Wednesday, December 24, 2008

The Power of Instruction & The Brain

What is wrong with this tiger's face? Humans strife for harmony in their social relationships as well as in their aesthetic appreciation of artistic expression. In our perception, perfect symmetry reflects absolute harmony. For that reason, we perceive a perfect sphere, that is the shape with greatest symmetry, as most aesthetically pleasing.

When we study this picture (courtesy anonymous), we are struck by the exquisite fur markings of the tiger's face. Black stripes boldly transect a background in mild shades of brown and white. On the cheeks, lines of black dots stand out on a very bright white close to the lips, transforming into light brown toward the nose. Our eye is caught by the side-to-side symmetry of the pattern. On each cheek, we notice four rows of black dots, increasing in number from top to bottom. Visible on occasion, a whisker rises from each dot.

The dot pattern seems entirely symmetrical on first glance. But close inspection tells us that this is not true for this tiger's face. On the animal's left we spot a solemn black dot between the first and the second row at the back. There is no dot at this location on the right side. We discovered a supernumerary whisker! To developmental biologists, this find is as significant as the extra finger or toe physicians observe on rare occasion in humans, a condition known as polydactyly.

The whiskers of a variety of mammals including cats, rodents, rabbits, pinnipeds and manatees are more than just hair. They rather constitute complex organs of touch. Whiskers are sinus hairs. In contrast to the hair of common fur, the follicles of sinus hairs contain large cavities filled with blood surrounding the hair shaft. While perhaps a dozen sensory nerve fibers innervate the hair follicles of common fur, hundreds of nerve fibers innervate the follicles of sinus hairs, ramifying into thousands of endings.

At least four different types of touch receptors have been identified in cat sinus hair follicles (Gottschaldt and Vahle-Hinz, 1981). Merkel cell receptors constitute one common type. They consist of cells on which sensory nerve endings attach and are distributed around and along the hair shaft. When the whisker is bent, the Merkel cells are compressed and the force is transduced into a rapid succession of electrical spikes in the sensory nerve endings. These signals are conducted along the nerve fibers and transmit the touch response via nerve cells in the brain stem, across the mid line to the thalamus in the diencephalon (interbrain) on the opposite side. From there, the signals are relayed into a region of cerebral cortex called primary somatic sensory area.

We may wonder how the somatic sensory system processes the tactile input from the supernumerary whisker. Are supernumerary whiskers inheritable? Does the genetic modification that precipitates the development of an extra whisker result in complementary change in the brain? Is the touch sensation from this whisker transmitted at all? If so, how is the additional sensory input accommodated?

Research in mice provides some answers. As in cats, the whiskers on the mouse's snout are cast in a remarkably conserved pattern. They are arrayed in five distinct rows that Thomas Woolsey and Hendrik Van der Loos (1970) called A to E from dorsal (top) to ventral (bottom). Four whiskers straddle the rows caudally, that is at the back. Woolsey and Van der Loos proposed to number the whiskers in each row in rising order, beginning caudally with 1. Rows A and B are shortest, consisting of only four whiskers, whereas rows C, D and E contain eight whiskers and more.

The snout of a mouse showing the five rows of whiskers (source: Jackson Lab.).
Furthermore and most intriguingly, the two researchers were able to show that cytoarchitectonic units, they called barrels, represent the whiskers on the snout topographically in primary somatic sensory cortex.

Mouse barrelfield (courtesy of H. Van der Loos)
The above picture shows a micrograph of an 80 micrometer-thick section cut tangentially through the left hemisphere of cerebral cortex of an albino mouse. The animal's left side is up (dorsal). The nose (rostral) is right. The section was stained for cell bodies with a blue dye. The barrels are clearly visible as nerve cell-dense rings surrounding cell-sparse centers.

In subsequent work, Woolsey and Van der Loos could demonstrate that the removal of whisker follicles at birth prevented the corresponding barrels from developing (Van der Loos and Woolsey, 1973). This was the first demonstration that the cerebral cortex was organized in structurally modular units and, most importantly, that the development of these structures was dependent on input from the sensory periphery.

After completion of these ground-breaking experiments, Hendrik Van der Loos moved from Johns Hopkins School of Medicine to Lausanne, Switzerland, where he assumed the directorship of the Institute of Anatomy at the medical school of the University of Lausanne. There, he met Josef Dörfl. Josef was going to be the first to knowingly observe a supernumerary whisker. He worked with Hendrik who continued in Switzerland to examine barrels and the factors that influence their development. One day, while they were reconstructing barrel patterns from histologically-stained sections through somatic sensory cortex, they haphazardly discovered a supernumerary barrel in row A. Josef was urged to check this animal's whiskerpads more closely.

Josef was an accomplished anatomist with a keen eye and well versed in examining whiskers. Unlike cats, mice whisk. They move their whiskers rhythmically to palpate objects. Josef was studying the anatomy of the mouse whiskerpad with micro-dissection, identifying the muscles that move the whiskers as well as the sensory and the motor innervation involved (Dörfl, 1982 and 1985). Examining the whiskerpad in question through a surgical microscope, Josef readily identified a supernumerary whisker near the nose at position A5 on the side opposite the cortical hemisphere with the extra barrel. The whisker was located exactly at the position that the extra barrel indicated! In future breeding, mice with supernumerary whiskers would predictably produce offspring with this trait.

This discovery constituted a decisive event laden with consequences. Josef, Hendrik and their colleagues had found an inheritable genetic modification adding a whisker follicle which then instructed the developing brain to add a whisker representation accordingly. Several thousand mice were bred. The effort eventually produced strains with a variety of supernumerary whisker phenotypes. There were mice with twin follicles, mice with fewer whiskers in rows A and B, mice with an extra row of whiskers between rows B and C, and mice with supernumerary whiskers in multiple locations.

In almost all cases, the barrels in somatic sensory cortex matched the whisker pattern. Hendrik's student Egbert Welker and his late wife Karin Van der Saag would spend years, compiling hereditary charts and comparing whisker follicles and follicular innervation with cortical barrel organization. They were instrumental in publishing the research. The effort led to a comprehensive series of publications in renowned scientific journals beginning with two articles in the Journal of Heredity (Van der Loos and others, 1984 and 1986), paving the way for Dr. Welker's thesis.

In the years that followed, Hendrik Van der Loos would end his life during a bout of deep depression and research on the mice with supernumerary whiskers would cease. Their embryos were frozen away, supplanted by vast numbers of genetically engineered mice strains that populate research universities worldwide at present. Nobody checks for supernumerary whiskers anymore.

We know today that the instruction from the whisker follicles for the development of barrels in somatic sensory cortex is crucially dependent on nerve cell activity mediated by the excitatory neurotransmitter glutamate. Genetic knock-out mice lacking glutamate receptors may not develop barrels and their equivalents in thalamus and brainstem, called barreloids and barrelettes, respectively. The neurotransmitter serotonin appears to play a facilitating role in cortex. High levels of serotonin impede proper barrel development in monoamine oxidase A-deficient knock-out mice. Most recently, the metabotropic glutamate receptor subtype mGluR5 has been identified as one pivotal player in the development of barrels, barreloids and barrelettes, affecting all stations of the somatic sensory system (Lu, 2008; Wijetunge and others, 2008). Other research suggests that mGluR5 receptors modulate the development of psychiatric disorders, and new drugs acting specifically at this type of receptor are being tested. I have written about these issues in my posts dated Apr. 1 and Oct.1, 2008.

Despite the advances in elucidating the molecular mechanisms underlying barrel development, the functional significance of these structures is obscure. The sensory inputs from a whisker terminate clustered inside the barrel representing this whisker. In accord, deflection of the whisker evokes the earliest nerve cell responses in the corresponding barrel. The separation of sensory input into discrete domains may, therefore, explain the observed intimate correspondence of functional and morphological whisker representations in somatic sensory cortex of mice. By contrast, cats do not contain barrels in their somatic sensory cortex. Yet, the nerve cells form functional topographic whisker maps as fine-grained as those observed in mice. Hence, barrels seem unnecessary for the separation of sensory input, and their role in the processing of tactile information remains to be elucidated.

On the other end of the whisker-to-barrel pathway, we do not understand to the day what mechanism precisely controls the induction of whisker follicles on the face and which factors determine their number and layout. Would not it be fascinating, if the same set of genes controlled the formation of this pattern in big felines as well as in tiny rodents?

  • Hendrik Van der Loos already suggested that two independent mechanisms influence the development of the whisker pad: one mechanism that controls whisker follicle formation and another that limits the area of maxillary skin available to follicle formation. Therefore, at least two separate molecular signaling pathways with their own sets of genes control the development of the whisker pattern. Sonic hedgehog and T-box genes may constitute candidates for follicle formation and number, respectively. Wilson and Conlon (2002) published an in-depth review on the role of the t-box family of genes in development and disease (04/22/09).
  • In her report for National Public Radio's Health Blog Shots with the title "Touring Memory Lane Inside the Brain" posted online today, Amy Standen describes recent advances in the visualization of cortical cell architecture (Micheva and others, 2010). A demonstration of results is presented in the animated journey along a coronal section through the anterior (nose-ward) mouse barrel cortex below. The movie covers a volume approximately 0.5 mm wide, 12 μm thick and 1.4 mm deep, stretching from the cortical surface (top) to the white matter and the striatum, an underlying subcortical structure (bottom). Nerve cell bodies are labeled green; their processes green and blue. Red labels the contacts between nerve cells known as synapses. The barrels are located above the agglomerations of red synapses at mid-depth (11/18/10).

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