Advancing Basic Science for Humanity
Fabrication and testing of implanted magnetic microcoils to stimulate neurons… Optogenetic-induced seizure model to enhance understanding of interneuron roles… New optical technique to identify voltage activity patterns in specific cell types…
Novel implantable magnetic microcoils stimulate cortical cells and consistently drive behavioral responses
Modulating neuronal activity through implanted devices, like electrodes and electrode arrays, offers potential relief from neurological disorders and opportunities for prosthetics that rely on brain-machine interfaces. Unfortunately, most implanted devices suffer from limitations including imprecise cellular activation (whether of cell types or passing fibers) and inflammation at the implant site. At Massachusetts General Hospital, Dr. Shelley Fried’s group proposes the use of novel magnetic microcoils – no larger than the width of a mouse cortical column – that can be safely and chronically implanted in the cortex to allow more targeted stimulation of cells to reliably elicit behaviors. To follow up on computational models of the coils, the group tested two coil designs in vitro, determining that the microcoils were most effective when oriented parallel to mouse pyramidal neurons and could stimulate regions as narrow as 60 µm at significantly reduced activation thresholds without spreading to passing axons. Dr. Fried’s team then implanted the coils in mouse motor and sensory cortices associated with whisking behaviors to illustrate the safety and efficacy of the designs when modulating whisker movement. These microcoils could offer a longer-lasting method of more selective cortical stimulation that can withstand the inflammation response relative to existing electrode-based technologies.The figure above compares the spread of excitation due to stimulation from a standard microelectrode (panels A and B) or a microcoil (panels C and D) in a cortical slice from a transgenic mouse. Neurons express a calcium indicator that exhibits increased fluorescence with increased spiking activity. Panels E and F depict how the asymmetric distribution of excitation from the microcoil allows for more targeted stimulation of neurons parallel to the coil, with actual calcium signals plotted in panel G.
Optogenetics enables identification of distinct roles for interneuron subtypes in mouse model of seizures
Extensive work on the mechanisms underlying seizures and epilepsy has identified a role for GABAergic interneurons, but models of spontaneous seizure activity lack the temporal control necessary to parse cell-type specific roles in the genesis and maintenance of seizures. At the University of California, San Francisco, Dr. Vikaas Sohol’s lab employed optogenetic and photometric imaging techniques to reliably and repeatedly induce seizure activity in mice with high temporal precision, simultaneously record electrical activity using electroencephalogram (EEG) and cell-type specific calcium signals, and manipulate the activity of selected subtypes of GABAergic interneurons to modulate seizure activity. The group expressed channelrhodopsin in mouse primary motor cortex to induce seizure activity using a sequence of optogenetic stimuli with temporal specificity. Simultaneous with optogenetic seizure induction, they used fiber photometry to record from labeled interneuron subtypes expressing parvalbumin (PV+), somatostatin (SOM+), or vasoactive intestinal peptide (VIP+), as well as excitatory neurons in neocortex. They demonstrated that all of the interneurons activate within one second following seizure onset, excitatory neurons exhibit an approximately 10 s delay, and calcium signaling varies across cell types during seizure activity. To explore causal roles for the different interneurons before and during seizures, the group again relied on optogenetics to systematically inhibit each subtype. Inhibition of VIP+ interneurons contralateral to the seizure induction site increased seizure threshold, reduced seizure probability, and reduced seizure duration, while inhibition of PV+ and/or SOM+ interneurons inconsistently modulated seizure activity. Overall, this novel, time-selective model of optogenetically-induced seizures revealed the anti-seizure effect of inhibiting VIP+ interneurons, providing a novel potential therapeutic target for anti-epileptic therapies.Panel A (left) depicts optogenetic induction of seizure activity (blue light from “a”) and optogenetic inhibition (red light from “a” and “b”) of a specific interneuron population (green) ipsilateral or contralateral to the seizure induction site. Bilateral head screws allow for simultaneous EEG recording. Panel A (right) outlines experimental paradigms to establish baseline seizure activity (top), optogenetic inhibition of ipsilateral interneurons (middle), or optogenetic inhibition of contralateral interneurons (bottom) during seizure initiation and maintenance. Panels B and C illustrated increased seizure probability from inactivation of PV+ and SOM+ interneurons, regardless of location, and decreased seizure probability from inactivation of contralateral VIP+ interneurons. Panel D charts the associated shifts in threshold to seizure initiation. Panel E displays a reduction in seizure duration when PV+ and SOM+ interneurons are inhibited, and when contralateral VIP+ interneurons are inhibited.
Transmembrane Electrical Measurements Performed Optically (TEMPO) records voltage activity of distinct cell types in freely moving animals with high sensitivity
Techniques to measure the dynamics of neural activity, including intracellular recordings and genetically-encoded calcium level indicators, have advanced significantly, but researchers struggle to distinguish both spiking and oscillatory contributions of multiple cell types in a given brain region with high temporal sensitivity – particularly in behaving animals. Dr. Mark Schnitzer’s team at Stanford University describe an optical imaging technique, TEMPO, that is 10 times more sensitive than calcium indicators, records voltage signals near the physical sensitivity limitsGraphical diagram comparing local field potential (LFP) voltage recording technique, which is unable to distinguish between interspersed neuronal subtypes, and TEMPO, which can selectively record transmembrane voltage dynamics from a single cell type (ex. dopamine receptor subtype 2 (D2) expressing medium spiny neurons.
of optical imaging, and can distinguish hyperpolarization states of interspersed cell types with high fidelity. First, Dr. Schnitzer and colleagues validated TEMPO by measuring cortical and hippocampal oscillatory activity in anesthetized and awake, behaving mice, reproducing data well-established in studies using electroencephalogram (EEG) and local field potential (LFP) recordings. Next, the researchers selectively monitored voltage dynamics of two medium spiny neuron subtypes using transgenic animals, revealing two forms of synchronized hyperpolarization they could connect to distinct brain states in freely behaving mice, all previously unresolved by EEG or LFP. Taken together, these data identify TEMPO as a highly sensitive method to record neural voltage dynamics in specific cell types in awake and behaving animals at significantly higher resolution than current recording techniques.Panels A and B illustrate the targeting of distinct neuronal subtypes (D1 or D2-expressing striatal neurons) with a genetically-encoded green voltage sensor and red reference in transgenic mice. Panel C illustrates two, distinct forms of hyperpolarization (marked with black dots: high-voltage spindles and temporally isolated events) that were measured by TEMPO in D1-expressing neurons while animals were at rest.
Recent meetings of the Multi-Council Working Group (MCWG) for the NIH BRAIN Initiative and the MCWG Neuroethics Division provided updates on BRAIN’s scientific progress, considerations of the neuroethical issues surrounding the science, and discussions on how to continue working toward the goals of the BRAIN 2025 report…
On February 14th, 2017, the Neuroethics Division of the Multi-Council Working Group (MCWG) to the National Institutes of Health (NIH) Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative® held its third in-person meeting. The Neuroethics Division serves as a resource of neuroethics expertise, to help navigate the unique ethical considerations entailed by BRAIN-supported research. The goal of the meeting was to review the Division’s previous efforts and strategize next steps. Dr. Walter Koroshetz, Director of the National Institute of Neurological Disorders and Stroke (NINDS), opened the meeting with a current snapshot of the NIH BRAIN Initiative. The Division has been busy during its first year, with its members having published various articles regarding the BRAIN Initiative and neuroethics, organized ongoing conversations with stakeholders (including BRAIN investigators and partner organizations) interested in neuroethics, provided a consultation to a BRAIN investigator, and produced the first in a series of neuroethics one-pagers for investigators. The Division focused on determining where to place its attention and resources moving forward.
To that end, the Division heard additional input from a few partner organizations, including the Kavli Foundation, Korean Brain Initiative, and Food and Drug Administration (FDA). NIH program officers in attendance engaged the Division in a discussion of how to prepare for potential future, unanticipated ethical concerns, while addressing pragmatic neuroethics questions prompted by current research projects. Division member Dr. Rafael Yuste from Columbia University advocated for developing ethical guidelines for novel neurotechnologies. The group discussed his proposal and the value of working to define neuroethical considerations that may be unique to the BRAIN Initiative. Additionally, the group discussed leveraging the annual BRAIN Initiative Investigators Meeting as an optimal venue to continue efforts to raise the profile of neuroethics, potentially with a plenary neuroethics talk. The Division also discussed the need for neuroethics training for the next generation of neuroscientists.
The following day, the MCWG convened to discuss the current state of the BRAIN Initiative and its future. The group included MCWG members, directors and staff from the 10 NIH Institutes and Center supporting the Initiative, representatives from the White House Office of Science and Technology Policy, National Science Foundation, FDA, Intelligence Advanced Research Projects Activity (IARPA), Kavli Foundation, Simons Foundation, and Allen Institute for Brain Science, and members from the MCWG Neuroethics Division. The meeting began with welcoming remarks from Dr. Alan Willard, Acting Deputy Director of NINDS and the MCWG Designated Federal Official, who announced the addition of Dr. Eve Marder from Brandeis University as an at-large member of the MCWG. Dr. Marder replaces Dr. Cori Bargmann, who resigned from the MCWG because of the additional demands on her time as the new leader of the Chan-Zuckerberg Initiative. The meeting included several updates from:
- NIH BRAIN Initiative by Dr. Koroshetz and Dr. Joshua Gordon, Director of the National Institute for Mental Health (NIMH)
- MCWG Neuroethics Division by co-chairs Prof. Hank Greely and Dr. Christine Grady
- IARPA by Dr. David Markowitz, who administers the MICrONS project
- Allen Institute for Brain Science by Dr. Christof Koch, President and Chief Scientific Officer
The meeting then had a discussion of evaluation of the NIH BRAIN Initiative, led by Dr. Paul Scott, Director of the NINDS Office of Science Policy and Planning, and by Dr. Meredith Fox, Director of the NIMH Office of Science Policy, Planning, and Communications. The MCWG suggested tracking the extent to which new technologies have been disseminated to the community, associated training efforts, the status of diversity in the BRAIN workforce, and the number of publications citing BRAIN support. The group also stressed the importance of ensuring that methodologies exist for monitoring and sharing tools and data created through the Initiative, and proposed using the NIH BRAIN Initiative and BRAIN Initiative Alliance websites to support disseminating such information.
Next, Dr. Greg Farber, the Director of NIMH’s Office of Technology Development and Coordination, and other program staff reported an analysis of how the current set of NIH BRAIN Initiative awards fit onto the roadmap outlined in the BRAIN 2025 report. Within the context of developing tools to understand brain cells and circuits, the MCWG discussed the merits of a funding opportunity specifically focused on non-neuronal brain cells. They also suggested the incorporation of an independent group to review the standards and software development tools that are being proposed in applications. Furthermore, the group considered how to better involve software engineers, computational scientists, and theorists, the importance of training programs for computational and quantitative methods, and the idea of re-forming the BRAIN Advisory Committee to assess the BRAIN Initiative as it enters its second phase (2020-2026).
The meeting concluded with a discussion about the annual BRAIN Initiative Investigators Meeting. Dr. Amy Adams, Director of the NINDS Office of Scientific Liaison, provided information on the third annual meeting that occurred on December 12-14, 2016. The next meeting is being planned for early April 2018. The MCWG offered suggestions to help make the next meeting even more successful, including reducing the number of parallel sessions and replacing the evening talks with poster sessions.
Overall, the MCWG meeting established that the BRAIN Initiative has had a strong beginning phase. As the Initiative approaches its next stage, it will become increasingly important to evaluate outcomes, support dissemination of the tools/technology/data, and bolster training so scientists can incorporate BRAIN tools and technologies and leverage the data sets. Recruitment of non-traditional neuroscientists and incorporation of interdisciplinary approaches remain an important aspect of BRAIN, and measures indicate that the field continues to grow.
The next MCWG and Neuroethics Division meetings are being planned for August 2017.
In December, the NIH BRAIN Initiative held its third annual BRAIN Initiative Investigators Meeting, gathering experts from around the globe to share their cutting-edge BRAIN-funded research. The attendees spent three days learning about each other’s work, forming new collaborations, and hearing from members of the BRAIN Initiative Alliance. The meeting featured many interesting panel discussions about the direction and implications of BRAIN Initiative research. On the second day of the meeting, a group of ethicists and scientists discussed some of the ethical implications of the technological advances emerging from BRAIN Initiative research. The panel included five members of the Neuroethics Division of the NIH BRAIN Multi-Council Working Group (MCWG), Drs. Christine Grady, Karen Rommelfanger, Rafael Yuste, Khara Ramos, Prof. Hank Greely, as well as Dr. Winston Chiong, a University of California, San Francisco (UCSF) professor and co-investigator on an NIH BRAIN grant. The neuroethics panel focused on defining neuroethics, why it is important to the scientific community, and how the scientific community might address neuroethical implications of their work.
Dr. Christine Grady provided an introduction to the session, discussing what neuroethics entails and how the NIH is working to integrate neuroethics in the BRAIN Initiative. Dr. Grady emphasized that neuroethics is distinct from research ethics and regulatory compliance. That is to say, neuroethics is a combination of both neuroscience and philosophy that explores the unexpected ethical consequences of neuroscience research. To follow up on this point, the director of the National Institute of Neurological Disorders and Stroke, Dr. Walter Koroshetz, spoke about the importance of integrating neuroethics into the BRAIN Initiative’s priorities. Current and future BRAIN Initiative research studies aim to elucidate, and potentially influence, the mechanisms that give rise to consciousness, our innermost thoughts, and our behaviors, thereby prompting novelThe neuroethics panel, with Dr. Rafael Yuste speaking.
social and ethical questions. Because of this, the NIH released a Request for Applications (RFA) to fund research on neuroethical issues associated with neurotechnological advances supported by the BRAIN Initiative. Dr. Koroshetz reminded the audience that unexpected ethical problems can potentially derail cutting-edge science, and neuroethics is a tool for anticipating and navigating ethical issues.
A common theme of the panel was how critical it is for neuroscientists to engage actively with study participants, philosophers, ethicists, and lawyers to continue to ensure that the ethical implications of their research are fully considered. Dr. Chiong described his experience as an ethicist embedded in a neurosurgical research team. He commented that the interdisciplinary nature of neuroscience results in researchers with diverse expertise working together. Though integrating different views can be challenging, the different perspectives can strengthen the work.
Overall, the panel provided attendees with food for thought and information about the neuroethics resources available to them. The audience actively discussed many of the issues raised during the panel and brought up several new issues they felt needed attention as well. Questions ranged from concerns about patient consent and autonomy, animal research ethics, the possible need for neuroethics guidelines to inform development and use of novel neurotechnologies, and how to engage properly with the public on neuroethics. Throughout the session many resources were described, such as individual university ethics offices, the option to consult with the MCWG Neuroethics Division, and expertise available through the International Neuroethics Society. These and other resources can equip scientists to navigate the novel ethical concerns that cutting-edge neuroscience research often raises. The main take–home message, summarized by Mr. Greely, is that neuroscientists should avail themselves of the expertise that neuroethicists offer.
Videocasts will be available for the NIH BRAIN Initiative Multi-Council Working Group and Neuroethics Division meetings on February 14th and February 15th…
The NIH BRAIN Initiative Multi-Council Working Group (MCWG), consisting of representatives from the 10 NIH Institutes and Centers participating in BRAIN, five at-large members, and ex officio representatives from DARPA, FDA, IARPA and NSF, provides ongoing oversight of the long-term scientific vision of the BRAIN Initiative, in the context of the evolving neuroscience landscape. The sixth meeting of the MCWG will occur on Wednesday, February 15th, 2017, at the NIH Porter Neuroscience Research Center (35 Convent Dr., Bethesda, MD 20892). The videocast may be accessed here.
The Neuroethics Division of the MCWG recommends overall approaches for how the NIH BRAIN Initiative might handle issues and problems involving ethics. The third meeting of the Neuroethics Division will occur on Tuesday, February 14th, 2017, at the NIH Porter Neuroscience Research Center (35 Convent Dr., Bethesda, MD 20892). The videocast may be accessed here.
Whole brain mapping of a sensorimotor response in the zebrafish… Novel optogenetics combination to control select cells in deep brain tissue at high resolution… Instability of neurons during stable song behavior in songbirds…
Whole-brain optogenetic mapping of a visual sensorimotor behavior in the larval zebrafish.
Mapping the interactions between neurons across numerous anatomically distinct brain regions is one of the main priorities of the BRAIN Initiative. This will provide a more comprehensive understanding of how the brain works. Previous studies in invertebrate animals, such as the fruit fly and the nematode, established the utility of these smaller model systems for recording and mapping complex interactions of neurons during behaviors. However, scaling up to the larger central nervous systems of vertebrate animals is difficult due to the orders of magnitude increase in cell numbers and complexity. In a recent article published in Cell, Dr. Florian Engert and colleagues used 2-photon calcium imaging to map neuronal responses across the entire zebrafish brain. First, the researchers created a computational model of sensorimotor responses based on larval zebrafish swimming behaviors elicited by a series of stimuli moving across the whole visual field. Approximately one-hundred thousand neurons can be simultaneously optically imaged in the translucent larval zebrafish brain, allowing investigation of the functional connections between different brain regions responsible for the observed complex swimming behaviors. The researchers found that a great deal of the brain became activated during the optomotor response to the motion stimuli. Comparison of the activity recorded within a given region of the brain to the computational model provided a neurological map of how the visual sensory signal is transformed into a motor output. By combining these models with a series of lesion experiments, the researchers confirmed that particular regions, including retinal arborization field 6, the pretectum, and posterior commissure, were necessary for the sensorimotor transformation. The use of whole-brain neuronal imaging within ever larger vertebrate model systems will enhance our ability to explore interactions between brain regions during increasingly complex behaviors.A) A dorsal view of the anatomy of the zebrafish larva. The labels represent the major anatomical regions of the zebrafish brain. B) The activity of all the imaged neurons in relation to the direction of the motion stimulus. The colors represent the preferred direction of each of the cells, as described by the circle in the bottom right.
Establishing stimulation parameters for red-shifted opsin, ReaChR, using 2-photon imaging to control cellular activity in deep brain structures.
Optogenetics, which enables activation or inhibition of select neurons via genetically incorporated light-sensitive ion channels, has dramatically enhanced our understanding of the brain in a short period of time. To date, use of this technology has been limited to relatively superficial brain tissue, due to increased scattering of light within deep brain structures. Recent efforts suggest that red-shifted opsins that react to longer wavelengths of light may help overcome this technical challenge. BRAIN awardees Drs. Hongkui Zeng and Valentina Emiliani pioneered work combining 2-photon stimulation with the red-shifted channelrhodopsin, ReaChR. In their Frontiers in Cellular Neuroscience article, the research team characterized stimulation parameters using an amplified laser for 2-photon holographic stimulation of ReaChR in cultured cells in vitro, as well as in mouse visual cortex neurons in vivo. After achieving nanoampere-scale current generation by stimulating ReaChR, the group illustrated reliable action potential (AP) generation following 15-micrometer diameter light stimulation for 10-milliseconds to neurons expressing ReaChR. To manage latency and variability in AP generation, the investigators found that increasing the laser power density by a factor of 1.5 consistently elicits an AP with millisecond temporal resolution. Finally, the researchers successfully evoked repeated AP trains in ReaChR-expressing pyramidal and fast-spiking cells using 10 light pulses at a range of frequencies. This first demonstration of control of AP generation via 2-photon stimulation of red-shifted ReaChR with high spatial and temporal resolution offers a novel method to control targeted cells in deep brain circuits.A) Expression of ReaChR opsin in mouse brain slice 2 weeks after viral injection. B) 2-photon microscopy of ReaChR in mouse visual cortex, 7-weeks after viral injection. D) Action potential evoked by 15-micrometer diameter 2-photon holographic stimulation (10 ms) of ReaChR expressing mouse cortical neuron.
Excitatory neurons display variable activity during stable song production in zebra finches.
Recording neural activity over long periods of time to determine how circuit activity gives rise to behavior is an important goal of the BRAIN Initiative. BRAIN awardee Dr. Timothy Gardner and his laboratory recently developed minimally invasive carbon fiber arrays for long-term neural recordings. In an article in Nature Neuroscience, Dr. Gardner and his team used the carbon fiber arrays, as well as newly designed head-mounted, miniature fluorescence microscopes, to study neural activity in conjunction with song production in zebra finches. The song of the zebra finch exemplifies a stable behavior, where the precise timing and acoustic structure of a song is well-maintained, making it a useful model for studying dynamic neural activity during a stereotyped action. The team recorded from the premotor nucleus, called the HVC, which contains inhibitory interneurons and several classes of excitatory projection neurons. In previous experiments, activity of multi-unit ensembles, or local field potentials (LFPs), was stable over minutes and hours during song production. In these sets of experiments, Dr. Gardner and his team showed that during song, the activity of LFPs, and that of single-unit, inhibitory neurons, were highly stable over the course of days, weeks, and/or months. In contrast, individual, excitatory projection neurons exhibited unstable activity over the course of days. Thus, the song motor pattern in the zebra finch and the activity of the HVC on the regional scale (i.e., LFPs) remained consistent, whereas individual excitatory projection neurons in the HVC drifted in activity patterns, including the probability of their bursting activity, and the likelihood of firing at all, during song. This study used novel recording and imaging techniques to demonstrate an important distinction between multi-unit and single-unit neural activity, as well as inhibitory versus excitatory neuronal activity, during a stable behavioral action.A) Images of song-related premotor nucleus neuronal activity, false-colored by the timing of max pixel intensity across 5 days for one songbird (top) and 4 days for a second songbird (bottom). B) Trial-averaged activity from all song-related neurons from 1 songbird, plotted over the course of five consecutive days. C) For the same animals shown in A), cells active on 3 consecutive days are combined into a single image in which color indicates neuron participation rather than timing (red = day 1, green = day 2, blue = day 3). Cells with activity on all 3 days appear in white. D) Electrophysiological recording of a projection neuron reveals a new song-related burst, which emerged over the course of 1 day. The blue arrows indicate the trials plotted at the bottom.