Xinjian Li1, Vania Y Cao2, Wenyu Zhang3, Surjeet S Mastwal4, Qing Liu5, Stephani Otte6, Kuan Hong Wang7. 1. Unit on Neural Circuits and Adaptive Behaviors, Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD, USA. Electronic address: xinjian.li@nih.gov. 2. Inscopix Inc., Palo Alto, CA, USA. Electronic address: vania@inscopix.com. 3. Unit on Neural Circuits and Adaptive Behaviors, Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD, USA. Electronic address: wenyu.zhang@nih.gov. 4. Unit on Neural Circuits and Adaptive Behaviors, Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD, USA. Electronic address: mastwals@mail.nih.gov. 5. Unit on Neural Circuits and Adaptive Behaviors, Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD, USA. Electronic address: qing.liu2@nih.gov. 6. Inscopix Inc., Palo Alto, CA, USA. Electronic address: stephani@inscopix.com. 7. Unit on Neural Circuits and Adaptive Behaviors, Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Bethesda, MD, USA. Electronic address: wkuan@mail.nih.gov.
Abstract
BACKGROUND: In vivo optical imaging of neural activity provides important insights into brain functions at the single-cell level. Cranial windows and virally delivered calcium indicators are commonly used for imaging cortical activity through two-photon microscopes in head-fixed animals. Recently, head-mounted one-photon microscopes have been developed for freely behaving animals. However, minimizing tissue damage from the virus injection procedure and maintaining window clarity for imaging can be technically challenging. NEW METHOD: We used a wide-diameter glass pipette at the cortical surface for infusing the viral calcium reporter AAV-GCaMP6 into the cortex. After infusion, the scalp skin over the implanted optical window was sutured to facilitate postoperative recovery. The sutured scalp was removed approximately two weeks later and a miniature microscope was attached above the window to image neuronal activity in freely moving mice. RESULTS: We found that cortical surface virus infusion efficiently labeled neurons in superficial layers, and scalp skin suturing helped to maintain the long-term clarity of optical windows. As a result, several hundred neurons could be recorded in freely moving animals. COMPARISON WITH EXISTING METHODS: Compared to intracortical virus injection and open-scalp postoperative recovery, our methods minimized tissue damage and dura overgrowth underneath the optical window, and significantly increased the experimental success rate and the yield of identified neurons. CONCLUSION: Our improved cranial surgery technique allows for high-yield calcium imaging of cortical neurons with head-mounted microscopes in freely behaving animals. This technique may be beneficial for other optical applications such as two-photon microscopy, multi-site imaging, and optogenetic modulation. Published by Elsevier B.V.
BACKGROUND: In vivo optical imaging of neural activity provides important insights into brain functions at the single-cell level. Cranial windows and virally delivered calcium indicators are commonly used for imaging cortical activity through two-photon microscopes in head-fixed animals. Recently, head-mounted one-photon microscopes have been developed for freely behaving animals. However, minimizing tissue damage from the virus injection procedure and maintaining window clarity for imaging can be technically challenging. NEW METHOD: We used a wide-diameter glass pipette at the cortical surface for infusing the viral calcium reporter AAV-GCaMP6 into the cortex. After infusion, the scalp skin over the implanted optical window was sutured to facilitate postoperative recovery. The sutured scalp was removed approximately two weeks later and a miniature microscope was attached above the window to image neuronal activity in freely moving mice. RESULTS: We found that cortical surface virus infusion efficiently labeled neurons in superficial layers, and scalp skin suturing helped to maintain the long-term clarity of optical windows. As a result, several hundred neurons could be recorded in freely moving animals. COMPARISON WITH EXISTING METHODS: Compared to intracortical virus injection and open-scalp postoperative recovery, our methods minimized tissue damage and dura overgrowth underneath the optical window, and significantly increased the experimental success rate and the yield of identified neurons. CONCLUSION: Our improved cranial surgery technique allows for high-yield calcium imaging of cortical neurons with head-mounted microscopes in freely behaving animals. This technique may be beneficial for other optical applications such as two-photon microscopy, multi-site imaging, and optogenetic modulation. Published by Elsevier B.V.
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