December 1, 2009 (Vol. 29, No. 21)

NIH Commits $30 Million to Mapping Circuitry of the Brain Using Imaging Technologies

The scope and sheer volume of innovative research, fresh insights, next-generation technologies, and promising therapeutic opportunities presented at the recent Society for Neuroscience’s annual meeting in Chicago was more than any one, normal functioning brain could absorb, even with all neurons firing and all synapses and circuits on high alert.

The meeting focused on themes ranging from brain development and physiology, neural function and dysfunction, synaptic plasticity, genetics, pathology, and learning and memory, to normal aging.  Presenters described how they are using light energy and light-responsive molecules to turn neurons on and off as part of a field of study called optogenetics. They also presented research on motor control, sensory mechanisms, cognition, and addiction.

Francis Collins, M.D., Ph.D., the new director of the NIH, urged attendees to “engage in advocacy” and “make the case for scientific research”—basic, translational, and clinical research—in the public arena. He described his vision for the NIH and took aim at the financial challenges that lie ahead.

Over the next two years, there is a $10 billion light at the end of the funding tunnel, due to the stimulus dollars appropriated to the NIH as part of the recent Recovery Act. Dr. Collins explained that $8.2 billion of that money will be allocated for scientific research. As of October 1, $5 billion had been awarded and 12,000 grants funded. Twenty-eight institutions and 1,885 investigators were first-time awardees.

Dr. Collins emphasized the NIH Blueprint for Neuroscience Research and the announcement in July that it would launch a $30 million Human Connectome Project (HCP) aimed at mapping the circuitry of the healthy adult human brain using imaging technologies. Selected projects will be funded at up to $6 million per year for five years.

The HCP represents one of three planned Blueprint Grand Challenges that will focus on neuroscience research to understand brain function and treat brain disorders. The other two will promote targeted drug development for neurological diseases and research on the neural basis of chronic pain disorders.

Among the many new technologies and products on display at the meeting were a host of imaging tools, including confocal and two-photon laser scanning microscopes, antibodies, labeling reagents, and proteins for neuroscience research, software tools, and functional assays and live-cell imaging systems for high-content screening applications.

A Bright Idea

An illuminating symposium chaired by Mark Schnitzer, Ph.D., assistant professor of biology and of applied physics at Stanford University, explored “New Technologies for Probing Brain Disease with Light: From Super-Resolution and in vivo Imaging to Optical Control of Circuits.” Light energy is helping researchers explore the healthy and diseased nervous system with technologies such as optogenetics, super-resolution imaging, and microscopy in live unanesthetized animals.

Guoping Feng, Ph.D., associate professor of neurobiology at Duke University’s Institute for Brain Sciences, described the development of cell type-specific optogenetic mice for use in probing neural circuitry and dysfunction. Xiaowei Zhuang, Ph.D., professor of chemistry and chemical biology and of physics at Harvard University, gave a presentation on the application of nanoscopic fluorescence imaging to study neurons and brain tissue.

Maiken Nedergaard, from the University of Rochester, described her work using in vivo two-photon imaging in astrocyte cell cultures containing luciferase to study the role these cells play in neuronal function and communication.

Dr. Schnitzer’s work using minimally invasive imaging in awake mice to study the brain’s “cellular orchestra”, aims to define large-scale patterns that underlie brain function. Light-scattering imaging techniques that rely on confocal and two-photon microscopy are limited by how deeply in the brain they are able to look. Dr. Schnitzer described an alternative technique called fluorescence microendoscopy that uses micro-optical probes to target specific brain regions. By introducing fluorescein into the plasma of mice and inserting probes into the hippocampal region, he demonstrated the ability to image capillary blood flow. “At the highest resolution you can see individual red blood cells.”

An important advantage of this relatively low-cost technology is the ability to re-image the same site in the same animal over time. He described a range of clinical applications in which microendoscopy could be relevant; in muscular dystrophy, for example, the probes can be inserted directly into the muscle to image the contractile behavior of individual muscle fibers. Using laser scanning just once can image the dynamics of sarcomere contractility over time.

Karl Deisseroth, M.D., Ph.D., associate professor in the departments of bioengineering and of psychiatry and behavioral sciences at Stanford University, showed how light-responsive proteins called opsins, derived from microorganisms such as algae, respond to specific wavelengths of light and can be used to control individual cell types in neural circuits, leaving others unaffected.

Channelrhodopsin-2 (ChR2), a positive stimulator (or “on switch”) for neurons that was isolated from Chlamydomonas reinhardtii, is an example of an opsin. Fiberoptic delivery of pulses of blue light at 470 nm into the brains of ChR2-expressing transgenic mice results in optogenetic stimulation, causing the light-gated ion channels to open and allowing sodium ions to flow across the membranes of ChR2-containing cells.

Feng Zhang, a graduate student in Dr. Deisseroth’s laboratory described the ongoing search for light-responsive proteins in microorganisms across diverse ecological environments, with the hope of finding defined populations of molecules that he described as “microbial antennae,” which will allow for targeted light stimulation of neurons to activate or deactivate specific neuronal circuits and synaptic connections.

Herbert Covington III, a postdoctoral fellow, described research performed in Eric Nestler, M.D., Ph.D.’s laboratory of molecular psychiatry at Mount Sinai School of Medicine, in which directed laser light was used to stimulate Ch2 in the medial prefrontal cortex of mice with a depression-like phenotype. This region of the brain exhibits altered function in humans suffering from major depression. Optogenetic stimulation of Ch2 was associated with antidepressant effects in the mice.

At the University of California, San Francisco, Garret Stuber, Ph.D., and colleagues are exploring the use of optogenetic control of brain reward circuitry to modulate addictive behaviors. They are using Ch2 and light stimulation in the amygdala to determine which synapses in the brain have reinforcing effects similar to drugs of abuse.

Preliminary results of studies on optical stimulation-driven memory recall performed by Michael Häusser, Ph.D., and co-workers at University College London show that Ch2 activation to provoke memory recall stimulates neural activity and plasticity only in brain cells initially involved in memory formation. The researchers used a mouse model of memory based on fear conditioning, in which the mice were conditioned to respond to a tone. They demonstrated that in mice injected in the hippocampus with the Ch2 construct, light activation of Ch2 could evoke fear memory recall and the same behavior the mice exhibit after hearing the tone.

MilliTrace CX Nestin GFP reporter human neural stem cells from Millipore expressing Nestin, GFP, and Sox-2.

Breaking Down Barriers

The blood brain barrier (BBB), which regulates the passage of compounds into and out of the brain by relying on tightly controlled cellular junctions, is essential for the normal functioning of the central nervous system. However, it presents a significant challenge for therapeutic drug delivery to the brain and inhibits the body’s ability to eliminate unwanted molecules that may build up in the brain as a result of disease.

Jean-Paul Castaigne, M.D., president and CEO of Angiochem, highlighted the large unmet medical need in the treatment of brain diseases, with the overall market estimated at >$65 billion, broken down into psychiatric disorders ($19.1 billion), Alzheimer’s disease ($6.4 billion), other neurodegenerative diseases ($10.7 billion), and brain cancer ($1.6 billion).

Angiochem designed its EPiC platform to develop drugs that target receptors present on cells that actively transport molecules across the BBB, such as low density lipoprotein (LDL) receptor-related protein (LRP). LRP is noteworthy for its ability to transport amyloid beta from the brain to the bloodstream. The company currently has an LRP-mediated taxane compound—a DNA replication inhibitor—to treat glioma, a highly malignant form of brain cancer, in Phase II trials. Other EPiC therapeutics are in preclinical development for the treatment of neurodegenerative diseases and obesity, and for neuroprotection in hypothermia.

Jan Drappatz, M.D., from the Dana-Farber Cancer Institute, presented data gathered from the more than 100 patients with recurrent glioma treated with the drug in two Phase I/II escalating-dose studies. He reported no evidence of CNS toxicity or immunogenicity after repeated dosing, as well as early findings of efficacy, with evidence of disease control in 60% of patients with primary brain cancer who received a therapeutic dose, and in 71% of patients with secondary (metastatic) brain cancer.

These encouraging results were supported by reductions in brain tumor size and reversal of neurological decline in several cases, and by substantial reductions in the size of metastatic tumors in a variety of other organs. Therapeutic concentrations of the drug were documented in brain tumor tissue removed from patients, demonstrating its ability to cross the BBB.

Berislav Zlokovic, M.D., Ph.D., from the University of Rochester, discussed the role of the BBB in the pathogenesis of Alzheimer’s disease (AD) and described a strategy for engineering a mutant form of the LRP1 receptor that could pull amyloid beta from the brains of affected patients.

In other work, Serge Rivest, Ph.D., from University Laval in Quebec, demonstrated that bone marrow-derived microglial cells—the main type of immune cell in the central nervous system—are present in the blood and are able to cross the BBB. In a mouse model of AD, these cells can associate with amyloid protein in the brain and prevent the formation of amyloid deposits, or eliminate those already present. He proposed the use of autologous microglial cells derived from bone marrow stem cells as a vehicle for gene therapy to treat AD.

John El Khoury, Ph.D., from Harvard Medical School, pointed out that the role of microglia in AD pathogenesis is not yet clear, with some evidence suggesting a protective effect and the ability to clear amyloid, while other data suggests a detrimental role. At the root of this mechanism is the interaction between the microglial cell and amyloid, and the process by which the protein alters microglial function and stimulates cytokine and chemokine production that compromises neuronal viability.

Kate Nautiyal, Ph.D., from Columbia University, showed a link between another type of immune cell, the mast cell, which mediates allergic and anaphylactic responses, to regulation of emotion and cognition. Mast cells can be found in the hippocampus and are able to cross the BBB. They appear to have a role in easing anxiety and promoting learning and may represent a novel target for treating mental health disorders.

In a symposium exploring the role of the BBB in neurodegeneration, presenters gave examples of BBB dysfunction in a variety of acute and chronic disorders, including stroke, dementia, Parkinson’s disease, and amyotrophic lateral sclerosis. This dysfunction may result in abnormal entry of molecules into the brain or insufficient clearance of toxic molecules. Research findings have demonstrated that altered blood flow resulting from diminished microcirculation in various brain regions likely precedes neuronal changes and clinical symptoms. Dramatic reductions in capillary volume have been demonstrated in mice before neurodegenerative changes occur, and the total brain capillary length is reduced in AD, for example. This neurovascular uncoupling appears to compromise the BBB and may prove to be a fruitful target for drug discovery.

Dual-channel fluorescence image of F-actin and nuclei distribution in bovine pulmonary artery endothelial cells obtained with Thorlabs’ Video-rate Confocal System.

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