Optogenetics

This animation illustrates optogenetics—a radical new technology for controlling brain activity with light. Ed Boyden, the co-inventor of this technology, is a professor at the MIT Media Lab and at the McGovern Institute for Brain Research, where he continues to develop new technologies for controlling brain activity. Video courtesy of MIT TechTV.

A Not-So-Short Circuit?

As neuroscientists look to the future of their field, they are beginning to delve into more complex factors that define our emotions and intentions.

Fifteen years after writing the influential book The Emotional Brain (1996), on the neurobiology of emotion, New York University neuroscientist Joseph LeDoux is rethinking his approach. “I’m not even using the word emotion anymore,” he says.

LeDoux and some of his contemporaries have instead shifted to studying the neurophysiology behind behaviors that are central to an organism’s or species’ existence. “I think it’s wrong to study joy or pleasure,” says LeDoux. “Those are abstractions of things that are happening at a much more basic level.” He’s more interested in asking questions like, “What’s in the brain that’s keeping the rat alive?” LeDoux argues that survival instincts—such as the desire for food or sex—are strongly conserved across species. Humans alone have abstracted these desires into words like love or hurt, which may not reflect the underlying biological impulse. “Behaviors are species-specific, but the fundamental function of the [neural] circuit is general,” says LeDoux. He thinks that studying these conserved circuits will be more helpful in revealing how we process the needs that we express as feelings. “I think we’ve only scratched the surface of emotion in the brain,” he says.

When Columbia University neuroscientist Eric Kandel went looking for the neurological circuit for memory in sea slugs, he turned to the defensive reflex of learned fear—one of the strongest and most easily made memories. His Nobel Prize-winning work to define the neural circuit involved in that behavior became a model for many others. But researchers are now beginning to look beyond the circuit.

From the simple to the complex
Defining neural circuits is still “the holy grail of neuroscience,” says Eve Marder at Brandeis University. But they are “absolutely necessary and completely insufficient” for understanding what dictates subtle changes in behavior, she adds. Mapping circuits in the relatively simple nervous systems of the crab and lobster, Marder has shown that something more defines behavior than just the cellular connections between input (some kind of stimulus), processing, and output (some kind of response). The time course of the synaptic events and their amplitude are also important, and these in turn are determined by the kinds of channels and receptors that the neurons have. Under normal conditions, all of her experimental animals performed rhythmic motor functions such as breathing or feeding movements comparably to one another. But with the introduction of environmental stressors—an increase in temperature, for example—certain configurations and concentrations of receptors and channels were better for controlling these behaviors and would likely confer a survival advantage in nature. Though the neuronal circuits rarely change, the receptors are constantly being recycled and presented again on the surface. “Each cell is constantly rebuilding itself,” Marder says, offering a new way of understanding “how you build a nervous system to have the potential for plasticity” and still retain the stability of the overall system.

Even without such temporal and environmental complications, studying the neuronal circuitry behind complex behaviors is not easy, especially in more complex animals. The sheer number of cells involved in a vertebrate brain circuit, for example, is a major hurdle. Rather than dissecting a one-to-one connection, researchers must consider connections between hundreds of one type of neuron and hundreds of another type. These “microcircuits,” as Gordon Shepherd at the Yale School of Medicine calls them, carry out the processing that happens in a particular brain region after a stimulus is received and before the signal is sent onward. Each microcircuit performs a particular function and unique task in the circuit. And yet the same microcircuits may be used for processing diverse inputs, such as smell, vision, or hearing. “The more we realize how similar they are and yet [how] fine-tuned for the particular kind of processing, the more we understand the basic principles about how the brain operates,” says Shepherd.

Advances in brain-imaging techniques and in optogenetics are allowing scientists to start teasing apart microcircuits in more complex brains. “We’re now approaching a point where we can do in vertebrates what used to only be possible with invertebrates,” says Marder. (See “The Birth of Optogenetics,” July 2011.)

From the lab to the clinic
Eberhard Fetz at the University of Washington works on an area of neuroscience that is perhaps closest to being translated into the clinic. His field of study, brain-machine interface (BMI), aims to repair lost function in stroke victims, paralysis patients, and amputees by implanting electrodes that record intention to act, as manifested by neuronal firing in the brain. These recorded signals are then converted by an external computer into movements—either of a cursor on a computer screen or of a robotic arm—bridging the gap between damaged neurons and motion.

Although the clinical applications are still a number of years away, Fetz says this type of research is also sparking new insights into basic neurology. For example, thinking about behaviors only in terms of the circuit involved—the cells that receive a stimulus, process it, and send instructions for action—implies that certain cells have been programmed to perform certain tasks. But when Fetz implants monkeys with electrodes that are part of a brain-machine interface, he does not even attempt to pinpoint the cellular circuit responsible for a behavior—say, the twitch of a wrist. Instead, the electrodes are implanted more or less at random in the area of the brain responsible for all movement, and signals are recorded only from the neurons in the vicinity of the electrode. Using only their vision as a guide, Fetz’s monkeys can learn to fire those neurons that are touching the electrode and twitch a muscle.

Studying circuits involved in a particular behavior has revealed much about the cellular and molecular changes that must occur in order to interpret the environment. Such studies have given experimenters a simpler framework from which to test their ideas. Now, as parts of that framework are challenged and reformed, researchers are coming ever closer to answering questions that had once only been the purview of poets and philosophers.

Indeed, at the core of brain research is the desire to fathom what happens when memory fails and information processing goes awry. The brain is “the area that is responsible for more disorders than any other organ of the body,” says Kandel. Understanding circuits is one solution, Marder adds, but “all the new techniques are bringing us to the point where, in the next 5 to 10 years, those problems will be solved. And then the really interesting work can start.”

by Edyta Zielinska is a Senior Editor at The Scientist.

Notable Papers

P. Fatt, B. Katz, “Spontaneous subthreshold activity at motor nerve endings,” J Physiol, 117:109-28, 1952.

A.L. Hodgkin, A.F. Huxley, “A quantitative description of membrane current and its application to conduction and excitation in nerve,” J Physiol, 117:500-44, 1952.

W.B. Scoville, B. Milner, “Loss of recent memory after bilateral hippocampal lesions,” J Neurol Neurosurg Psychiatry, 20:11-21, 1957.

B. Katz, R. Miledi, “The timing of calcium action during neuromuscular transmission,” J Physiol, 189:535-44, 1967.

E.R. Kandel, J.H. Schwartz, “Molecular biology of learning: modulation of transmitter release,” Science, 218:433-43, 1982.

Saturn

Saturn is the most beautiful planet in our Solar System. Famous for its bright yet ethereal rings, the gas giant has over sixty natural satellites in orbit around it – and one artificial satellite: NASA’s Cassini spacecraft, which provides many of the results and images that will be showcased in this talk. We shall explore the weather observed in the atmosphere of Saturn, the curious structures that develop within the rings, and its wide variety of moons – from smog-shrouded Titan, two-sided Iapetus, to busy Prometheus, and the icy plumes erupting from frozen Enceladus.

Outreach Officer at the Instituteof Astronomy and Fellow of Emmanuel College, University of Cambridge, Professor Carolin Crawford is one of Britain's foremost science communicators.

An advise for my advisor

If you want to build a ship, don't drum up people together to collect wood and don't assign them tasks and work, but rather teach them to long for the endless immensity of the sea.
- Antoine de Saint-Exupery

Feynman on Scientific Method

Physicist Richard Feynman explains the scientific and unscientific methods of understanding nature.

Quantum Levitation

Don Eigler: Moving Atoms, one-by-one

Stimulated Emission Depletion (STED) Microscopy

Superresolution microscopy using stimulated emission depletion (STED) creates sub-diffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation. The suppression of fluorescence is achieved through stimulated emission that occurs when an excited-state fluorophore encounters a photon that matches the energy difference between the ground and excited state. Upon interaction of the photon and the excited fluorophore, the molecule is returned to the ground state through stimulated emission before spontaneous fluorescence emission can occur. Thus, the process effectively depletes selected regions near the focal point of excited fluorophores that are capable of emitting fluorescence.

STED microscopy operates by using two laser beams to illuminate the specimen. An excitation laser pulse (generally created by a multiphoton laser) is closely followed by a doughnut-shaped red-shifted pulse that is termed the STED beam. Excited fluorophores exposed to the STED beam are instantaneously returned to the ground state by means of stimulated emission. The non-linear depletion of the fluorescent state by the STED beam is the basis for superresolution. Even though both laser pulses are diffraction-limited, the STED pulse is modified to feature a zero-intensity point at the center of focus with strong intensity at the periphery. When the two laser pulses are superimposed, only molecules that reside in the center of the STED beam are able to emit fluorescence, thus significantly restricting emission. This action effectively narrows the point spread function and ultimately increases resolution beyond the diffraction limit. To generate a complete image, the central zero is raster-scanned across the specimen in a manner similar to single-photon confocal microscopy, as illustrated in the tutorial. STED microscopy is capable of 20 nanometer (or better) lateral resolution and 40 to 50 nanometer axial resolution.

source: zeiss

Photoactivated Localization Microscopy (PALM)

Photoactivated localization microscopy (PALM) is a superresolution technique that dramatically improves the spatial resolution of the optical microscope by at least an order of magnitude (featuring 10 to 20 nanometer resolution), which enables the investigation of biological processes at close to the molecular scale. The technique relies on the controlled activation and sampling of sparse subsets of photoconvertable fluorescent molecules, either synthetic or genetically-encoded. This interactive tutorial explores the sequential steps involved in creating a PALM image.

Using photoactivatable fluorescent proteins, it is possible to selectively switch on thousands of sparse subsets of molecules in a sequential manner. The basic principle behind PALM is to start with the vast majority of the molecules in the inactive state (in effect, not contributing fluorescence emission). A small fraction (less than 1 percent) is photoactivated or photoconverted using a brief pulse of ultraviolet or violet light to render that subset fluorescent. The activated molecules are then imaged and localized to produce nanometer-level precision coordinates, followed by removal from the larger set of unactivated molecules by photobleaching. In the next step, a second fraction of molecules is photoactivated, localized, and eliminated by photobleaching. The process is repeated many thousands of times until the molecular coordinates of all labeled molecules are obtained. The PALM image is a composite of all the single molecule coordinates. As new fluorescent probes for PALM are developed, the photoconversion and readout wavelengths are likely to ultimately span the entire ultraviolet, visible, and near-infrared spectral regions.

source: zeiss

A unique strategy for overcoming the diffraction barrier employs photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution. This approach relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. Thus, very closely spaced molecules that reside in the same diffraction-limited volume are temporally separated. Merging all of the single-molecule positions obtained by repeated cycles of photoactivation followed by imaging and bleaching produces the final superresolution image. Techniques based on this strategy are often referred to as probe-based superresolution, and were independently developed by three groups in 2006 and given the names photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). All three methods are based on the same principles, but were originally published using different photoswitchable probes.

source: zeiss