FY2020 Annual Report

Optical Neuroimaging Unit
Professor Bernd Kuhn



In the FY 2020 the Optical Neuroimaging Unit continued the projects of the previous years. Research projects focused on imaging neuronal activity in the brain of awake mice.

1. Staff

  • Dr. Bernd Kuhn, Professor
  • Dr. Christopher J. Roome, Staff Scientist
  • Dr. Sigita Augustinaite, Staff Scientist
  • Dr. Shinobu Nomura, Postdoc
  • Dr. Claudia Cecchetto, Postdoc
  • Dr. Leonidas Georgiou, Junior Research Fellow
  • Soumen Jana, Ph.D. student
  • Lina Koronfel, Ph.D. student
  • Mohamed Mostafa Eltabbal, PhD student
  • Isabel Anai Echeverria Oviedo. PhD student
  • Cedric Galetzka, PhD student
  • Miles Desforges, PhD student (co-supervised by Kenji Doya)
  • Charlotte Denman, PhD student (Junghyun Jo Team)
  • Thato Mokhothu, PhD student (co-supervised by Kazumasa Tanaka)
  • Gaston Sivori, PhD student (so-supervised by Tomoki Fukai)
  • Dr. Kazuo Mori, Technician
  • Yohanna Sanicola, Research Unit Administrator
  • Sara Parnell, Intern student
  • Qiyi Qian, Intern student

2. Collaborations

2.1 High-resolution imaging of barrel cortex through VSD and LFP recordings

  • Description: Combining high-resolution electical recording with electrode arrays and voltage imaging
  • Type of collaboration: Joint research
  • Researchers:
    •  Associate Professor Stefano Vassanelli, Padua University, Italy

2.2 Imaging average membrane potential with ANNINE-6 in layer 1 of barrel cortex of the mouse

  • Description: Imaging voltage from cortical layer 1 in mice; synthesis of new voltage-sensitive dyes
  • Type of collaboration: Joint research
  • Researchers:
    •  Dr. Eugene Khaskin, Science and Technology Group, OIST

2.3 Motion correction of in vivo 2P imaging data

  • Description: Development of algorythms for motion correction of 2P imaging data
  • Type of collaboration: Joint research
  • Researchers:
    • Philipp Flotho, MSc., Systems Neuroscience & Neurotechnology Unit, Saarland University, Germany

2.4 Quantitative dynamics of endogenous noradrenergic transmission in the brain

  • Description: Combined Optogenetic Approaches Reveal Quantitative Dynamics of Endogenous Noradrenergic Transmission in the Brain
  • Type of collaboration: Joint research
  • Researchers:
    • Prof. Bertrand Lambolez, Neuroscience Paris Seine—Institut de Biologie Paris Seine (NPS—IBPS), France
    • Dr. Régine Hepp, Neuroscience Paris Seine—Institut de Biologie Paris Seine (NPS—IBPS), France

2.5 Subthreshold voltage oscillation  in the inferior olive

  • Description: Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro
  • Type of collaboration: Joint research
  • Researchers:
    • Assistant Prof. Marylka Yoe Uusisaari, OIST
    • Dr. Kevin Dorgans, OIST

2.6 Development of a genetic targeting mechanism for the pure electrochromic voltage-sensitive dye ANNINE-6

  • Description: Synthesis of new voltage-sensitive dyes and in vivo testing of labeling
  • Type of collaboration: Joint research
  • Researchers:
    •  Dr. Eugene Khaskin, Science and Technology Group, OIST

3. Activities and Findings

3.1 Voltage Imaging of Cortical Oscillations in Layer 1 with Two-Photon Microscopy

Membrane voltage oscillations in layer 1 (L1) of primary sensory cortices might be important indicators of cortical gain control, attentional focusing, and signal integration. However, electric field recordings are hampered by the low seal resistance of electrodes close to the brain surface. To study L1 membrane voltage oscillations, we synthesized a new voltage-sensitive dye, di1-ANNINE (anellated hemicyanine)-6plus, that can diffuse into tissue. We applied it with a new surgery, leaving the dura intact but allowing injection of large quantities of staining solution, and imaged cortical membrane potential oscillations with two-photon microscopy depth-resolved (25–100 μm below dura) in anesthetized and awake mice. We found delta (0.5–4 Hz), theta (4–10 Hz), low beta (10–20 Hz), and low gamma (30–40 Hz) oscillations. All oscillations were stronger in awake animals. While the power of delta, theta, and low beta oscillations increased with depth, the power of low gamma was more constant throughout L1. These findings identify L1 as an important coordination hub for the dynamic binding process of neurons mediated by oscillations.


3.2 Complementary Ca2+ Activity of Sensory Activated and Suppressed Layer 6 Corticothalamic Neurons Reflects Behavioral State

Layer 6 (L6) corticothalamic neurons project to thalamus, where they are thought to regulate sensory information transmission to cortex. However, the activity of these neurons during different behavioral states has not been described. Here, we imaged calcium changes in visual cortex L6 primary corticothalamic neurons with two-photon microscopy in head-fixed mice in response to passive viewing during a range of behavioral states, from locomotion to sleep. In addition to a substantial fraction of quiet neurons, we found sensory-activated and suppressed neurons, comprising two functionally distinct L6 feedback channels. Quiet neurons could be dynamically recruited to one or another functional channel, and the opposite, functional neurons could become quiet under different stimulation conditions or behavior states. The state dependence of neuronal activity was heterogeneous with respect to locomotion or level of alertness, although the average activity was largest during highest vigilance within populations of functional neurons. Interestingly, complementary activity of these distinct populations kept the overall corticothalamic feedback relatively constant during any given behavioral state. Thereby, in addition to sensory and non-sensory information, a constant activity level characteristic of behavioral state is conveyed to thalamus, where it can regulate signal transmission from the periphery to cortex.

OIST Press Release

Layer 6 pyramidal neurons in the primary visual cortex of mouse.

3.3 Combined Optogenetic Approaches Reveal Quantitative Dynamics of Endogenous Noradrenergic Transmission in the Brain

Little is known about the real-time cellular dynamics triggered by endogenous catecholamine release despite their importance in brain functions. To address this issue, we expressed channelrhodopsin in locus coeruleus neurons and protein kinase-A activity biosensors in cortical pyramidal neurons and combined two-photon imaging of biosensors with photostimulation of locus coeruleus cortical axons, in acute slices and in vivo. Burst photostimulation of axons for 5–10 s elicited robust, minutes-lasting kinase-A activation in individual neurons, indicating that a single burst firing episode of synchronized locus coeruleus neurons has rapid and lasting effects on cortical network. Responses were mediated by β1 adrenoceptors, dampened by co-activation of α2 adrenoceptors, and dramatically increased upon inhibition of noradrenaline reuptake transporter. Dopamine receptors were not involved, showing that kinase-A activation was due to noradrenaline release. Our study shows that noradrenergic transmission can be characterized with high spatiotemporal resolution in brain slices and in vivo using optogenetic tools.

3.4 Imaging PKA activity in vivo

In the CNS, protein kinase A (PKA) is controlled by neuromodulators through G-protein-coupled receptors, therefore it is thought to be involved in multiple brain functions. However, PKA activity has not been observed with cellular or subcellular resolution in vivo. 

In this study we imaged PKA activity of cortical neurons in awake mice.  A genetically encoded single GFP-based PKA sensor, GakdYmut, was expressed after adeno-associated viral gene transfer. Mice were head fixed on a cylindrical treadmill and were allowed to walk ad libitum during imaging using two-photon microscopy through a chronic cranial window.

In somatosensory cortex, PKA activity rose in dendrites and somata of neurons in layer II/III and V with the onset of locomotion, and then reached a maximal amplitude after walking offset. PKA activity in dendrites and somata peaked 20 ± 9 seconds (n = 685) and 20± 6 seconds (n = 372), respectively, after the offset of locomotion, with maximal amplitudes of ΔF/F = 14%.   PKA activity from labeled dendrites (43%) and somata (22%) showed synchronous responses greater than 2.5% ΔF/F (20s time window), in the same field of view. Similar PKA activity patterns were detected in the posterior parietal cortex, but not in the anterior cingulate cortex. Simultaneous imaging of PKA and calcium with the red calcium indicator protein rGECO revealed that PKA activation is independent of calcium influx due to spiking or bursting. We will try to identify neuromodulators which affect locomotion-related PKA activity in cortical neurons using pharmacology and electrophysiology.

3.5 Dendritic coincidence detection in Purkinje neurons of awake mice

Dendritic coincidence detection is fundamental to neuronal processing yet remains largely unexplored in awake animals. Specifically, the underlying dendritic voltage–calcium relationship has not been directly addressed. Here, using simultaneous voltage and calcium two-photon imaging of Purkinje neuron spiny dendrites, we show how coincident synaptic inputs and resulting dendritic spikes modulate dendritic calcium signaling during sensory stimulation in awake mice. Sensory stimulation increased the rate of postsynaptic potentials and dendritic calcium spikes evoked by climbing fiber and parallel fiber synaptic input. These inputs are integrated in a time-dependent and nonlinear fashion to enhance the sensory-evoked dendritic calcium signal. Intrinsic supralinear dendritic mechanisms, including voltage-gated calcium channels and metabotropic glutamate receptors, are recruited cooperatively to expand the dynamic range of sensory-evoked dendritic calcium signals. This establishes how dendrites can use multiple interplaying mechanisms to perform coincidence detection, as a fundamental and ongoing feature of dendritic integration in behaving animals.

OIST Press Release

3.6 Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

3.7 Chronic Cranial Window for Imaging Cortical Activity in Head-Fixed Mice

Chronic cranial window surgery is a critical procedure for in vivo imaging in neuroscience. Here, we describe our surgical protocol with several subtle improvements that increase the success rate significantly. The window allows high-quality imaging in head-fixed behaving mice within the first week after the surgical procedure and remains clear for months. We used this procedure to prepare mice for intrinsic signal imaging and two-photon imaging of layer 6 neurons in visual cortex.

3.8 GRACE: hiGh-Resolution imAging of the barrel CortEx through VSD and LFP recordings

The aim of this research project is to develop an innovative and advanced dual approach to study the barrel cortex in mice, combining Voltage Sensitive Dye (VSD) imaging and high-resolution electrical recordings. The research will be carried out between Japan and Italy: at OIST (Okinawa, Japan) during the initial outgoing phase and at the Department of Biomedical Sciences of the University of Padova during the returning phase.

Sensory-evoked activity in the neocortex is known to manifest in the form of propagating waves but up to now there are no studies directed towards a high-resolution mapping of these waves in the barrel cortex in vivo. VSD imaging will be performed simultaneously with high-resolution electrical mapping of Local Field Potentials (LFPs) through CMOS-based implantable neural probes developed within an EU project coordinated by UNIPD (CyberRat; FP7, #216528). Once fully established in OIST, this dual method will be transferred to UNIPD and will allow the study of neuronal signal propagation through a 3D architecture in living tissue with simultaneous high-resolution optical and electrical recordings.

3.9 Imaging neuron glia interaction in awake mice

One of the most exciting modern hypothesis in neuroscience is that astrocytes respond to neuronal signals with fast (<500ms) calcium transients that in turn can influence neuronal activity. However, it is controversial whether astrocytes respond reliably to neuronal signals in vivo. We developed a method that allows us to simultaneously record the activity of cortical astrocytes and thalamocortical axons at high contrast in the awake mouse. The method exploits the poorly characterized anterograde trans-cellular transfer properties of adeno-associated viruses (AAVs). We found that AAVs can transfer via axons to both neurons and astrocytes. This property allows us to express genetically encoded calcium indicators in axons and sparse contacting astrocytes and image their interactions with two-photon microscopy through a cranial window. We are investigating how different behavioral states (i.e. whisker stimulation, running, resting, sleeping, stress) influence the communication between axons and astrocyte microdomains in the somatosensory cortex of mice. Our findings challenge the spatial specificity of AAVs and allow us to record astrocyte-neuron interactions at high contrast under physiological conditions.


4. Publications

4.1 Journals

  1.  Flotho, P.*, Thinnes, D., Kuhn, B. , Roome, C.J., Vibell, J.F., & Strauss, D.J. (2021) Fast Variational Alignment of non-flat 1D Displacements for Applications in Neuroimaging. Journal of Neuroscience Methods.

  2. Augustinaite, S.* & Kuhn, B. (2020) Chronic cranial window for imaging cortical activity in head-fixed mice. STAR protocols. DOI: https://doi.org/10.1016/j.xpro.2020.100194

  3. Dorgans, K.*, Kuhn, B., & Uusisaari, M.Y.* (2020) Imaging subthreshold voltage oscillation with cellular resolution in the inferior olive in vitro. Frontiers in Cellular Neuroscience. DOI: https://doi.org/10.3389/fncel.2020.607843

  4. Roome, C.J.* & Kuhn, B.* (2020) Coincidence detection in Purkinje neuron dendrites of awake mice. eLife2020;9:e59619 DOI: 10.7554/eLife.59619

    OIST Press Release

  5. Nomura, S.*, Tricoire, L., Cohen, I., Kuhn, B., Lambolez, B.*, & Hepp, R.* (2020) Combined optogenetic approaches reveal the dynamics of endogenous catecholaminergic transmission in the brain. iScience 23:11. DOI: https://doi.org/10.1016/j.isci.2020.101710

  6. Augustinaite, S.* & Kuhn, B.* (2020) Complementary Ca2+ activity of sensory activated and suppressed layer 6 corticothalamic neurons reflects behavioral state. Current Biology 30:20, p.3945-3960.E5.  DOI: https://doi.org/10.1016/j.cub.2020.07.069

    OIST Press Release

  7. Dalphin, N.*, Dorgans, K., Khaskin, E., & Kuhn, B.* (2020) Voltage imaging of cortical oscillations in layer 1 with two-photon microscopy. eNeuro DOI: https://doi.org/10.1523/ENEURO.0274-19.2020

  8. Kuhn, B., Picollo, F., Carabelli, V., & Rispoli, G.* (2020) Advanced real-time recordings of neuronal activity with tailored patch pipettes, diamond multi-electrode arrays, and electrochromic voltage-sensitive dyes. Pflügers Archiv - European Journal of Physiology.

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

Nothing to report


5. Intellectual Property Rights and Other Specific Achievements

Nothing to report


6. Meetings and Events

6.1 ICIIBMS 2020

  • 5th International Conference on Intelligent Informatics and BioMedical Sciences (ICIIBMS) 
  • Date: November 18-20, 2020
  • Venue: Naha, Okinawa
  • Organized by researchers of the University of the Ryukyus, the Okinawa National College of Technology, and OIST
  • 48 participants (4 on site, 44 online) from 8 different countries

7. Other

7.1 Planning of ICIIBMS 2021

  • The 6th International Conference on Intelligent Informatics and BioMedical Sciences (ICIIBMS) is in the planning phase.
  • Date: November 25-27, 2021
  • Venue: Oita, Japan
  • Organized by researchers of the Okinawa National College of Technology and OIST

6.2 Onna-son/OIST Children’s School of Science 2020

  • Canceled due to Covid-19
Pyrite, Okinawa