Whiteboard Methods: GFP-Fusion and Immunocytochemistry

Check out my new video explaining two of the most widely-used imaging methods in the field!


Now Hear This (Part II)

We have all heard of the “One-Hit Wonder”; when a seemingly inspired artist is able to create a masterpiece but only once in their career. The work is often played and remembered as a pop sensation, but the remaining body of work is totally underwhelming in comparison or even non-existent in the case that the artist ceases to write new pieces following the success of the acclaimed song.

Alfonso Corti

Alfonso Corti

But in science? The same phenomenon can occur. A brilliant mind publishing field-defining work promptly disappearing from the major journals following the famed discovery? This is certainly the case for Alfonso Corti, the man who nearly single-handedly uncovered the mysterious cellular basis for our understanding of sound. The diligent, detailed study of the mammalian cochlea is unrivaled in its precision. His studies on the topic may have only lasted one season in the spring of 1850 at Wurzburg, but the body of work is so complete that one could hardly argue against his proposed mechanism of sound transduction. However, less than a year after the publication of his studies on the cochlea, Conti’s inherited estate following his father’s death forced him into the busy career path of a nineteenth century European aristocrat and he never returned to his careful studies.

What exactly is the focus of Conti’s season of intense observation? In part one we looked at the physical logic of sound and the overarching themes of the cochlea’s ability to dissect sounds. Now we will examine the cellular basis for this phenomenon in the Organ of Conti, a structure within the cochlea.

Recall that a cross-section of the cochlea reveals three separate compartments and the wavelike movements of the separating membranes create stress on small structures called hair cells. The hair cells are organized in a row of single cells closer to the attachment point of the basilar membrane called “Inner Hair Cells”, and a triple row further out appropriately named the “Outer Hair Cells”. The hair cells, just like Merkel cells, are specialized skin cells, not neurons, with the specific purpose of signaling changes in mechanical force. These cells are easily distinguished by a tuft of hair-like structures, called Stereocilia, arranged on the top layer in order shortest to tallest (kind of like the signal bars on any cell phone).corti2

When stress is created between the two cochlear membranes, the tallest hair on each hair cell, tethered to the tectorial (top) membrane is pulled away from the bundle of other cilia. However, the other cilia are then yanked in the same directions by a thin connection called the Tip Link connecting one end of each cilium to the sidewall of the next tallest one. It is believed that the pulling of these tip links in response to stress from sound waves opens gated ion channels to flood these cells with charged particles necessary for signaling the underlying neurons.

These responses are extremely fast (occurring in under a millisecond) and it is due to this responsiveness that our cochlea is able to so accurately discriminate between different sounds. The same process occurs every time you tune in to listen to the one-hit wonders of today.

Now Hear This! (Part I)

Of all of the great inventors, engineers, designers, and architects, Nature is by far my favorite. She designs an enormous diversity of elegant solutions to very complicated environmental challenges based on only a few simple concepts. Life on earth adapts by accumulating mutations, or changes, in the genetic sequence. Over enormous periods of time and many generations, these slight changes enable our bodies to build better and better structures for specific uses. Often times, the outcome of this process is astoundingly simple.

One of the great challenges faced by nature in the design of us, human beings, is the ability to detect and understand the differences between sounds around us. We are surrounded by objects and other organisms each creating their own sounds, and the ability to process sound is an ability that has greatly aided our survival as a species. The ability to hear, in combination our other senses, is used for orientation as well as discovery and avoidance of dangerous predators or hazards.

But what is sound really? From a physics standpoint, sound travels as vibrations in the molecules of the air around us. These vibrations are understood as waves, where air molecules bunch up and spread out alternatively like ripples over water. Sound waves of higher pitch, or frequency, have peaks occurring in much closer groupings than low base-like sounds with peaks much more spread out. What you know as volume or loudness, is interpreted as the Amplitude, or the maximum height of each wave (interpreted from the maximum density of air particles, but this is beyond what you need to know).Pressure Wave

Understanding the physical basis of sound now, you can begin to ponder a device to detect it. Not only would you need a way to capture the range of sounds, but you need to transform them into understandable signals. Our brains integrate signals from sensory receptors in the form of neural firing. The most impressive piece of the story is what happens in the space of time between sound reaching our ears and our brains perceiving it.

What you probably already know is that the cuplike shape of mammalian ears is for focusing sound into your ear canal, where it is amplified up to twenty-two times the original intensity and funneled towards the eardrum. Tiny changes in the position of this membrane coordinated by the frequency of sounds hitting the eardrum push a series of three bones, each connected to the next to confer the eardrum’s movement to one of the most astounding sensory organs in the human body, the cochlea.

Choclea Diagram - Dollos, 1992The cochlea resembles a snail shell, spiraling inwards to its end, the apex. When the last of the three inner ear bones makes contact with the base at what’s called the oval window, mechanical waves mimicking the original sound waves from the ear canal travel the length of the cochlea.

This is where the story gets really interesting. Here in this organ is the site of transduction from mechanical waves to neural pulsing. The natural process of evolution has rendered a method for determining both of the two qualities of sound (frequency and amplitude) that is so perfectly simple I can’t help but be in awe.

Imagine if we were able to unroll the cochlea from its packed spiral into a straight line. At the base, the middle ear bones press a copy of the mechanical sound waves onto the outer membrane of the cochlea. These representative sound waves then travel the length of the cochlea all the way to the apex. Now, if we cut across the width of the unrolled organ, you would notice that the inside of our “shell” has three compartments. The outer two fluid-filled compartments oppose one another and are separated from the center by thin membranes. The center chamber is lined with rows small hair-like cells responsible for signaling to the underlying neurons (more on this in the next installment).

The bottom, or basilar membrane running the length of the cochlea actually changes shape to match the movements of the incoming sound wave. It’s the thinness of this membrane that is the key. When pressure at the peak of the wave builds, the basilar membrane responds by forming a physical wave!

Now you have to wonder why the cochlea is divided into so many compartments, right? Well, the separation between these compartments is the secret to dissecting the qualities of sound. Recall that the frequency of sound is marked by the distance separating peaks in air pressure. The hair cells of the cochlea can actually determine the frequency of sound by the distance from the base to the peak of the wave formed by the basilar membrane. High-frequency sounds, such as screaming, have short distances separating peaks and will peak closer to the base of the cochlea while low baritone sounds with long separations between peaks will peak further towards the apex. This peak remains vibrating up and down in its location along the basilar membrane until the sound eventually dies out.

Since the hair cells in the middle chamber of the cochlea are rooted on top of the basilar membrane and situated under the other, tectorial membrane, they transmit signals to neurons when they are compressed or stretched between the two. This change in compression between the two membranes, called “shear stress”, gives the brain an accurate picture of the location of sound wave, therefore, the frequency, and the height between the two membranes interpreted as loudness.

In essence, our bodies have engineered an incredibly simple device to first modify sound waves into mechanical waves and then allow for interpretation of the qualities of the incoming sound. It does so by creating a tonographic (“sound map”) image of incoming sound waves. This double membrane system situated in the spiraled cochlea of the inner ear is the remarkably elegant design rendered by nature to address the problem of deciphering auditory information.

To be continued in Part II…

A Touchy Subject

More often than not when learning science, we are taught to ignore the context underlying major discoveries. We are told to memorize details without seeing the big picture; however, the stories behind these great advances can be extremely compelling. To me anyways, one of the most intriguing trends in the stories underlying scientific exploration is that often times the most critical advances in understanding come not from rigorous experimentation (as so many of us are misled to believe), but from insights and revelations formed via intense observation of natural processes.

The greatest scientific minds throughout history are remembered for predictions rooted in realizations that had never occurred to anyone else before them. Based only on the movements of stars in the sky, Copernicus postulated the structure of our solar system. Newton hypothesized gravity by observing falling objects and Darwin created the Theory of Evolution by collecting specimens and fossils separated in form by time and geography. In the same manner, a great German anatomist once predicted the cellular basis for the sensation of touch.


Friedrich Sigmund Merkel

In 1864, after bureaucracy put a stop to his adventurous dream to become a sailor in the German navy, a youthful Friedrich Sigmund Merkel began studying to be a physician. The son of intelligent and wealthy parents, Friedrich’s free thinking was encouraged in his pursuit of a scientific education. Early in his learning, however, he firmly assured his mother that out of all of the sciences, he would never become an anatomist; a statement completely invalidated by his lasting impact on the field he so adamantly rejected early on.

The ambitious scientist’s fascination by the invention of the microscope lead to many revelations. He published works on the iris musculature of the eye, papers about the connections of the chest muscles, and manuscripts outlining benefits of microscope use for anatomy. Then, in 1875, Merkel first observed and described “Tastzellen”, or touch cells. Merkels’ newly discovered cells would make an enormous impact on the physiology of touch, or mechanosensation, but his original term has been substituted for a new name. “Merkel Cells” forever commemorate the enormous impacts of the famous Anatomist.

In mammals, Merkel cells are dispersed throughout all of the skin. While, in the majority of the body, the cells surround the base of hair follicles; the areas that are highly sensitive to touch use Merkel cells concentrated in tiny lumps called touch domes. In both cases, these specialized skin cells form connections with sensory neurons projecting to the brain. While it is true that your skin is the largest body organ, containing over 30 billion cells, very few of them actually connect with the nervous system in any way. Most just form protective layers which will eventually be shed off of your body.

In a manner resembling other great scientific discoveries outlined above, having only the observation that his cells form synaptic connections with underlying neurons, our insightful Friedrich Merkel predicted that his tastzellen were likely responsible for the sensation of touch.

Modern science now defines several classes of mechanosensation including painful touch, heavy pressure, vibration, stretch, and more. Other cell types have been explored for roles in these sensations, and evidence has been built for the Merkel cell’s involvement in the discrimination of “light touch”. These cells likely give us the basis for discriminating between textures and finding small edges.

Hair Follicle and Touch Dome Merkel Cell Complexes Updated Merkel Cell Drawing

How can these skin-derived, or epithelial, cells send all this information to our brains? Non-neuron cells are known to play parts in the tongue for taste detection, and also in the inner ear in our ability to hear. The common feature of these signaling epithelial cells is their ability to quickly change their electrical charge, or depolarize, in the same way that our neurons do. However, unlike neurons, epithelial cells do not form complex signaling networks or project to the brain or spine. When the correct stimulus is present, these cells open channels in the surrounding membranes to allow charged ions to flood the cell. This rapid change is carried all the way to the site where the receptor cell meets a sensory neuron, at which point; the depolarization is carried along the length of the neuron towards the brain.

Now, a major question is begging to be asked. How do these receptor cells, in our case, Merkel cells, discriminate between different types of stimuli? Each receptor cell must have a specialized receptor molecule on its cell membrane, either acting as an ion channel itself or else with the ability to cause a separate ion channel to open. In many cases of sensory transduction, vision, taste, smell, these ion channels are well known. However, in most cases of touch mechanosensation, the identity of these critical players was not known. What we do know is that both isolated Merkel cells and the ones in living tissue display electrical current changes when slightly moved by a probe. This means that a molecule on the membrane of these cells must respond to mechanical force by allowing charged ions to enter the cell.

The Piezo family, a family of large membrane proteins, is one of the largest proposed ion channels with over 30 regions of the protein passing through the membrane (most have only 7 such regions). Over the last year, groups around the world have been investigating the role of one such Piezo protein family member, named Piezo2, as the ion channel involved in Merkel Cell touch sensation in mice.

Mice typically prefer textured ground conditions in their cages. When given a choice between a textured surface and smooth ground, mutant mice without working Piezo2 protein don’t show the same preference. By tagging one end of the protein with a fluorescent marker, they can use microscopes to observe Piezo2 expressed only at the tips of the neurons closest to the Merkel cells and in the Merkel cells themselves. Studies of electrical current changes, or electrophysiology, in Merkel cells and the sensory neurons demonstrate that the mutant mice cells do not respond to mechanical force as do the cells with working Piezo2.

The enormous body of evidence continues to build for Piezo2’s responsibility as the mechanically activated ion channel in mice. While studies in mice are not always indicative of answers for humans, our two species’ versions of the proteins are fairly similar. Future studies will determine whether or not the protein plays the same role in the human being, but if so, the identity of the channel responsible could have major impacts on the medicine of touch and pain. It is astounding to me that just now, 150 years after Friedrich Sigmund Merkel began his studies as a physiologist, we are finally beginning to understand the molecular basis for his most important prediction and the cells that bear his name.