FY2018 Annual Report

Optical Neuroimaging Unit
Associate Professor Bernd Kuhn

Chris Roome, Sigita Augustinaite;
Claudia Cecchetto, Miles Desforges;
Isabel Anai Echeverria Oviedo, Shinobu Nomura, Eltabbal Mohamed Mostafa Kamal;
Lina Mohamed Koronfel, Hiroko Chinone, Kazuo Mori, Leonidas Georgiou;
Soumen Jana, Bernd Kuhn, Neil Dalphin

Photo by Emiko Asato

Abstract

In the FY 2018 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, Associate Professor
Dr. Christopher J. Roome, Staff Scientist
Dr. Sigita Augustinaite, Staff Scientist
Dr. Shinobu Nomura, Postdoc
Ray X. Lee, Ph.D. student
Neil Dalphin, Ph.D. student
Leonidas Georgiou, Ph.D. student
Soumen Jana, Ph.D. student
Lina Koronfel, Ph.D. student
Dr. Kazuo Mori, Technical Assistant
Hiroko Chinone, Research Unit Administrator

2. Collaborations

2.1 Post-traumatic behavioral development in adult mice

Description: Behavioral study with fine scale analysis of post-traumatic behavioral development in adult mice
Type of collaboration: Joint research
Researchers:
- Associate Professor Greg Stevens, OIST and Vrije Universiteit Amsterdam

2.2 Imaging cortical activity during spontaneous behavior

Description: Calcium imaging of cortical neurons in different regions and layers during spontaneous behavior
Type of collaboration: Joint research
Researchers:
- Associate Professor Greg Stevens, OIST and Vrije Universiteit Amsterdam

2.3 High-resolution imaging of the 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

2.4 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

3. Activities and Findings

3.1 Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice

Spatio-temporal maps of dendritic signalling and their relationship with somatic output is fundamental to neuronal information processing, yet remain unexplored in awake animals. Here, we combine simultaneous sub-millisecond voltage and calcium two-photon imaging from distal spiny dendrites, with somatic electrical recording from spontaneously active cerebellar Purkinje neurons (PN) in awake mice. We detect discrete 1-2 ms supra-threshold voltage spikelets in the distal spiny dendrites during dendritic complex spikes. Spikelets and their calcium correlates are highly heterogeneous in number, timing and spatial distribution within and between complex spikes. Back-propagating simple spikes are highly attenuated. Highly variable 5-10 ms voltage hotspots are localized to fine dendritic processes and are reduced in size and frequency by lidocaine and CNQX. Hotspots correlated with somatic output but also, at high frequency, trigger purely dendritic calcium spikes. Summarizing, spatio-temporal signalling in PNs is far more complex, dynamic, and fine scaled than anticipated, even in resting animals.

Double-labelling individual neurons for combined voltage and calcium two-photon imaging in awake mice. (a) 5-mm glass cover slip with silicone access port (Roome & Kuhn 2014). (b) A chronic cranial window with access port on the vermis of the cerebellum allows access to the brain with a pipette (schematically indicated). (c) ANNINE-6plus dissolved in pure ethanol at 3 mM concentration. (d) Sketch of the setup with a mouse mounted on a treadmill under a two-photon microscope. An electrode is used to fill single neurons by electroporation and to electrically record from their soma. A behavioral camera allows detailed observation of the pupil, the vibrissa, and the face of the mouse. (e-h) A patch pipette filled with ANNINE-6plus/ethanol solution is used to label single GCaMP6f expressing neurons by electroporation in the anesthetized mouse. (i) During the imaging experiment the mouse is fully awake, sitting on a treadmill and monitored with behavioral a camera. 24 hours after labelling a Purkinje neuron with ANNINE-6plus, the dye has spread out evenly, as can be seen in (j) the cross section of the Purkinje neuron dendrite as an overlay of the green channel (GCaMP6f) and the red channel (ANNINE-6plus) and in (k) the reconstruction of the Purkinje neuron in the red channel (ANNINE-6plus). The dotted line indicates the line scan position used in Fig. 4 and 5. It is also possible to fill other neurons with ANNINE-6plus by electroporation, as, for example, (l) cortical layer 2/3 pyramidal neurons shown as overlaid z-projection of the green channel (GCaMP6f) and the red channel (ANNINE-6plus).

Simultaneous voltage and calcium imaging of Purkinje neuron dendrites and somatic recording in the awake mouse. (a) A line scan at 2 kHz was taken along the Purkinje neuron dendrites (scan position shown in Fig. 3j) to record a voltage spatio-temporal map in an awake mouse. The spatially averaged dendritic voltage (red trace) clearly shows suprathreshold dendritic complex spikes (black triangles). (b) The corresponding dendritic calcium spatio-temporal map and spatially averaged dendritic calcium (green trace) shows large calcium transients for every dendritic complex spike. (c) The access port also allowed simultaneous extracellular electrical recordings from the soma (black trace) while imaging voltage and calcium transients from the dendrites. Simple spikes (somatic Na+ spikes) result in a current sink at the soma, while complex spikes (dendritic Ca2+ spikes) result in a dominant current source signal at the soma. (d) Different parts of the dendritic tree show a different number of spikelets during the same complex spike event. The number of spikelets correlate with the amplitude of the calcium transients in each part of the dendritic tree. Open arrowheads indicate spatially localized low activity, filled arrowheads show high activity. Spatially localized dendritic spikelets during complex spikes correlate with a local boost in the dendritic calcium transient (small arrowheads). (Roome & Kuhn 2018)

Sub- and suprathreshold dendritic signaling in awake mice. (a) Dendritic voltage spatio-temporal maps show epochs of low and high frequency subthreshold ‘hotspot’ events in Purkinje neuron dendrites (scan position shown in Fig. 3j). White arrow heads indicate single hotspot events. (b) The corresponding calcium spatio-temporal map does not show any correlated calcium transients except following suprathreshold complex spikes and dendritic spikes indicated in (a) by filled and open triangles, respectively. (c) By thresholding and additional spatio-temporal selection criteria, a spatio-temporal hotspot map can be generated. (d) Hotspot activity correlates with the simple spike (SS) activity at the soma. (e) Spatially averaged dendritic voltage (red) and calcium (green) recorded at 2kHz, reveal rapid (1-2 ms) and variable suprathreshold dendritic spikelets during complex spikes (filled triangle). Extracellular somatic recordings (black) were used to identify the somatic output signals; simple spikes (SS: black binary trace) and complex spikes (CS: red binary trace). Non-climbing fiber evoked suprathreshold dendritic calcium spikes (open triangles) were detected in the awake mouse which enhance local calcium influx and showed no coincident sodium influx (simple spike) at the soma. (Roome & Kuhn 2018)

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

Membrane voltage oscillations in layer 1 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 layer 1 membrane voltage oscillations, we synthesized a new voltage-sensitive dye, di1-ANNINE-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 to 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 layer 1. These findings identify layer 1 as an important coordination hub for the dynamic binding process of neurons mediated by oscillations.

3.3 Two-photon imaging of layer 6 corticothalamic feedback in a behaving mouse

Layer 6 (L6), the deepest lamina of cerebral cortex, is one of the key structures regulating behavior state related information processing within cortex and various subcortical areas. However, very little is known about the functional significance of different L6 circuits in vivo. In this project, we focus on primary visual cortex L6 feedback projections to visual thalamus (dorsal lateral geniculate nucleus, dLGN) which regulate visual signal transmission from retina to cortex. We perform calcium imaging of retrogradely marked L6 corticothalamic (CT) neurons with 2P microscopy in a head-fixed Ntsr1-cre mouse. We record neuronal activity from the same neurons for several hours and / or repeat during different days while presenting visual stimuli and monitoring the activity of a mouse. In this way, we can study the corticothalamic feedback during different behavior states, ranging from full alertness to sleep.

3.4 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.

3.5 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.

3.6 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.

3.7 Social relationship and brain hemisphere-specific correlates explain stress incubation in adult mice

While stress reactions can emerge long after the triggering event, it remains elusive how they emerge after a protracted, seemingly stress-free period, during which stress incubates. Here, we study the behavioral development in mice isolated after observing an aggressive encounter inflicted upon their pair-housed partners and compared the results with those in multiple control paradigms. Mice isolated following the acute witnessing social stress gradually developed a wide range of long-term differences, compared to control, of their spontaneous behaviors, social interactions, and physiological conditions, including hemisphere-specific structural differences in multiple cerebrocortical areas. We developed a spatially resolved fine-scale behavioral analysis and applied it to standard behavioral tests. It reveals that the seemingly sudden emergent behavioral differences developed gradually. Interestingly, these behavioral differences were not observed if the aggressive encounter happened to a stranger mouse, suggesting that social relationship underlies the stress development in this paradigm, which may have important disease-relevant links.

3.8 Imaging Ca²⁺ concentration and pH in nanopores/channels of protein crystals

Protein crystals are nanoporous materials. Despite this important characteristic, little is known about the conditions in the pores, also called channels. Here, we describe a method to study the calcium concentration and pH in the nanopores of thaumatin and lysozyme crystals. We load the crystal nanopores with fluorescent indicators and then perfuse the crystals with solutions of different calcium concentrations and pH while reading out the crystal’s fluorescence intensity with confocal microscopy. By calibrating the fluorescence signal we can determine the calcium concentration and pH in the nanopores. For the pH in thaumatin nanopores measured with the ratiometric pH sensor SNARF-1 we find a -0.7 pH shift compared to the bath pH corresponding to a 5-fold higher proton concentration. This is similar to the -0.3 pH shift found in lysozyme nanopores. With single-wavelength probes we find that the calcium concentration in thaumatin crystal nanopores is the same as in the bath while it is 0.24 times lower in lysozyme nanopores. Summarizing, our experiments show that calcium concentration and pH in the nanopores of protein crystals can deviate significantly from that in the bath. In general, the described method can be applied for testing a wide range of ion or small molecule concentrations in transparent nanoporous materials not only with ratiometric but also with single wavelength fluorescent indicators.

**Figure 4** Protein crystals can be labeled with synthetic calcium sensitive fluorescent dyes (left). This allows measuring the calcium concentration in the nanopores of protein crystals.

4. Publications

4.1 Journals

C.J. Roome* and B. Kuhn* (2018) Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice. Nature communications, DOI: 10.1038/s41467-018-05900-3
K. Mori* and B. Kuhn* (2018) Imaging Ca²⁺ concentration and pH in nanopores/channels of protein crystals. J.Phys.Chem.B 122, 42, 9646-9653
S. Augustinaite and P. Heggelund (2018) Short-term synaptic depression in the feedforward inhibitory circuit in the dorsal lateral geniculate nucleus. Neuroscience 384, 76-86.

4.2 Books and Other One-Time Publications

Nothing to report

4.3 Oral and Poster Presentations

S. Augustinaite (2019) Two-photon imaging of the deepest cortical layer (layer 6) in the behaving mouse. Internal Seminar at OIST
R.X. Lee and B. Kuhn (2018) Neuronal dynamic framework of cerebral cortical networks for spontaneous behaviors. ICIIBMS 2018, Bangkok, Thailand
S. Augustinaite and B. Kuhn (2018) V1 layer 6 corticothalamic feedback encodes behavioral state by complementary activity of two neuronal populations. Resonance Biology for Innovative Bioimaging, Meeting of Bioimaging for Young Researchers, OIST
C.J. Roome and B. Kuhn (2018) Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice. Resonance Biology for Innovative Bioimaging, Meeting of Bioimaging for Young Researchers, OIST
N. Dalphin and B. Kuhn (2018) Simultaneous voltage and calcium imaging of mouse barrel cortex, in vivo. Resonance Biology for Innovative Bioimaging, Meeting of Bioimaging for Young Researchers, OIST
S. Nomura and B. Kuhn (2018) Imaging Protein Kinase A Activity in Somatosensory Cortex of Behaving Mice. Resonance Biology for Innovative Bioimaging, Meeting of Bioimaging for Young Researchers, OIST
L. Georgiou and B. Kuhn (2018) AAV mediated trans-synaptic tagging of astrocytes: A novel tool for studying neuron-astrocyte interactions. Society for Neuroscience Annual Meeting, San Diego, CA, USA
S. Augustinaite and B. Kuhn (2018) V1 layer 6 corticothalamic feedback encodes behavioral state by complementary activity of two neuronal populations. Society for Neuroscience Annual Meeting, San Diego, CA, USA
S. Nomura and B. Kuhn (2018) Imaging cortical Protein Kinase A activity in dendrites and somata of awake mice. FENS 2018, Berlin, Germany.
Eva Berlot, Noah Steinberg, Tyler Manning, and Lina Koronfel (2018) Analyzing the Effect of Noise Correlations on Decoding Neuronal Recording. Summer School in Computational Sensory-Motor Neuroscience, University of Minnesota, Minneapolis, MN, USA.

4.4 Invited Talks

OIST Minisymposium The 16th International Membrane Research Forum, OIST. Bernd Kuhn: Membrane voltage imaging with the pure electrochromic probe ANNINE-6. Organized by Prof. A. Kusumi, K. Kono (OIST). March 20, 2019.
5th Core-to-Core International Symposium "3D Lab-Exchange Program”, OIST. Bernd Kuhn: Voltage imaging from single neurons in awake animals. Organized by Prof. T. Inoue (Waseda University). February 27, 2019.
Photonics Workshop, OIST. Bernd Kuhn: Imaging neuronal activity with two-photon microscopy in awake mice. Organized by Prof. J. Hayase (KEIO University). November 30, 2018.
Resonance Biology for Innovative Bioimaging, Meeting of Bioimaging for Young Researchers “Chanpuru”, OIST. Bernd Kuhn: Two-photon microscopy: Principles and in vivo applications. Organized by Drs. K. Otomo, H. Ishii (Hokkaido University). October 29, 2018.
Merocyanine 540 - 45+1 Years of Voltage Imaging, Woods Hole, MA, USA. Bernd Kuhn: Simultaneous spatio-temporal dendritic voltage/calcium mapping and somatic recording from Purkinje neurons in awake mice. Organized by Profs. L.B. Cohen, D. Zecevic, B.M. Salzberg, August 30-September 1, 2018.
OIST-KAIST Joint Symposium, OIST. Bernd Kuhn: Simultaneous spatio-temporal dendritic voltage/calcium mapping and somatic recording from Purkinje neurons in awake mice. Organized by Profs. A. Shen, B. Kuhn (OIST). August 20, 2018.
Molecular Biology Department, Princeton University, Princeton, NJ, U.S.A. Bernd Kuhn: Adeno-Associated Virus (AAV) transfer from axons to astrocytes. Invited by Prof. S.J. Flint. June 16, 2018.
Princeton Neuroscience Institute, Princeton University, Princeton, NJ, U.S.A. Bernd Kuhn: Simultaneous spatio-temporal dendritic voltage/calcium mapping and somatic recording from Purkinje neurons in awake mice. Invited by Prof. S.S.-H. Wang. June 15, 2018.

5. Intellectual Property Rights and Other Specific Achievements