A sum of destructions

Pablo Picasso once said “To me painting is a sum of destructions. I paint a motif, then I destroy it.” Unknowingly, he had an intuition about visual processing. In fact, our current understanding is that retinas, quite like Picasso, break an image into its parts. The first man to lay the foundation of this idea was Haldan Keffer Hartline, a contemporary of Picasso. Professor GG Bernhard used this quote to present Hartline in the Nobel prize award ceremony 1967.

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I spy, I spy with my little eye

For the last couple of years I have been studying the retinal circuits of mice. While it is amazing how similar visual circuitry is among many species, I am always fascinated by surprising unique strategies that have developed in this system. The human visual system (from the retina to visual cortex) is a remarkable network that can see colors, adapt to a wide range of light intensities, perceive depth and distance, and much more. It is perfectly put-together such that each part contributes to a specific function: the lens focuses the image on the retina, different photoreceptors allow for color detection, our two frontal eyes allow for depth perception through parallax. The visual system of some animals has found other strategies to achieve the same functions, sometimes even using the same tools in new ways!

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TBT: Responses of Neurons of Primary Visual Cortex of Awake Unrestrained Rats to Visual Stimuli

In my research on the rat visual system, I have been designing an apparatus that would allow me to record neuronal responses to visual stimuli in freely moving rats. Most visual neuroscience experiments are now performed on restrained animals, who are usually treated with different drugs to suppress movement (anesthetics, muscle relaxants). But as anyone who has tried reading while falling asleep knows, just because your eyes are open does not mean that information is getting through to the brain. It makes more sense to study how neurons respond to images when the research subject is awake and paying attention.

While few researchers are studying vision in unrestrained rats today, I was surprised to find that the basic setup I have been working on for my experiments had already been created — in 1980’s Soviet Russia.

Working at the Moscow State University, Sergei Girman wanted to study the visual system in freely moving animals. So Girman chose to perform his experiments on rats, noting two features that made them convenient to use -  “the eyes in this animal are relatively immobile,” making it easy to know where they are looking (researchers go through a lot of trouble training a monkey to look at computer monitors in visual experiments), "while the visual analyzer is well developed” (analyzer being perhaps the fashionable word of the time to refer, in this case, to the visual areas of the brain).

The goal of Girman’s 1985 paper was to compare the responses of neurons in primary visual cortex in awake and attentive rats compared to those in restrained, anesthetized rats.

He outfitted the rats with a metal platform, glued to the head, that contained electrodes to record neural activity.  He then trained the animals to enter a vestibule that would constrain the head, so that when the visual stimuli were presented on a computer screen, the head would always be in the same position. While this may sound gruesome, by today’s experimental standards Girman’s procedure was quite original.

At the time, most experiments in visual neurophysiology were done in one long session - researchers would prepare the animal, insert electrodes and present visual stimuli for hours on end; at the end of the session, the electrodes were removed and the animal euthanized (in some cases the experiment could be repeated, but the electrodes would be placed in different brain areas each time). Girman was interested, in part, in recording the activity of neurons for long periods of time. If a neuron responds to particular visual stimuli today, would it respond to the same stimuli in the same way again, tomorrow? To be able to answer questions like this, Girman needed to construct an apparatus that would stay on the animal’s head for months without causing so much damage that the body would reject the implant.

One of Girman's rats with electrodes and head implant attached.

Today, researchers routinely record neuronal activity in rodents for months at a time (techniques such as calcium imaging allow one to examine the activity of the same neurons from one day to the next), but the surgical procedure of attaching head implants is quite drastic. In most cases, the animals are scalped (nonviolently, of course), holes are drilled in the skull, and after the electrodes are inserted into the brain, the scalp is replaced with glue (usually dental cement). Researchers take great care to perform such procedures in a sterile environment to reduce the risk of infections. Inevitably, however, after months (or in lucky cases, perhaps a year), the implants fall off.

Girman’s original solution to this problem was to not scalp the animals in the first place. Instead, he would only make holes large enough for the electrodes to pass through (0.12 mm, according to his paper). Then, he would create a platform for the electronic equipment by threading stiff metal wires under the scalp. While this sounds like a less invasive solution, it must be quite difficult to perform (although I haven’t tried it in my own experiments yet).

Girman’s papers are quite fascinating, partially because of his unique methods, which make me wonder if they really do work better than today's established techniques. Is it just the no one read Girman’s work (which was originally published in Soviet Russian journals and translated later)? Or is Girman's idea of keeping the scalp intact really not all that better than removing it?

It is both encouraging and frustrating to learn about obscure research techniques: the wheel does get reinvented over and over, but perhaps we learn something new each time.

Girman, S. V. (1985). Responses of neurons of primary visual cortex of awake unrestrained rats to visual stimuli. Neuroscience and Behavioral Physiology, 15(5), 379–386.

(Please tweet us @harvardneurosci if you have trouble accessing the paper)

 

 

Luck in Science

One of the most fascinating results in brain research - one that revolutionized neuroscience, launching it into the modern age - came from David Hubel and Torsten Wiesel in a series of papers in the early 1960’s. Hubel and Wiesel were awarded the Nobel Prize in 1981 for finding that the brain breaks down visual scenes into elementary components, dedicating networks of neurons to compute simple features that are eventually built up again into ever more complex representations.

As we wait to learn who this year’s Nobel Prize winners are, I can’t help but wonder how those women and men come upon their fascinating findings. From Galileo’s revolutionary work in astronomy to Oswald Avery’s determination of DNA as the molecule of heredity, the scientific method has been the unrivaled system of discovery in a world full of mysteries. While there is no doubt that the scientific method is still producing incredible new knowledge, it is not clear why some scientists are more successful than others at utilizing that method. If all scientists - supposedly intelligent, driven people - employ the same general strategy, why are some so much better at discovery than others?

In their account of their 25-year collaboration, Brain and Visual Perception, Hubel and Wiesel describe the chance situation that contributed to their initial result:

“Suddenly, just as we inserted one of our glass slides into the ophthalmoscope, the cell seemed to come to life and began to fire impulses like a machine gun. It took a while to discover that the firing had nothing to do with the small opaque spot [i.e. the intended stimulus]—the cell was responding to the fine moving shadow cast by the edge of the glass slide as we inserted it into the slot... People hearing the story of how we stumbled on orientation selectivity might conclude that the discovery was a matter of luck.”

 

My own experience in neurophysiology has produced some seemingly lucky results. As a technician in the lab of Tim Gardner at Boston University, I worked to develop a system to record the activity of large numbers of neurons in singing zebra finches using minimally invasive carbon fiber electrodes that promised to outperform traditional metal electrodes because of their small size and biocompatibility.

Our strategy seemed straightforward, but I kept running into a problem - after coating the carbon fibers with insulating plastic, the fibers’ electrical resistance went through the roof, making it practically impossible to see electrical activity from the neurons.

After taking several images of the fibers’ tips under an electron microscope, I realized that the problem lay in the way that I had been cutting the fibers: instead of a nice carbon core surrounded by a layer of plastic, like a pencil’s graphite cased in wood, the tips more closely resembled a chewed straw or gnarled tree, with the plastic practically swallowing the carbon in a frayed mess. The scissors I was using had been crushing my fibers, leaving almost no carbon surface exposed to record neural activity!

I needed a way to cut the fibers cleanly. This was a stage of wild exploration: the first idea featured a hacked hard-drive that was supposed to grind the plastic off the carbon tips; after that failed, I embedded the fibers in wax and cut them on a machine normally used to cut slices of brain (a distant cousin of the deli slicer). When that didn’t pan out, I nearly burned down the lab by trying to torch carbon tips that were just barely protruding from the same wax embedding I had used before; in the excitement of the prospect of success, I forgot that the wax was based in ethanol, and watched my latest idea go up in flames.

The torching wasn’t such a bad idea though - I simply needed to experiment using a non-flammable insulator. The answer was water. I poured a little bath for my carbon electrodes and immersed them in the water, with the tips sticking out above the water surface; I then ran my torch over the water and measured their resistance. The tips emerged clean, the resistance low and as an added bonus the tips had tapered to a fine point, making insertion into the brain much easier!

This modest scientific success seems to have come from a combination of perseverance and exploration, two seemingly contradictory tactics - a sort of focused play. Was it a matter of luck that I eventually stumbled onto a suitable method? Hubel and Wiesel analyze the matter of luck in their first success:

“While never denying the importance of luck, we would rather say that it was more a matter of bullheaded persistence, a refusal to give up when we seemed to be getting nowhere. If something is there and you try hard enough and long enough you may find it; without that persistence, you certainly won't. It would be more accurate to say that we would have been unlucky that day had we quit a few hours before we did... But just as important as stubbornness, in getting results, was almost certainly the simplicity, the looseness, of our methods of simulation.”

 

   

While it may be impossible to predict who will succeed in science or which experiments are going to be worthwhile, we shouldn’t rely on blind luck for success. Louis Pasteur wrote that chance favors the prepared mind. That adage might work not just for school examinations, but for the uncharted land of science as well. Perhaps what we can take from Hubel and Wiesel’s reflections is that chance favors the open yet persevering mind, those who ask interesting questions and don’t give up until the results are in hand.