brain (Fig. 1B). For example, trim to 200 µm when recording from a layer two-thirds into the mouse cortex. 15. Place the optic fiber transiently into a manipulator and use the site covered with the metal tube for fixation. 16. Thirty minutes after multicell bolus loading of the calcium-sensitive dye (e.g., OGB-1), insert the tip of the optic fiber gently into the brain through the skull opening. The optimal depth corresponds to the site with the highest fluorescence intensity. See Troubleshooting.
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In Vivo Recording Through an Optical Fiber
17. Apply Tetric dental cement around the metal tube onto the skull (Fig. 1C) and harden it with UV light for 20 sec. The fiber can now be released from the micromanipulator. 18. For the recording of calcium signals, increase the laser beam to an intensity such that it is just below the level in which bleaching is detected (typical range is 0.01–0.1 mW at the tip of the optical fiber). See Troubleshooting.
Calcium Recording and ChR2 Activation
19. For combined calcium recordings and optogenetic activation, apply the procedures described in the preceding steps to animals expressing optogenetic actuators (e.g., ChR2) in the brain area of interest (see Fig. 2). ChR2 stimulation can be performed in combination with fluorometric calcium recordings and also without calcium recordings.
20. For combined calcium recording–ChR2 stimulation experiments, increase the excitation light intensity 20–80 times for the period of stimulation. Because of the saturation of the light detected by APD, calcium recordings are not possible during ChR2 stimulation.
TROUBLESHOOTING Problem (Step 16): There is little or no fluorescence after implanting the optical fiber. Solution: Aside from a failure in dye injection (see In Vivo Two-Photon Calcium Imaging Using
Multicell Bolus Loading [Garaschuk and Konnerth 2010]), this problem is mainly caused by A
B
Optical fiber
1% 1 sec
C
D
E
0.5 sec
Layer 5 neurons expressing ChR2
2.3 mW pulse power
% Response
1%
100 90 80 70 60 50 40 30 20 10 0
2
3
5
20
50
300
Pulse duration (msec) 0.03 mW baseline power
FIGURE 2. (A) (Left) Photograph of a mouse brain indicating the site of posthoc sectioning at the level of the visual cortex. Scale bar, 2 mm. (Right) Section through the primary visual cortex indicating the OGB-1 AM-stained region (green in false color) and the position of the optical fiber. (B) Calcium transient detected in the primary visual cortex evoked by a 50-msec light flash applied to the contralateral eye in an anesthetized adult mouse. (C ) Schematic showing the tip of an optic fiber implanted into a stained cortical region above neurons expressing ChR2 (red). (D) Calcium transient evoked by optogenetic stimulation of ChR2-expressing layer 5 neurons. The optical fiber was implanted at a depth of 500 µm in the visual cortex of an adult transgenic mouse. Light pulses of 488 nm wavelength and 50 msec duration, with a power of 2.3 mW, were used for stimulation. (E) Graph depicting, for constant excitation light intensity, the relationship between the duration of the stimulus pulse and the response probability. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084145
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placement of the fiber tip outside of the stained area, especially when recording from deep brain regions. To avoid this problem, take care to insert the staining pipette and the optical fiber exactly at the same position and with the same angle, by optimizing the use of the stereotaxic device. Another reason for low fluorescence levels is bleeding into the tissue after damage of a blood vessel, either with the staining pipette or with the optical fiber. Blood vessel rupture may be partially avoided by lowering the pipette and the fiber very slowly. Problem (Step 18): The traces are too noisy. Solution: This problem is often caused by heartbeat-mediated pulsation of a large blood vessel located
close to the tip of the optical fiber. Minor changes in the depths of the fiber tip may lead to major improvements. Problem (Step 18): There are movement artifacts. Solution: Artifacts can occur in anesthetized animals by strong breathing excursions or, in awake
animals by fast movements. Proper fixation of the fiber in the metal tube and on the skull helps to minimize movement artifacts. DISCUSSION
In many instances (e.g., when recording oscillatory wave activity from simultaneously active cells), useful information can be extracted from monitoring the activity of large neuronal populations. We therefore developed the fluorescence detection system described here (see also Adelsberger et al. 2005). When combined with multicell bolus loading of calcium indicators (Stosiek et al. 2003; In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading [Garaschuk and Konnerth 2010]), it allows the in vivo recording of brain Ca2+ signals from a local cluster of coactive neurons. Such recordings may be performed in any brain region in both anesthetized and nonanesthetized animals. Importantly, the recordings can be readily combined with the stimulation of light-activated ion channels, such as ChR2 (Boyden et al. 2005), allowing the monitoring of the local calcium signals evoked by optogenetic stimulation. The system we describe here includes a thin multimode fiber that is inserted to the skull of the mouse. This optical fiber is used for the detection of fluorescence signals from all stained cells and the surrounding neuropile within the volume illuminated by the excitation light (Fig. 1D). For twophoton imaging recordings, the calcium signals detected by the optical fiber predominantly represent the action potential activity in the stained region (Stosiek et al. 2003; Kerr et al. 2005). From combined two-photon imaging and optical fiber experiments, we estimate that about 20 coactive neurons located near the tip are sufficient for producing a detectable calcium signal. Note that a variant of this approach has been used for the detection of calcium signals from the upper dendrites of layer 5 cortical neurons (Murayama et al. 2007; see also Fiber-Optic Calcium Monitoring of Dendritic Activity In Vivo [Murayama and Larkum 2012]). The original application of this approach was concerned with the monitoring of spontaneous calcium waves in the cortex of awake newborn mice (Adelsberger et al. 2005). An example of a population calcium signal evoked by sensory stimulation in the adult mouse is shown in Figure 2. The optical fiber was implanted in the primary visual cortex (Fig. 2A). Stimulation of the contralateral eye with a white light flash (Fig. 2B) evoked a large calcium transient lasting for 2–3 sec. The transient occurred with a delay of 80 msec, which corresponds closely to the range of the delays observed with electrical recordings (Porciatti et al. 1999). For the illustration of calcium recordings combined with optogenetic stimulation, we used mice expressing ChR2 under the control of the Thy1 promoter in cortical layer 5 neurons (Arenkiel et al. 2007). Figure 2C shows the experimental arrangement of an optical fiber placed in the stained cortical region after multicell bolus loading. The calcium signals were monitored by using low light levels (0.03 mW) from the 488-nm excitation laser. For ChR2 stimulation, the excitation light intensity was increased for 50 msec to a level of 2.3 mW. The resulting calcium transients had time courses and 1078
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In Vivo Recording Through an Optical Fiber
amplitudes that were comparable with those evoked by sensory stimulation. This experimental configuration was extremely robust and allowed many trials of repeated stimulations and recordings. For example, in Fig. 2E we illustrate the result of such a long-lasting experiment, showing that for constant laser intensity, the response probability was proportional to the duration of the stimulus.
ACKNOWLEDGMENTS
This work was supported by the Bundesministerium für Bildung und Forschung (NGFN-2) and the Deutsche Forschungsgemeinschaft (International Graduate School 1373). REFERENCES Adelsberger H, Garaschuk O, Konnerth A. 2005. Cortical calcium waves in resting newborn mice. Nat Neurosci 8: 988–990. Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, Augustine GJ, Ehlers MD, Feng G. 2007. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54: 205–218. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. 2005. Millisecondtimescale, genetically targeted optical control of neural activity. Nat Neurosci 8: 1263–1268. Flecknell P. 2000. Laboratory animal anesthesia. Academic, New York. Garaschuk O, Konnerth A. 2010. In vivo two-photon calcium imaging using multicell bolus loading. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5482.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084145
Kerr JN, Greenberg D, Helmchen F. 2005. Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci 102: 14063–14068. Murayama M, Larkum M. 2012. Fiber-optic calcium monitoring of dendritic activity in vivo. Cold Spring Harb Protoc doi: 10.1101/pdb .prot067835. Murayama M, Perez-Garci E, Lüscher H-R, Larkum ME. 2007. Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J Neurophysiol 98: 1791–1805. Porciatti V, Pizzorusso T, Maffei L. 1999. The visual physiology of the wild type mouse determined with pattern VEPs. Vision Res 39: 3071–3081. Stosiek C, Garaschuk O, Holthoff K, Konnerth A. 2003. In vivo two-photon calcium imaging using multi-cell bolus loading (MCBL). Proc Natl Acad Sci 100: 7319–7324.
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