Kavli Blog

BRAIN Initiative team pushes the limits of functional magnetic resonance imaging (fMRI) for human brain… A novel tool to manipulate gene function in specific cell types… Understanding the functional diversity of retinal bipolar cells…

Advancements to functional imaging technique result in ultra-high resolution capture of human cortical columns

Despite numerous advances in fMRI technology, most components are optimized for the entire body. This makes safe, ultra-high resolution (UHR) imaging of columnar organization throughout the cortex of the human brain nearly impossible. At the University of California, Berkeley, Dr. David Feinberg and colleagues applied updates to magnetic gradients, receiver arrays, and pulse sequences of simultaneous multi-slice echo planar imaging fMRI to achieve UHR imaging of human ocular dominance columns. Focusing particularly on a prototype receiver array (8 channels with 4 cm diameter coils), the group systematically describes the changes necessary to achieve the higher signal-to-noise ratio required to attain ~0.5 mm imaging resolution in 3 dimensions. Finally, the researchers display their updated UHR system compared to commercially available technology when mapping ocular dominance columns in three subjects shown visual stimuli in the scanner. The group notes their findings are part of a growing set of 3D imaging studies, moving to leverage fMRI to understand neural circuitry by revealing activity of distinct cell populations in different cortical layers. They postulate that this 3D imaging technique could eventually progress from 0.5 mm to 300-400 µm resolution fMRI of the entire human brain.

Brief fMRI scans from a visual activation paradigm in a human subject reveal enhanced cortical activation at 0.5 mm spatial resolution in the prototype receiver array (8-channel with 4 cm diameter loops) compared to commercially available technology (32-channel). The prototype array accurately measured activation at 0.45 mm resolution as well, further illustrating improved signal-to-noise ratio.

New technique developed for controlling gene function in distinct cell types in the fruit fly

The ability to manipulate genes in specific cell types is critical for understanding circuit function and dysfunction. Unfortunately, there are limitations for the currently available tools, including off-target effects (i.e., modifying genes other than the targeted gene), incomplete inactivation of the targeted gene, requiring cell division (which restricts the time during development when the gene can be targeted), and incompatibility with model systems like the fruit fly Drosophila melanogaster, which is a principle model for studying neural development and function. At Stanford University, Dr. Thomas Clandinin and colleagues have developed a tool called FlpStop, which can completely disrupt targeted genes, as well as rescue gene expression of the disrupted genes, in differentiated and undifferentiated cells in Drosophila. FlpStop uses endogenous mechanisms within cells and a process called insertional mutagenesis to completely inactivate/disrupt the normal function of a gene. The FlpStop insertion is tagged with a fluorescent protein so that mutant cells can be visualized, and it can be inserted in both non-disrupting and disrupting orientations. The team successfully disrupted gene function in six out of eight of the genes that were tested and confirmed that the non-disrupting orientation did not interfere with gene function in any case. They successfully labeled the FlpStop inserted genes in three different cell types in the Drosophila visual system (Mi1, Tm1, and T4). Finally, the group effectively combined FlpStop with in vivo calcium imaging. The findings suggest that FlpStop represents a promising and powerful new tool for investigating gene function in specific cell types.

(a) Schematic of the experimental design for testing the effectiveness of the FlpStop insertion. Drosophila Gal4 driver lines were used, whereby three distinct cell types in the visual system were targeted: (b) Mi1, (c) Tm1, and (d) T4. The full expression of each Gal4 driver line is labeled green while the combination of Gal4 (in green) and the successfully-expressed FlpStop gene (in red) together appear yellow, demonstrating 70%-93% overlap depending on the cell type.

Studying the functional diversity of bipolar cells improves understanding of neuronal processing in the visual system

One core goal of the BRAIN Initiative is to better understand brain circuitry and the role of different cell types in these brain circuits, in both healthy and diseased brains. A relevant area of focus is retinal neurons and their ability to encode visual stimuli for the brain. While the anatomy and genetics of retinal bipolar cells are well characterized, their functional diversity is incompletely understood. In an article in Nature, BRAIN awardee Dr. Thomas Euler and colleagues used two-photon imaging to examine the effects of amacrine cell activity (a type of retinal interneuron) on the output of bipolar cells in the mouse retina. Through exposing different areas of the retina to light, the researchers found that functionally opposite signals, such as those used to describe ON and OFF bipolar cells, exist at the same layer of the retina, suggesting that the retina structure is more complicated than previously thought. Furthermore, inhibition of amacrine cell activity led to increases in the functional diversity of bipolar cells. The team determined that a bipolar cell’s output is determined by a combination of excitatory input to the dendrite and amacrine cell input to the axon, which ultimately allows for temporal encoding in the visual system. These important findings give us a better understanding of the visual system and of the mechanisms through which different neuronal cell types communicate with one another.

Local (gray) and full-field (black) output responses of bipolar cells in both control and drug conditions. TPMPA/Gbz blocks GABAergic amacrine cell activity, while strychnine blocks glycinergic amacrine cell activity. Blocking amacrine cell activity led to opposite responses from bipolar cells compared to control conditions.

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 limits

Graphical 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:

  1. NIH BRAIN Initiative by Dr. Koroshetz and Dr. Joshua Gordon, Director of the National Institute for Mental Health (NIMH)
  2. MCWG Neuroethics Division by co-chairs Prof. Hank Greely and Dr. Christine Grady
  3. IARPA by Dr. David Markowitz, who administers the MICrONS project
  4. 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.