Periodic Reporting for period 2 - GRACE (hiGh-Resolution imAging of the barrel CortEx through VSD and LFP recordings)
Reporting period: 2020-09-01 to 2021-08-31
The aim of this project (EU funded, Horizon 2020 Framework Programme, Project #796177 — GRACE) is to develop an innovative and advanced dual approach to study the barrel cortex, combining two-photon imaging and high-resolution electrical recording, in order to investigate in depth the spatiotemporal dynamics of whisker-evoked cortical activity in anaesthetised and awake head-restrained mouse.
The barrel cortex in rodents is part of the primary somatosensory cortex and a well-known example of topographic mapping, where each whisker on the snout of the animal is mapped onto a specific cortical area, called a barrel. Thanks to its unique functional organisation, this system offers excellent conditions to investigate neural mechanisms at the base of sensory coding, processing, and plasticity. Sensory-evoked activity in the barrel cortex is known to manifest as 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.
Thanks to the chronic cranial window designed and patented by prof. Kuhn at OIST, Voltage Sensitive Dye and Calcium imaging will be performed simultaneously with Local Field Potentials (LFP) recordings through high-resolution implantable neural probes for the first time. Once fully established, this multimodal technique will allow the study of neuronal signal propagation through a 3D architecture in living tissue with simultaneous high-resolution optical and electrical recordings. This action will promote a close collaboration and a two-way transfer of scientific knowledge between Padova (UNIPD) and Okinawa (OIST), enhancing the scientific relations between the European Research Area and Japan. This project will also contribute to the design and development of advanced neuroprostheses which will allow a bidirectional communication with brain microcircuits through high-resolution LFP recordings and patterned electrical stimulation of neural populations. Moreover, the results of this research will have a strong impact on basic neuroscience of population coding, providing novel insights on the propagation of sensory information within the barrel cortex and offering a new advanced technique to study the brain in the upcoming research projects.
A methodological paper about the new technique and the first results are published in Frontiers in Neuroscience – Brain Imaging Methods with the title "Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice" (Authors: C. Cecchetto*, S. Vassanelli, B. Kuhn*, http://doi.org/10.3389/fnins.2021.741279(opens in new window) Associated data are available on Dryad at https://doi.org/10.5061/dryad.dbrv15f23(opens in new window)).
The barrel cortex in rodents is part of the primary somatosensory cortex and a well-known example of topographic mapping, where each whisker on the snout of the animal is mapped onto a specific cortical area, called a barrel. Thanks to its unique functional organisation, this system offers excellent conditions to investigate neural mechanisms at the base of sensory coding, processing, and plasticity. Sensory-evoked activity in the barrel cortex is known to manifest as 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.
Thanks to the chronic cranial window designed and patented by prof. Kuhn at OIST, Voltage Sensitive Dye and Calcium imaging will be performed simultaneously with Local Field Potentials (LFP) recordings through high-resolution implantable neural probes for the first time. Once fully established, this multimodal technique will allow the study of neuronal signal propagation through a 3D architecture in living tissue with simultaneous high-resolution optical and electrical recordings. This action will promote a close collaboration and a two-way transfer of scientific knowledge between Padova (UNIPD) and Okinawa (OIST), enhancing the scientific relations between the European Research Area and Japan. This project will also contribute to the design and development of advanced neuroprostheses which will allow a bidirectional communication with brain microcircuits through high-resolution LFP recordings and patterned electrical stimulation of neural populations. Moreover, the results of this research will have a strong impact on basic neuroscience of population coding, providing novel insights on the propagation of sensory information within the barrel cortex and offering a new advanced technique to study the brain in the upcoming research projects.
A methodological paper about the new technique and the first results are published in Frontiers in Neuroscience – Brain Imaging Methods with the title "Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice" (Authors: C. Cecchetto*, S. Vassanelli, B. Kuhn*, http://doi.org/10.3389/fnins.2021.741279(opens in new window) Associated data are available on Dryad at https://doi.org/10.5061/dryad.dbrv15f23(opens in new window)).
The main aim of GRACE project, in vivo dual simultaneous recording of two-photon imaging and LFP signals in the mouse barrel cortex, was achieved at OIST (Japan) and in UNIPD (Italy) using both viral expressed Calcium indicators and Voltage Sensitive Dyes along with implantable neuro probes. A commercial sensor having a linear 32-channels recording array on the tip was inserted in the mouse brain under the objective of the two-photon imaging setup and LFP signals were recorded simultaneously with imaging data, both during anaesthesia and awake state, while the mouse was free to walk, run or rest on a custom-made treadmill. Once inserted in the brain, the needle of the probe covered 4 out of 6 cortical layers.
Along with simultaneous LFP recordings, two-photon calcium or VSD imaging was acquired at 3 depths, corresponding to layer I, II and IV of the mouse barrel cortex via full-frame scans or box scans (having a much higher imaging frequency).
With this system, both spontaneous and evoked activity could be acquired: during the stimulation epochs, the whiskers of the mouse were deflected by means of air-puffs delivered with random timing, with an average interstimulus interval of about 10 seconds.
Methods for analysing the signals with custom MATLAB and ImageJ routines were designed and implemented during the whole data acquisition period, in parallel with data collection. These algorithms were used to load raw data, remove motion artefacts (frequently generated by the breathing or running of the animal), select Regions of Interest (ROIs) and compute stimulus-locked averages of evoked responses and frequency spectra of Ca2+, VSD and LFP signals. A preliminary analysis was first focused on comparing the shapes of evoked responses in LFP and VSD signals recorded from different cortical layers and different mice and on the frequency content of spontaneous activity traces recorded by both the acquisition systems at the same cortical depths, both during anaesthesia and awake state.
A methodological paper about the new technique and the first results was published in Frontiers in Neuroscience – Brain Imaging Methods with the title "Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice" (Authors: C. Cecchetto*, S. Vassanelli, B. Kuhn*, https://doi.org/10.3389/fnins.2021.741279(opens in new window) Associated data available on Dryad at https://doi.org/10.5061/dryad.dbrv15f23(opens in new window)).
Along with simultaneous LFP recordings, two-photon calcium or VSD imaging was acquired at 3 depths, corresponding to layer I, II and IV of the mouse barrel cortex via full-frame scans or box scans (having a much higher imaging frequency).
With this system, both spontaneous and evoked activity could be acquired: during the stimulation epochs, the whiskers of the mouse were deflected by means of air-puffs delivered with random timing, with an average interstimulus interval of about 10 seconds.
Methods for analysing the signals with custom MATLAB and ImageJ routines were designed and implemented during the whole data acquisition period, in parallel with data collection. These algorithms were used to load raw data, remove motion artefacts (frequently generated by the breathing or running of the animal), select Regions of Interest (ROIs) and compute stimulus-locked averages of evoked responses and frequency spectra of Ca2+, VSD and LFP signals. A preliminary analysis was first focused on comparing the shapes of evoked responses in LFP and VSD signals recorded from different cortical layers and different mice and on the frequency content of spontaneous activity traces recorded by both the acquisition systems at the same cortical depths, both during anaesthesia and awake state.
A methodological paper about the new technique and the first results was published in Frontiers in Neuroscience – Brain Imaging Methods with the title "Simultaneous two-photon voltage or calcium imaging and multi-channel LFP recordings in barrel cortex of awake and anesthetized mice" (Authors: C. Cecchetto*, S. Vassanelli, B. Kuhn*, https://doi.org/10.3389/fnins.2021.741279(opens in new window) Associated data available on Dryad at https://doi.org/10.5061/dryad.dbrv15f23(opens in new window)).
During the first part of the project, the innovative dual technique was set up, allowing the simultaneous recording of in-plane VSD imaging and in-depth LFP signals from different cortical layers through implantable probes for the first time. The main target by the end of the project is to transfer this dual method to UNIPD and to collect a reliable dataset in order to be able to deeply investigate the neuronal coding in the barrel cortex using this new powerful technique.
At the end of the project, we validated the combined recording of neuronal activity in vivo through two-photon voltage or calcium imaging and LFP recordings in the mouse somatosensory cortex, confirming the possibility of using the two techniques simultaneously with highly reduced optical noise and no photo-induced interferences in the electrical recordings, both at OIST and at UNIPD.
The potential impacts for future research in neuroscience are numerous: this multimodal technique can be learnt and used in the coming future by other researchers investigating different aspects and regions of the brain.
This newly developed method will hopefully open a new range of possibilities for many research groups studying neuronal networks all over the world.
We envision that this platform could be used and expanded in many awake and behavioural experiments targeting different brain regions, possibly with chronically implanted neuro probes and flexible connectors, which will make it easier to perform repeated experiments on the same animal.
Most importantly, we hope our dataset will help theorists to explain how current sinks and sources connect measured LFP signals to average membrane potential changes measured with the pure electrochromic VSD ANNINE-6plus, and how these voltage and current changes trigger calcium changes.
In the long term, the results of this project will also contribute to the development of new patentable and marketable neural interfaces.
These advanced devices will allow the neuromodulation of brain microcircuits by efficient ‘read-out’ (via high-resolution LFP recordings) and ‘write-in’ (by means of patterned electrical stimulation of neuronal groups) of information from and to the brain.
At the end of the project, we validated the combined recording of neuronal activity in vivo through two-photon voltage or calcium imaging and LFP recordings in the mouse somatosensory cortex, confirming the possibility of using the two techniques simultaneously with highly reduced optical noise and no photo-induced interferences in the electrical recordings, both at OIST and at UNIPD.
The potential impacts for future research in neuroscience are numerous: this multimodal technique can be learnt and used in the coming future by other researchers investigating different aspects and regions of the brain.
This newly developed method will hopefully open a new range of possibilities for many research groups studying neuronal networks all over the world.
We envision that this platform could be used and expanded in many awake and behavioural experiments targeting different brain regions, possibly with chronically implanted neuro probes and flexible connectors, which will make it easier to perform repeated experiments on the same animal.
Most importantly, we hope our dataset will help theorists to explain how current sinks and sources connect measured LFP signals to average membrane potential changes measured with the pure electrochromic VSD ANNINE-6plus, and how these voltage and current changes trigger calcium changes.
In the long term, the results of this project will also contribute to the development of new patentable and marketable neural interfaces.
These advanced devices will allow the neuromodulation of brain microcircuits by efficient ‘read-out’ (via high-resolution LFP recordings) and ‘write-in’ (by means of patterned electrical stimulation of neuronal groups) of information from and to the brain.