SPINE Volume 39, Number 15, pp E865-E869 ©2014, Lippincott Williams & Wilkins
BASIC SCIENCE
Channelrhodopsin-2-Expressed Dorsal Root Ganglion Neurons Activates Calcium Channel Currents and Increases Action Potential in Spinal Cord Yi Zhang, MD,* Jing Yue, MD, PhD,† Midan Ai, PhD,‡ Zhigang Ji, PhD,§ Zhiguo Liu, PhD,¶ Xuehong Cao, MD, PhD, and Li Li, MD, PhD§
Study Design. We used optogenetic techniques in spinal cord and dorsal root ganglion (DRG) neuron studies. Objective. This study investigated changes in channelrhodopsin-2 (ChR2) expression in the spinal cord and DRG neurons using optogenetic techniques. The results show the possibility of using optogenetics to treat neuropathic pain. Summary of Background Data. Previous studies have shown that activated ChR2 induces an increase in DRG neuron action potential. Methods. Western blot analysis was used to measure ChR2 protein levels in the spinal cord and DRG neurons or rats intrathecally injected with ChR2 lentivirus. Electrophysiology recording was used to detect differences in action potential levels in the spinal cord and calcium channel currents in the DRG neurons. Results. Our studies showed that ChR2 expression increased the action potential in the spinal cord and increased calcium channel currents in DRG neurons. Conclusion. We successfully expressed the ChR2 protein in the spinal cord and DRG neurons. We also found that ChR2 increased From the Departments of *Anesthesiology and †Reproductive Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China; ‡Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; §Department of Neurology, Baylor College of Medicine, Houston, TX; ¶Institution of Nutrition, Wuhan Polytechnic University, Changqing Garden, Wuhan, People’s Republic of China; Institution of Nutrition, Wuhan Polytechnic University, Changqing Garden, Wuha, People’s Republic of China; and Children’s Nutrition Research Center, Baylor College of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX. Acknowledgment date: November 12, 2013. Revision date: March 6, 2014. Acceptance date: March 20, 2014. The device(s)/drug(s) is/are FDA-approved or approved by corresponding national agency for this indication. National Natural Science Foundation of China funds (grant no. 81370246) were received to support this work. Relevant financial activities outside the submitted work: fees for participation in review activities such as data monitoring boards, statistical analysis, end point committees, and the like; payment for writing or reviewing the manuscript; and provision of writing assistance, medicines, equipment, or administrative support. Yi Zhang and Jing Yue made the equal contribution to this project. Address correspondence and reprint requests to Li Li, MD, PhD, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, MS NB302 Houston, TX 77030; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000373 Spine
the action potential in the spinal cord and activated the calcium channel in DRG neurons. These findings support the research possibilities of using optogenetic studies to improve treatment for neuropathic pain. Key words: ChR2, DRG, spinal cord, pain. Level of Evidence: N/A Spine 2014;39:E865–E869
O
ptogenetics is a recent advance that is now widely used in the neuroscience field.1–3 Two opsins commonly used in optogenetics are channelrhodopsin-2 (ChR2), a light-sensitive protein that induces intracellular cation flow when a blue light is activated, and halorhodopsin (NpHR), which may inactivate cells by hyperpolarizing the cells when a yellow light is activated.4 Most optogenetic research has focused on the central nervous system; therefore, little is known about the optogenetic function in the peripheral nervous system, which comprises the nerves and ganglia outside of the brain and spinal cord. The dorsal root ganglion (DRG) contains the main afferent neurons to the central nervous system: the mechanosensory afferent fiber and the pain and temperature afferent fiber.5–7 The main functions of DRG neurons are sensitivity to pain, temperature, and touch pressure. Several studies have reported that ion channels change in DRG neurons and the spinal cord when a person experiences pain.8,9 However, little is known about ChR2-wrought changes in the spinal cord and DRG neurons. Moreover, ion channels activity were widely reported in pain, especially in sodium and calcium channel. Here we sought to determine whether optogenetics could be applied to neurons in the peripheral nervous system as a novel method for relieving pain. In our study, we transfected ChR2 into the spinal cord and DRG neurons to investigate cell activation from ChR2 expression.
MATERIALS AND METHODS We bought ChR2 cDNA (pcDNA3.1/hChR2-enhanced yellow fluorescent protein) from Addgene (20939; Cambridge, MA). We used HindIII and XbaI to cut the ChR2 cDNA to yield 1781 base ChR2 protein. HindIII and XbaI were bought www.spinejournal.com
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
from New England Biolabs (Ipswich, MA). Using a previously obtained lentivirus (LV)-making protocol,10 we used pLenti6/V5 DEST (including green fluorescent protein promoter) gateway methods (V496-10; Invitrogen, Grand Island, NY) to produce ChR2 lentivirus (LV-ChR2). Finally, we used the HT1080 cell line to determine the virus titration. For virus injection, we intrathecally injected 250-g Sprague-Dawley rats with 10 μL of LV-ChR2 (the titer was approximately 1 × 107/transducing unit). After 14 days, the rats were killed and their spinal cords and DRG neurons were removed to use in the electrophysiology experiments. LV-LacZ (Invitrogen) was used as a negative control to confirm ChR2 expression. One month after injecting LV into the rats, DRG neurons and spinal cord section would be used for experiments. A previously described method to dissociate DRG neurons from the spinal cord was used.11 We quickly removed L5–L6 DRGs from the rats and placed the DRGs in Dulbecco’s modified Eagle’s medium (Invitrogen). Trypsin (type I, 0.2 mg/mL; Sigma-Aldrich, St. Louis, MO) and collagenase (type I, 1 mg/ mL; Sigma-Aldrich) were used for enzyme digestion at 34°C for 40 minutes. We used 10% fetal bovine serum to stop the trypsin-induced digestion. We then plated the neurons onto coverslips precoated with 50 μg/mL of poly-L-lysine (SigmaAldrich). Recording was processed in 1 to 2 days after DRG dissociation. To quantify the virus transfection efficiency,12 DRG neurons were labeled with Cy3-conjugated anti-NeuN (1:200
dilution, ABN78C3; Millipore, Billerica, MA). LSM5 Pascal confocal imaging system (Zeiss, Shanghai, China) was used to take the images. Images of the size 120 × 120 μm were obtained and then cell number was counted according to immunochemistry results (n = 18). For spinal cord slice recording, we removed the spinal cord from the lumbar segment at the L5–L6 level and transferred it to ice-cold artificial cerebrospinal fluid, using 95% O2 and 5% CO2 as a buffer to bubbling. We cut 300-μm spinal cord slices, which we used for electrophysiological recording after 1 hour of recovery in the artificial cerebrospinal fluid. After being fire polished, 2- to 4-MΩ glass capillaries were used to pull electrodes for the electrophysiology recordings. We used an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany) to obtain the DRG neuron signal recording. The extracellular solution consisted of 140mM tetraethylammonium, 2mM MgCl2, 3mM BaCl2, 10mM glucose, and 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH, 7.4; osmolarity, 320 mOsm/L). The pipette internal solution consisted of 120mM CsCl, 1mM MgCl2, 10mM HEPES, 10mM ethylene glycol tetra-acetic acid, 4mM magnesium adenosine triphosphate, and 0.3mM sodium guanosine-5´triphosphate (pH, 7.2; osmolarity, 300 mOsm/L). To record spinal cord firing, we used an extracellular solution consisting of 145mM NaCl, 2.5mM KCl, 2mM CaCl2, 1mM MgCl, 10mM HEPES, and 10mM glucose (pH, 7.3; osmolarity, 325 mOsm/L). The intracellular solution consisted of 140mM
A
B
bright field
GFP
DRG
10 μm
Mechanosensory afferent fiber
Pain and 20 temperature um afferent fiber
100 μm GFP
NeuN
20 μm
merge GFP/NeuN cell number (%)
C
150
100
50
0 NeuN
GFP
Figure 1. ChR2 expression in DRG neurons. A, GFP-ChR2 is expressed in recently harvested DRG tissue. Scale is 100 μm. B, ChR2 is expressed in dissociate DRG neurons what will be used for electrophysiology. Scale is 10 μm. C, Immunostained with anti-NeuN to show that some DRG neurons were colocoted with GFP and NeuN together. Scale is 20 μm. ChR2 indicates channelrhodopsin-2; DRG, dorsal root ganglion: GFP, green fluorescent protein.
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
Figure 2. ChR2 expression in spinal cord and DRG neurons as measured by Western blot analysis. ChR2 was expressed only in the spinal cords (A) or DRGs (B) LV-ChR2–transfected rats. LacZ was used as a negative control to confirm the ChR2 expression. Glyceraldehyde 3-phosphate dehydrogenase was used as a positive control to confirm successful protein extraction and equal protein quantity loading. ChR2 indicates channelrhodopsin-2; LV, lentivirus.
potassium gluconate, 4mM magnesium-adenosine 5´-triphosphate, 10mM HEPES, and 1.1mM ethylene glycol tetra-acetic acid (pH, 7.2; osmolarity, 300 mOsm/L). Signals were measured by an amplifier (MultiClamp 700B; Molecular Devices, Sunnyvale, CA), and the pCLAMP 10 software (Molecular Devices) was used for analysis. Venus fluorescence was used to confirm ChR2 expression before the electrophysiology. ChR2 channels were activated by light pulses (10-ms pulse width) delivered at 3 Hz for 5 to 10 minutes with a transistor-transistor logic-driven 473-nm blue laser (Laserglow Technologies, Ontario, Canada). DRG tissues were sonicated in radio immunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA) that included a cocktail protease inhibitor (Sigma-Aldrich).
Tissues were centrifuged at 16,000g for 10 minutes at 4°C, and then the supernatant was collected as total protein for the protein quantitation. We ran 20-μg total protein in 4% to 20% sodium dodecyl sulfate polyacrylamide electrophoresis gradient gel (Bio-Rad, Hercules, CA) and transferred the protein onto a polyvinylidene difluoride membrane (ImmobilonP; Millipore). The blot was incubated in the primary antibody: anti–green fluorescent protein (1:1000 dilution; MBL International, Woburn, MA); anti–glyceraldehyde 3-phosphate dehydrogenase (1:1000 dilution; Millipore); or anti-LacZ (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Glyceraldehyde 3-phosphate dehydrogenase was used as a positive control to confirm the protein was extracted successfully and to confirm equal protein quantity loading. The seconded was donkey anti-mouse immunoglobulin G (Jackson Immuno Research Labs, West Grove, PA).
Statistical Analysis The calcium channel current data were analyzed by using the PulseFit software program (HEKA Instruments). The action potential data were analyzed by using Clampfit 10 (Molecular Devices). Virus efficiency and the protein value data were analyzed using Image J (http://imagej.nih.gov/ij/). The Student t test was used to compare 2 groups; mean ± standard error P < 0.05 was considered statistically significant.
RESULTS
Figure 3. The action potential increased in LV-ChR2–expressing spinal cords when a blue light pulse was used. No firing was shown in the LacZ-expressing spinal cord. ChR2 indicates channelrhodopsin-2; LV, lentivirus. Spine
The DRG tissues, which were immediately removed from the rats, clearly showed LV-ChR2 expression (Figure 1A). The DRG neurons used for the electrophysiology recordings also showed ChR2 expression (Figure 1B). Moreover, we used immunostaining to quantify the virus efficiency, there are about 60% cells were successful to be infected by virus (Figure 1C). Because of the thickness (300 μm) of the spinal cord tissues, it was difficult to obtain photographs of spinal cord. Western blot analysis results showed that ChR2 was expressed in the spinal cord (Figure 2A) and DRG neurons (Figure 2B) and was almost no expression in the negative control (the LV-LacZ–transfected DRG neurons). These results www.spinejournal.com
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
Figure 4. Calcium channel currents increased significantly in ChR2-expressing DRGs when a blue light pulse was off. No change in the calcium channel currents was seen in the LV-LacZ–transfected DRG neurons. ChR2 indicates channelrhodopsin-2; DRG, dorsal root ganglion; LV, lentivirus.
show that LV-ChR2 was successfully expressed in the spinal cord and DRG neurons. Next, we investigated the action potential in the spinal cord. Under the voltage clamp, the blue light-emitting diode light pulse evoked rapid membrane depolarization in an intensity-dependent manner. The action potential had significantly appeared after this light pulse stimulation in the ChR2-expressed spinal cord compared with that in the LacZexpressed spinal cord (Figure 3). Under the whole-cell voltage clamp, calcium channel currents increased significantly after blue light pulse stimulation (Figure 4). When the light was off, calcium channel currents were nearly 2 times reduced than currents measured while the light was on.
DISCUSSION Until now, most optogenetic projects have focused on the central nervous system, with little research focusing on the peripheral nervous system; however, neuropathic pain has been shown to result in action potential and calcium channel current activation.8,9 To find out a more effective way to relieve light-sensitive proteins the pain, we use optogenetics, this advanced research method to investigate the changes action potation and calcium channel after light stimulation. ChR2, one of the famous light-sensitive proteins, is activated when blue light is on. When a ChR2 channel in cell membrane is activated, a large influx of positively charged sodium ions occurs. Because the ion channel is nonselective to induce the ions influx, this strongly suggests that positive charge ions, as Na+, Ca2+, H+ (note, however, that the channel is nonselective and thus is also permeable to potassium, calcium, and hydrogen ions). Primarily because of this inward Na+ current, ChR2 activation causes a rapid depolarization of the cell membrane and a strong excitatory effect on the cell. To our knowledge, our study is the first to investigate such changes in the calcium channel in ChR2-expressed DRG neurons. We showed that ChR2 expression changed the action potential in the spinal cord, which the calcium channel currents changed in ChR2-activated DRG neurons, and that ChR2 induces cell activation and intracellular calcium influx upon the use of a blue light pulse. These results further prove that exposing light-sensitive proteins to specific wavelengths of light may induce calcium increase, neurotransmitter E868
release, or membrane depolarization in spinal cord and DRG neurons.13,14 As stated previously, activation of the calcium channels in DRG neurons will exacerbate neuropathic pain. Thus, ChR2 is not a good tool to be used in patients with neuropathic pain. However, NpHR hyperpolarizes the cells to reduce neural activity when yellow-light pulses are used and, therefore, may be a good tool for treating neuropathic pain.4 Further studies of the function of NpHR in neuropathic pain treatment are warranted.
CONCLUSION Overall, we successfully expressed the ChR2 protein in the spinal cord and DRG neurons. We also found that ChR2 increased the action potential in the spinal cord and activated the calcium channel in DRG neurons. These findings support the research possibilities of using optogenetic studies to improve treatment for neuropathic pain.
➢ Key Points The LV system can be used to deliver green fluorescent protein-ChR2 protein for expression in the spinal cord and DRG neurons. When a blue light pulse is used, neuron firing is greater in the ChR2-expressed spinal cord than in the negative control (the LacZ-expressed spinal cord). Calcium channel currents are greater in ChR2activated DRG neurons than in non–ChR2activated DRG neurons.
References
1. Tonnesen J, Parish CL, Sorensen AT, et al. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model. PLoS One 2011;6:e17560. 2. Deisseroth K. Optogenetics. Nat Methods 2011;8:26–9. 3. Yizhar O, Fenno LE, Davidson TJ, et al. Optogenetics in neural systems. Neuron 2011;71:9–34. 4. G N Tan A, Farhatnia Y, et al. Channelrhodopsins: visual regeneration and neural activation by a light switch. New Biotechnol 2013;30:461–74. 5. Tucker BA, Mearow KM. Peripheral sensory axon growth: from receptor binding to cellular signaling. Can J Neurol Sci 2008;35:551–66.
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BASIC SCIENCE 6. Treede RD. Transduction and transmission properties of primary nociceptive afferents. Ross Fiziol Zh Im I M Sechenova 1999;85:205–11. 7. Sameda H, Takahashi Y, Takahashi K, et al. Dorsal root ganglion neurones with dichotomising afferent fibres to both the lumbar disc and the groin skin. A possible neuronal mechanism underlying referred groin pain in lower lumbar disc diseases. J Bone Joint Surg Br 2003;85:600–3. 8. Staats PS, Yearwood T, Charapata SG, et al. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 2004;291:63–70. 9. Schmidtko A, Lotsch J, Freynhagen R, et al. Ziconotide for treatment of severe chronic pain. Lancet 2010;375:1569–77. 10. Jiang YQ, Wang XL, Cao XH, et al. Increased heat shock transcription factor 1 in the cerebellum reverses the deficiency of
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Changes in ChR2-Expressed DRG Neurons • Zhang et al
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Purkinje cells in Alzheimer’s disease. Brain Res 2013;1519: 105–11. Li L, Cao XH, Chen SR, et al. Upregulation of Cavbeta3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain. J Biol Chem 2012;287:6002–13. Tu J, Yang F, Wan J, et al. Light-controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats. GLIA 2014;62: 106–121. Miesenbock G, Kevrekidis IG. Optical imaging and control of genetically designated neurons in functioning circuits. Annu Rev Neurosci 2005;28:533–63. Fiala A, Suska A, Schluter OM. Optogenetic approaches in neuroscience. Curr Biol 2010;20:R897–903.
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BASIC SCIENCE
Channelrhodopsin-2-Expressed Dorsal Root Ganglion Neurons Activates Calcium Channel Currents and Increases Action Potential in Spinal Cord Yi Zhang, MD,* Jing Yue, MD, PhD,† Midan Ai, PhD,‡ Zhigang Ji, PhD,§ Zhiguo Liu, PhD,¶ Xuehong Cao, MD, PhD, and Li Li, MD, PhD§
Study Design. We used optogenetic techniques in spinal cord and dorsal root ganglion (DRG) neuron studies. Objective. This study investigated changes in channelrhodopsin-2 (ChR2) expression in the spinal cord and DRG neurons using optogenetic techniques. The results show the possibility of using optogenetics to treat neuropathic pain. Summary of Background Data. Previous studies have shown that activated ChR2 induces an increase in DRG neuron action potential. Methods. Western blot analysis was used to measure ChR2 protein levels in the spinal cord and DRG neurons or rats intrathecally injected with ChR2 lentivirus. Electrophysiology recording was used to detect differences in action potential levels in the spinal cord and calcium channel currents in the DRG neurons. Results. Our studies showed that ChR2 expression increased the action potential in the spinal cord and increased calcium channel currents in DRG neurons. Conclusion. We successfully expressed the ChR2 protein in the spinal cord and DRG neurons. We also found that ChR2 increased From the Departments of *Anesthesiology and †Reproductive Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China; ‡Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; §Department of Neurology, Baylor College of Medicine, Houston, TX; ¶Institution of Nutrition, Wuhan Polytechnic University, Changqing Garden, Wuhan, People’s Republic of China; Institution of Nutrition, Wuhan Polytechnic University, Changqing Garden, Wuha, People’s Republic of China; and Children’s Nutrition Research Center, Baylor College of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX. Acknowledgment date: November 12, 2013. Revision date: March 6, 2014. Acceptance date: March 20, 2014. The device(s)/drug(s) is/are FDA-approved or approved by corresponding national agency for this indication. National Natural Science Foundation of China funds (grant no. 81370246) were received to support this work. Relevant financial activities outside the submitted work: fees for participation in review activities such as data monitoring boards, statistical analysis, end point committees, and the like; payment for writing or reviewing the manuscript; and provision of writing assistance, medicines, equipment, or administrative support. Yi Zhang and Jing Yue made the equal contribution to this project. Address correspondence and reprint requests to Li Li, MD, PhD, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, MS NB302 Houston, TX 77030; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000373 Spine
the action potential in the spinal cord and activated the calcium channel in DRG neurons. These findings support the research possibilities of using optogenetic studies to improve treatment for neuropathic pain. Key words: ChR2, DRG, spinal cord, pain. Level of Evidence: N/A Spine 2014;39:E865–E869
O
ptogenetics is a recent advance that is now widely used in the neuroscience field.1–3 Two opsins commonly used in optogenetics are channelrhodopsin-2 (ChR2), a light-sensitive protein that induces intracellular cation flow when a blue light is activated, and halorhodopsin (NpHR), which may inactivate cells by hyperpolarizing the cells when a yellow light is activated.4 Most optogenetic research has focused on the central nervous system; therefore, little is known about the optogenetic function in the peripheral nervous system, which comprises the nerves and ganglia outside of the brain and spinal cord. The dorsal root ganglion (DRG) contains the main afferent neurons to the central nervous system: the mechanosensory afferent fiber and the pain and temperature afferent fiber.5–7 The main functions of DRG neurons are sensitivity to pain, temperature, and touch pressure. Several studies have reported that ion channels change in DRG neurons and the spinal cord when a person experiences pain.8,9 However, little is known about ChR2-wrought changes in the spinal cord and DRG neurons. Moreover, ion channels activity were widely reported in pain, especially in sodium and calcium channel. Here we sought to determine whether optogenetics could be applied to neurons in the peripheral nervous system as a novel method for relieving pain. In our study, we transfected ChR2 into the spinal cord and DRG neurons to investigate cell activation from ChR2 expression.
MATERIALS AND METHODS We bought ChR2 cDNA (pcDNA3.1/hChR2-enhanced yellow fluorescent protein) from Addgene (20939; Cambridge, MA). We used HindIII and XbaI to cut the ChR2 cDNA to yield 1781 base ChR2 protein. HindIII and XbaI were bought www.spinejournal.com
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
from New England Biolabs (Ipswich, MA). Using a previously obtained lentivirus (LV)-making protocol,10 we used pLenti6/V5 DEST (including green fluorescent protein promoter) gateway methods (V496-10; Invitrogen, Grand Island, NY) to produce ChR2 lentivirus (LV-ChR2). Finally, we used the HT1080 cell line to determine the virus titration. For virus injection, we intrathecally injected 250-g Sprague-Dawley rats with 10 μL of LV-ChR2 (the titer was approximately 1 × 107/transducing unit). After 14 days, the rats were killed and their spinal cords and DRG neurons were removed to use in the electrophysiology experiments. LV-LacZ (Invitrogen) was used as a negative control to confirm ChR2 expression. One month after injecting LV into the rats, DRG neurons and spinal cord section would be used for experiments. A previously described method to dissociate DRG neurons from the spinal cord was used.11 We quickly removed L5–L6 DRGs from the rats and placed the DRGs in Dulbecco’s modified Eagle’s medium (Invitrogen). Trypsin (type I, 0.2 mg/mL; Sigma-Aldrich, St. Louis, MO) and collagenase (type I, 1 mg/ mL; Sigma-Aldrich) were used for enzyme digestion at 34°C for 40 minutes. We used 10% fetal bovine serum to stop the trypsin-induced digestion. We then plated the neurons onto coverslips precoated with 50 μg/mL of poly-L-lysine (SigmaAldrich). Recording was processed in 1 to 2 days after DRG dissociation. To quantify the virus transfection efficiency,12 DRG neurons were labeled with Cy3-conjugated anti-NeuN (1:200
dilution, ABN78C3; Millipore, Billerica, MA). LSM5 Pascal confocal imaging system (Zeiss, Shanghai, China) was used to take the images. Images of the size 120 × 120 μm were obtained and then cell number was counted according to immunochemistry results (n = 18). For spinal cord slice recording, we removed the spinal cord from the lumbar segment at the L5–L6 level and transferred it to ice-cold artificial cerebrospinal fluid, using 95% O2 and 5% CO2 as a buffer to bubbling. We cut 300-μm spinal cord slices, which we used for electrophysiological recording after 1 hour of recovery in the artificial cerebrospinal fluid. After being fire polished, 2- to 4-MΩ glass capillaries were used to pull electrodes for the electrophysiology recordings. We used an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany) to obtain the DRG neuron signal recording. The extracellular solution consisted of 140mM tetraethylammonium, 2mM MgCl2, 3mM BaCl2, 10mM glucose, and 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH, 7.4; osmolarity, 320 mOsm/L). The pipette internal solution consisted of 120mM CsCl, 1mM MgCl2, 10mM HEPES, 10mM ethylene glycol tetra-acetic acid, 4mM magnesium adenosine triphosphate, and 0.3mM sodium guanosine-5´triphosphate (pH, 7.2; osmolarity, 300 mOsm/L). To record spinal cord firing, we used an extracellular solution consisting of 145mM NaCl, 2.5mM KCl, 2mM CaCl2, 1mM MgCl, 10mM HEPES, and 10mM glucose (pH, 7.3; osmolarity, 325 mOsm/L). The intracellular solution consisted of 140mM
A
B
bright field
GFP
DRG
10 μm
Mechanosensory afferent fiber
Pain and 20 temperature um afferent fiber
100 μm GFP
NeuN
20 μm
merge GFP/NeuN cell number (%)
C
150
100
50
0 NeuN
GFP
Figure 1. ChR2 expression in DRG neurons. A, GFP-ChR2 is expressed in recently harvested DRG tissue. Scale is 100 μm. B, ChR2 is expressed in dissociate DRG neurons what will be used for electrophysiology. Scale is 10 μm. C, Immunostained with anti-NeuN to show that some DRG neurons were colocoted with GFP and NeuN together. Scale is 20 μm. ChR2 indicates channelrhodopsin-2; DRG, dorsal root ganglion: GFP, green fluorescent protein.
E866
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
Figure 2. ChR2 expression in spinal cord and DRG neurons as measured by Western blot analysis. ChR2 was expressed only in the spinal cords (A) or DRGs (B) LV-ChR2–transfected rats. LacZ was used as a negative control to confirm the ChR2 expression. Glyceraldehyde 3-phosphate dehydrogenase was used as a positive control to confirm successful protein extraction and equal protein quantity loading. ChR2 indicates channelrhodopsin-2; LV, lentivirus.
potassium gluconate, 4mM magnesium-adenosine 5´-triphosphate, 10mM HEPES, and 1.1mM ethylene glycol tetra-acetic acid (pH, 7.2; osmolarity, 300 mOsm/L). Signals were measured by an amplifier (MultiClamp 700B; Molecular Devices, Sunnyvale, CA), and the pCLAMP 10 software (Molecular Devices) was used for analysis. Venus fluorescence was used to confirm ChR2 expression before the electrophysiology. ChR2 channels were activated by light pulses (10-ms pulse width) delivered at 3 Hz for 5 to 10 minutes with a transistor-transistor logic-driven 473-nm blue laser (Laserglow Technologies, Ontario, Canada). DRG tissues were sonicated in radio immunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA) that included a cocktail protease inhibitor (Sigma-Aldrich).
Tissues were centrifuged at 16,000g for 10 minutes at 4°C, and then the supernatant was collected as total protein for the protein quantitation. We ran 20-μg total protein in 4% to 20% sodium dodecyl sulfate polyacrylamide electrophoresis gradient gel (Bio-Rad, Hercules, CA) and transferred the protein onto a polyvinylidene difluoride membrane (ImmobilonP; Millipore). The blot was incubated in the primary antibody: anti–green fluorescent protein (1:1000 dilution; MBL International, Woburn, MA); anti–glyceraldehyde 3-phosphate dehydrogenase (1:1000 dilution; Millipore); or anti-LacZ (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Glyceraldehyde 3-phosphate dehydrogenase was used as a positive control to confirm the protein was extracted successfully and to confirm equal protein quantity loading. The seconded was donkey anti-mouse immunoglobulin G (Jackson Immuno Research Labs, West Grove, PA).
Statistical Analysis The calcium channel current data were analyzed by using the PulseFit software program (HEKA Instruments). The action potential data were analyzed by using Clampfit 10 (Molecular Devices). Virus efficiency and the protein value data were analyzed using Image J (http://imagej.nih.gov/ij/). The Student t test was used to compare 2 groups; mean ± standard error P < 0.05 was considered statistically significant.
RESULTS
Figure 3. The action potential increased in LV-ChR2–expressing spinal cords when a blue light pulse was used. No firing was shown in the LacZ-expressing spinal cord. ChR2 indicates channelrhodopsin-2; LV, lentivirus. Spine
The DRG tissues, which were immediately removed from the rats, clearly showed LV-ChR2 expression (Figure 1A). The DRG neurons used for the electrophysiology recordings also showed ChR2 expression (Figure 1B). Moreover, we used immunostaining to quantify the virus efficiency, there are about 60% cells were successful to be infected by virus (Figure 1C). Because of the thickness (300 μm) of the spinal cord tissues, it was difficult to obtain photographs of spinal cord. Western blot analysis results showed that ChR2 was expressed in the spinal cord (Figure 2A) and DRG neurons (Figure 2B) and was almost no expression in the negative control (the LV-LacZ–transfected DRG neurons). These results www.spinejournal.com
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BASIC SCIENCE
Changes in ChR2-Expressed DRG Neurons • Zhang et al
Figure 4. Calcium channel currents increased significantly in ChR2-expressing DRGs when a blue light pulse was off. No change in the calcium channel currents was seen in the LV-LacZ–transfected DRG neurons. ChR2 indicates channelrhodopsin-2; DRG, dorsal root ganglion; LV, lentivirus.
show that LV-ChR2 was successfully expressed in the spinal cord and DRG neurons. Next, we investigated the action potential in the spinal cord. Under the voltage clamp, the blue light-emitting diode light pulse evoked rapid membrane depolarization in an intensity-dependent manner. The action potential had significantly appeared after this light pulse stimulation in the ChR2-expressed spinal cord compared with that in the LacZexpressed spinal cord (Figure 3). Under the whole-cell voltage clamp, calcium channel currents increased significantly after blue light pulse stimulation (Figure 4). When the light was off, calcium channel currents were nearly 2 times reduced than currents measured while the light was on.
DISCUSSION Until now, most optogenetic projects have focused on the central nervous system, with little research focusing on the peripheral nervous system; however, neuropathic pain has been shown to result in action potential and calcium channel current activation.8,9 To find out a more effective way to relieve light-sensitive proteins the pain, we use optogenetics, this advanced research method to investigate the changes action potation and calcium channel after light stimulation. ChR2, one of the famous light-sensitive proteins, is activated when blue light is on. When a ChR2 channel in cell membrane is activated, a large influx of positively charged sodium ions occurs. Because the ion channel is nonselective to induce the ions influx, this strongly suggests that positive charge ions, as Na+, Ca2+, H+ (note, however, that the channel is nonselective and thus is also permeable to potassium, calcium, and hydrogen ions). Primarily because of this inward Na+ current, ChR2 activation causes a rapid depolarization of the cell membrane and a strong excitatory effect on the cell. To our knowledge, our study is the first to investigate such changes in the calcium channel in ChR2-expressed DRG neurons. We showed that ChR2 expression changed the action potential in the spinal cord, which the calcium channel currents changed in ChR2-activated DRG neurons, and that ChR2 induces cell activation and intracellular calcium influx upon the use of a blue light pulse. These results further prove that exposing light-sensitive proteins to specific wavelengths of light may induce calcium increase, neurotransmitter E868
release, or membrane depolarization in spinal cord and DRG neurons.13,14 As stated previously, activation of the calcium channels in DRG neurons will exacerbate neuropathic pain. Thus, ChR2 is not a good tool to be used in patients with neuropathic pain. However, NpHR hyperpolarizes the cells to reduce neural activity when yellow-light pulses are used and, therefore, may be a good tool for treating neuropathic pain.4 Further studies of the function of NpHR in neuropathic pain treatment are warranted.
CONCLUSION Overall, we successfully expressed the ChR2 protein in the spinal cord and DRG neurons. We also found that ChR2 increased the action potential in the spinal cord and activated the calcium channel in DRG neurons. These findings support the research possibilities of using optogenetic studies to improve treatment for neuropathic pain.
➢ Key Points The LV system can be used to deliver green fluorescent protein-ChR2 protein for expression in the spinal cord and DRG neurons. When a blue light pulse is used, neuron firing is greater in the ChR2-expressed spinal cord than in the negative control (the LacZ-expressed spinal cord). Calcium channel currents are greater in ChR2activated DRG neurons than in non–ChR2activated DRG neurons.
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