Thursday, December 3, 2009

H.M.'s Brain

The philosopher Martin Heidegger reasoned in his treatise "Sein und Zeit" that our sense of time confers our sense of being (Heidegger, 1976). Henry Gustav Molaison could attest to that. In order to relieve Henry from severe bouts of epileptic seizures, a part of his temporal lobes had been removed when he was in his late twenties.

The seizures subsided. However, as an unwanted consequence of the surgery, he had lost essential elements of his memory. He could not remember who he was and had to discover his identity anew every day. One day late in his adult life, Henry was caught curiously examining his face in a mirror. He called out in amazement, "I am not a boy!" In Heidegger's words, Henry Molaison had lost his sense of time and, with that, his sense of being.

As patient H.M., Henry volunteered for scientific research throughout his life, becoming one of the most intensely examined cases in psychology. He willed his brain to research. Yesterday a year ago, he passed away of respiratory complications at the age of 82. On the first anniversary of his passing, the histological processing of his brain was begun at the University of California San Diego. The university's Brain Observatory provides live online footage of the sectioning of the frozen whole brain on a cryotome until Dec. 4, 2009. The brain is cut coronally, that is in the transverse plane. Seventy micrometer-thin sections are collected for histology at equally-spaced intervals. At the speed maintained thus far, we shall reach the site of surgery tomorrow.

The ensuing histological examination of his brain will complete Henry's scientific journey as he willed. The findings may provide important insights into the histochemistry of memory, constituting the keystone in the arch of knowledge that H.M. allowed us to build.

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  • At 5:00 am (CST), we are in the midst of the temporal lobe of H.M.'s brain. If you look closely at the bottom of the brain (ventral, on the left hand side of the observer), you see cauliflower-shaped protrusions on both sides. These are the temporal lobes. There is a void inside the structures. Parts of the hippocampus seem absent (12/04/09).
  • At about noon, in section 1672 (46,344 micrometers), we can see a cavity in left lingual gyrus (in the hemisphere closest to the observer) (12/04/09).
  • At 5:00 pm (CST), we are entering the occipital lobes; at 10:00 pm we passed the middle of the occipital lobes (12/04/09). 
  • We should be able to detect signs of nerve cell and nerve fiber degeneration in the brain regions that were connected to the brain tissue that was removed or damaged otherwise during surgery, particularly in the structures of the limbic system. Degenerating nerve fibers can be rendered decades after the damage and ameboid microglia may reside in the affected locations (Miklossy and others, 1991). According to von Monakow's concept of diaschisis, the affected regions can be far-flung and distant from the site of immediate damage, comprising the opposite cerebral and cerebellar hemispheres. That is why H.M.'s whole brain needed to be sectioned, and 2,401 sections were collected (12/05/09).
  • The footage covering the sectioning does not seem to be available on The Brain Observatory's site at this time. You may find a CBS report broadcast Dec. 3, 2009, instructive (01/01/10):
  • Brain sections are prepared for histology with two distinctly different methods. One way requires that the tissue is embedded in hot paraffin wax or plastic. The tissue is sectioned at room temperature on a bench top with a microtome or ultramicrotome, respectively. Paraffin embedding is widely used in routine pathology. Plastic is used to embed specimen to be cut into ultra-thin sections for electron microscopy. The other method of tissue preparation requires that the specimen are frozen. Frozen tissue has to be stored and sectioned at temperatures below 0 ℃.

    With either method of preparation, the tissue is commonly first soaked in solutions of formaldehyde, glutaraldehyde or a mixture of both. The aldehydes help bond cellular protein, preserving cellular structure during the further processing steps. H.M.'s brain was then soaked in highly concentrated sucrose solutions and embedded in dextran. Sucrose and dextran are sugars. The sucrose prevents ice crystals from forming in the cells when the tissue is frozen. The growing crystals would otherwise burst the cells. The dextran helps preserve the integrity of the sections, preventing tissue from breaking off the perimeter of the specimen during cutting. Once the brain is fixed and cryo-protected, it can be cut into sections that are only tens of micrometers thick on a microtome on which only the stage holding the brain is kept at subzero temperatures. The sections are picked up from the microtome's knife with a brush and can be processed freely floating in solutions containing the substances needed for the chosen histological staining procedure.

    There are numerous histological staining procedures. Most comprise chemical reactions. Others involve antibodies that bind specifically to cellular proteins or fluorescent dyes that fill particular cellular components. Among the oldest procedures is the silver impregnation method named after  Camillo Golgi, rendering the cell bodies of nerve cells and a good part of their processes (dendrites) and protrusions (dendritic spines). This method helped Ramón Cajal to divide the cerebral cortex into six layers, a distinction that is still used today. Golgi and Cajal were awarded the Nobel Prize for their contributions. The most commonly employed methods, however, are the Nissl stain for cell bodies and the myelin stain that stains the fatty sheeth around nerve cell fibers in the brain's white matter. These methods will certainly be used with some sections of H.M.'s brain. After staining, the sections are mounted on glass slides, dehydrated in solutions of increasing alcohol concentration, embedded in a clear organic solvent-based glue and covered with thin glass coverslips.

    The methods of tissue preparation described above cannot be used when the procedures require that fixation-sensitive cellular proteins remain functional or when water-soluble, diffusible substances are to be localized in the tissue. In that case, the tissue must remain unfixed and immersion in sucrose cannot be used. The tissue has to be shock frozen at -70 ℃ and lower, and the sections must be kept frozen during sectioning. To fulfill this need the whole microtome is encased in a refrigerated cabinet. The instrument is then called a cryostat. Ten or 20 micrometer-thick sections are cut and picked up on chilled glass slides and quickly dried on a hotplate outside the cabinet. The subsequent procedures must consequently be carried out on the slide-mounted sections. This method is used for the autoradiography of diffusible radioactively-labeled tracers. For autoradiography, the sections are exposed to x-ray films or radiation-sensitive semiconductor chips that produce gray-scale images of the distribution of the tracer in the sections. The optical densities in the images called autoradiograms are proportional to the concentration of the tracer in the tissue and can be measured.

    In order to trace metabolites in whole bodies, Sven Ullberg and colleagues at Uppsala University, Sweden, engineered a microtome sufficiently sturdy to cut thin sections through the whole body of small animals. The microtome was placed it in a chest freezer with windows in the lid (Larsson and Ullberg, 1981). The instrument is called a whole-body cryotome and was commercially available from the Swedish company LKB. I was privileged to use a LKB cryotome 30 years ago for functional imaging studies (Melzer, 1984). The picture below shows a whole rat embedded length-wise in dextran and frozen in liquid nitrogen at -173 ℃. The dextran block with the rat is mounted on the sliding stage of the cryotome for sectioning.
    The block of dextran with the rat resembles that with H.M.'s brain in the footage of the Brain Observatory which misled me initially. But as I pointed out, H.M.'s brain was fixed and protected with sucrose. Only the microtome stage had to be cooled. No freezer chest was needed. Jacopo's hands stayed warm.

    One great advantage of whole body sectioning is that the sections render the organs in their actual position inside the body. The picture below shows a the block surface of the head of a sandrat embedded in dextran and cut transversely at the level of the midbrain.
    The structure at the center is the brain. The glassy cavities on both sides of the brain are the inner and middle ears. The middle ear cavities of sandrats are remarkably enlarged compared with other rodents, serving as a low pass filter. The animal had been exposed to sound for functional imaging. Brain activation images are shown in my post entitled "The Quest for the Infrasound Acoustic Fovea" and dated Oct. 12, 2009.

    Since the brain was preserved accurately as it was situated in the scull, we were able to pinpoint brain structures with stereotaxic precision, a goal that may be achieved with structural magnetic resonance imaging today. That method did not exist then.

    In addition to functional imaging, fresh tissue tissue sections permit us to examine the distribution of radioactively-labeled ligands that bind to specific receptors on cell surface or probes that identify sequences of genetic code. These procedures can not be applied to the sections of H.M.'s brain. However, even with the histological techniques available a treasure of knowledge will be uncovered from the brain that Harvey Molaison kindly willed.

    In the past three decades most departments of anatomy have been transformed into departments of cell biology for good reason. However, gazing through a microscope at the multitude of diversely shaped nerve cells in a Golgi-stained section through cerebral cortex still remains as fascinating as gazing through a telescope at the myriad of stars in the night sky. The neuroanatomical methods that make this experience possible resemble in many ways forms of art, though rather of an artisan than of an artist. Perhaps, the work on Harvey's brain will rekindle interest in this type of expertise, paving the way for a renaissance of neuroanatomy (01/02/10).
  • The Allen Institute for Brain Science in Seattle, WA, compiled the first comprehensive atlas of local gene expression in the mouse brain five years ago and has now released a similar atlas for the human brain (04/13/2011):


Thursday, November 19, 2009

Advances in Spongiform Encephalopathy

Well into the 1970s, members of the Fore tribe of Papua New Guinea were afflicted by a devastating neuro-degenerative epidemic known as kuru. The disease shares considerable similarity with spongiform encephalopathy first described by the German physician/scientists Hans Gerhard Creutzfeldt and Alfons Maria Jakob in the 1920s, CJD for short. Often more than a decade after infection, the afflicted develop conspicuous trembling and severe motor dysfunction as a consequence of progressive nerve cell death in the central nervous system. The video below portrays the symptoms of this horrible disease graphically. Some readers may find the footage disconcerting.

As a consequence of nerve cell degeneration, the brain tissue shrinks in places. Microscopic holes pockmark the tissue like a sponge in histological preparations.

Kuru may have been acquired through the inheritance of defective genes. Isolated tight-knit island communities like the tribes of Papua New Guinea are known to be particularly vulnerable to inheritable genetic defects because of their limited gene pools. Indeed, genetic mutations seem to cause spongiform encephalopathy. Recently, Wang and others (2008) provided evidence for the underlying molecular mechanisms.

Peculiarly, among the Fore of Papua New Guinea particularly women and children developed kuru. The Fore were known to ingest the bodies of deceased loved ones during funeral rites in order to capture their life forces. Women and children ate mainly brain parts. Perhaps the origin of the disease was passed along with the brain tissue. In support of this idea, the late American Nobel Prize-laureate Daniel Carleton Gajdusek proved that kuru was indeed an infectious disease that could be transmitted even to other primates. Curtailing cannibalism on the islands would drastically reduce the occurrence of the disease.

Years later, Stanley Prusiner would win the Nobel Prize for ultimately identifying the molecular basis of the transmitted variant of spongiform encephalopathy. The infectious agent was not an organism. Nerve cells incorporated misfolded proteins known as prions that attached to native forms of functional cellular proteins, forcing them to undergo a similar conformational change. Like a snowball, the dysfunctional protein grows forming amyloid plaques that eventually destroy the cells.

Misfolded prions are fairly stable, temperature resistant molecules, remaining active even in soil outdoors for decades. Scrapies, which causes spongiform encephalopathy among sheep and goats, is believed to be contracted through fodder contaminated with urine and feces from infected animals.  Bovine spongiform encephalopathy (BSE), popularly known as Mad Cow Disease, has been shown to be transmitted by protein-enriched power feed contaminated with prions. Beef from infected cattle may cause CJD in people.

Remarkably, however, some highly-exposed Fore remained unaffected by kuru. In this week's issue of the New England Journal of Medicine, researchers at University College of London report that they found a new variant of the gene PRNP encoding prion protein that is unique to the resilient tribal members and may improve resistance to the disease (Mead and others, 2009). The newly discovered genetic variant, called G127V, appears to render the native prion protein less pliable to conformational change induced by misfolded protein. It was detected in half of the women who were homozygotes for an already known resilience factor. That is, they possessed identical variants of PRNP on both chromosomes carrying the gene. By contrast, tribespeople who succombed to kuru did not possess this variant, and it is absent from the global human population unexposed to the disease.

This discovery does not only open new avenues for the treatment of spongiform encephalopathy, but moreover further affirms genotypal variation and phenotypal selection as fundamental evolutionary forces that affect all living things as Charles Darwin proposed in his theory published in "On the Origin of Species" almost to the day 150 years ago. Darwin would have been delighted to learn about this finding, particularly in the year of his 200th birthday.

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Wednesday, November 4, 2009

Constantin von Monakow & Brain Plasticity

Constantin von Monakow was born in Bobresovo, Poland at that time, on this day in 1853.  He was to become one of the most eminent neuroanatomists, neurologists and psychiatrists of his age.  Below I attempt to highlight some of his achievements, relying mainly on Mario Wiesendanger's informative article in the Comptes Rendus Biologies (Wiesendanger, 2006).

Von Monakow spent most of his life in and near Zürich, Switzerland, where his family moved in his youth. He studied medicine, worked with Eduard Hitzig at one of the first psychiatric research clinics in continental Europe, the Burghölzli. The principal investigators at the clinic led by Auguste Forel had fully recognized the role of cerebral organization in mental illness.  Hitzig and Gustav Theodor Fritsch were among the first to localize function in the cerebral cortex with electrical stimulation electrodes.  Hitizig hired von Monakow as assistant and taught him histological methods.  At a brief visit with the famous anatomist Bernhard von Gudden in Munich, he familiarized himself further with silver impregnation methods that permitted him to visualize degenerating nerve fibers. Injury to brain tissue, precipitated by hemorrhagic bleeding, stroke or trauma, results in the degeneration of the nerve cell connections between the affected location with other parts of the brain.  Tracing degenerating nerve fibers, therefore, could be used to examine pathways among brain structures.

Von Monakows independent research would begin under small and unusual circumstances as attending physician at St. Pirminsberg, a psychiatric asylum near Bad Ragaz, a spa not far from Zürich.  One day, while inspecting the premises, he discovered a brand new microtome stowed away in an unused closet.  He knew how to use the microtome and turned the pantry into a small histological laboratory to get a series of experiments off the ground that would uncover the brain's visual pathway.  The faculty of the University of Zürich accepted the publication of the results of these experiments as qualification for the right to teach (habilitation), and he started to give lessons at the university without salary, while attempting to maintain a private practice for neurology and general medicine in the city.  The practice barely supported him. Yet he managed to continue his research in a small laboratory he sat up for himself.

Eventually in 1894, von Monakow received a call from the University of Innsbruck, Austria, to assume a chair in psychiatry.  The offer compelled the government of Zürich to make a counterproposal which von Monakow happily accepted, although the appointment was only at the level of associate professor. Despite the lower rank, the new post permitted him to continue his research in his laboratory, which became known as the Brain Institute and serve as the director of a psychiatric policlinic.  Finally, 17 years after he graduated from medical school, he had garnered a stable position, securing financial support for his research and a stable income.

Von Monakow worked full-time at the university until his retirement at the age of 70, and continued another four years as honorary professor and director of his laboratory, which became known as the Brain Institute. During his years as professor, he maintained an active journal club, enjoying regular visits from a number of eminent scientists and physicians. Among others, the fourth director of the Burghölzli, Auguste Forel, and the famed neuroanatomist and neurologist Constantin von Economo were in attendance as much as the founder of psychotherapy, Carl Gustav Jung, whose long-hidden self-analysis, known as The Red Book, has just been published. 

Constantin von Monakow passed away in 1930 at the age of 77, and his former responsibilities were divided among several successors.  In 1962, Konrad Akert succeeded in recreating Von Monakow's Brain Institute as The Institute for Brain Research, which has remained a vibrant site for world-class neuroscience research to the day. 

Constantin von Monakow was a prolific writer and published a long list of scientific articles, book chapters, books, hand books and anatomical brain atlases.  His work entitled "Die Lokalisation im Grosshirn und der Abbau der Funktion durch kortikale Herde" (Localization in the Cerebral Cortex and Loss of Function Produced by Cortical Lesions) arguably became one of his most notable contributions.  In this book, he laid out comprehensively the principles of chronogenic localization and diaschisis.

Von Monakow's observations suggested that the cortical and subcortical components of a brain pathway cooperate in precisely timed sequence to synergistically effect coordinated brain function, though the components may not necessarily be situated near each other.  He called the cooperative, yet distributed localization of brain function chronogenic localization.  His concept ran counter to the widely-held belief at the time that brain function was localized in circumscribed centers dedicated to particular tasks.  Phrenology represented the most extreme and popular variation of that idea.  History would prove von Monakow right.  The functional brain imaging studies conducted today commonly render multiple loci of activation in cerebral cortex when the participants execute a task, confirming that brain functions are indeed carried out by distributed nerve cell networks involving a number of cortical areas.

For example, using functional magnetic resonance imaging, my colleagues and I observed numerous foci of activation scattered across cerebral cortex when people with severe visual disability read Braille with their fingertips (Melzer and others, 2001). Intriguingly, cortical areas in the occipital lobes were involved that process visual information in sighted people. The animation below shows these foci in a 36 mm slice through cerebral cortex. The poles of the occipital lobes point to the bottom at the finish.

As another significant finding, von Monakow observed that acute damage to one component of a brain system immediately depressed the function of the system's unharmed components, disabling their coordinated cooperation.  He called this immediate loss of adequate control over integrated brain function diaschisis.

Von Monakow noted furthermore that with time functionality would return, though a residual deficit almost always remained. Differences in the timing of the recovery of various aspects of the lost function would bare its chronogenic localization.  The recovery would help identify each component's contribution to the integrated function.  Nerve cell plasticity would permit the unharmed components to reorganize and reintegrate. However, he could only speculate on the underlying cellular mechanisms.

Von Monakow proposed that the increased use of existing, undamaged collateral nerve cell connections within cerebral cortex stimulated the development of new connections, recruiting nerve cells in other cortical areas for the recovery of lost function. Furthermore, he recognized that exercise benefited recovery, suggesting that exercise-related stimulation facilitated the growth of new, more extensive nerve cell connections. As a consequence novel behaviors would be learned to compensate for residual deficits.

In own research, my colleagues and I observed that nerve cells in the vicinity of a stroke lesion in the cerebral cortex respond to sensory input only at short latency in the days after the infarct.  By contrast, responses at long latency were suppressed (Melzer and others, 2006). Inputs from intracortical nerve cell connections are thought to drive the long latency responses.  The observed suppression is entirely consistent with von Monakow's diaschisis.

Von Monakow's concept of distributed nerve cell networks underlying brain function was astoundingly modern.  His ideas of recovery of function were profoundly forward looking.  More than a century after their inception, his ideas are still inspiring intense research.

  • Melzer P, Maguire MJ, Ebner FF (2006) Rat barrel cortex as a model for stroke analysis. Soc Neurosci Abst:583.13.
  • Melzer P, Morgan VL, Pickens DR, Price RR, Wall RS, Ebner FF (2001) Cortical activation during Braille reading is influenced by early visual experience in subjects with severe visual disability: a correlational fMRI study. Hum Brain Mapp 14:186-195.
  • Monakow von C (1914) Die Lokalisation im Grosshirn und der Abbau der Funktion durch kortikale Herde. J.F. Bergmann, Wiesbaden.
  • Wiesendanger M (2006) Constantin von Monakow (1853-1930): a pioneer in interdisciplinary brain research and a humanist. C R Biol 329:406-418.
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Friday, October 23, 2009

Free Will Exists: Morat Fribourg, 2009

This year's race from Morat to Fribourg, held Oct. 3 in its 76th edition (erratum: My count was one off, when I posted first), was won again by Helen Musyoka (1:01:29 h) and John Mwangangi (0:52:37 h), both from Kenya. Congratulations!

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Thursday, October 22, 2009

Absolutes, Relatives, Brain Imaging & Steroids

The functional brain imaging methods most commonly used in humans today are functional magnetic resonance imaging (fMRI) using blood oxygen-level dependent (BOLD) signals and the subtractive water method with positron emission tomography (PET). Both procedures record changes in local cerebral blood flow from a baseline. Local cerebral blood flow is associated with energy demands of activated nerve cells. The cells consume glucose sugar and oxygen to process information which cannot be stored in the brain and must be supplied on demand with the blood stream. Hence, blood flow increases with increased nerve cell activity. In the simplest conception of both procedures known as block design, measurements acquired over several minutes of mental activation are compared with measurements acquired during equivalent epochs of rest. The statistically significant difference is considered the result of cerebral activation.

In conventional fMRI, blood oxygen-level dependent signals are used to detected changes in blood flow (Ogawa and others, 1993). With the subtractive water method (Fox and others, 1984), water labeled with 15O, a radioactive positron-emitting isotope of oxygen, is used as tracer that freely diffuses into the brain tissue. The local concentration of the tracer is commensurate with local blood flow and can be imaged in a PET scanner. However, calibration for absolute blood flow is wrought with difficulty and has not found wide-spread application. Because the blood flow is not quantified, the differences between the compared mental states remain relative. That is, cerebral activation is usually expressed in percent difference from the state used as reference or in units of statistically significant difference (statistical parametric mapping).

By contrast, the cerebral glucose consumption is more directly related to nerve cell activity than cerebral blood flow. The deoxyglucose method of Sokoloff and others (1977) permits us to measure the local cerebral rate of glucose utilization. Deoxyglucose is an analogue to glucose that accumulates in the brain tissue commensurate with glucose consumption. Tagging deoxyglucose with 18F, a radioactive positron-emitting isotope of fluorine, the tracer's accumulation in the brain can be imaged with PET. The [18F]fluorodeoxyglucose method, therefore, provides a snapshot of the brain's energy consumption (Reivich and others, 1979). Although this snapshot needs 90 minutes to develop because of the tracer kinetics involved, the procedure constitutes an indispensable tool for the detection of long-term, pseudo-stationary changes in absolute cerebral metabolic activity as a consequence of disease or trauma. Below, I discuss one example.

After the collapse of the regime of Nicolae Ceauşescu at the end of 1989, U.S. parents began to adopt children from Romanian orphanages. The children had been kept in circumstances of great depravity, producing profound behavioral problems similar to autism (American RadioWorks report, 2006). Visiting scientists reported behavioral patterns resembling those the eminent American psychologist Harry Harlow had so aptly described in primates raised in isolation and with surrogates.

Although the adoptees were brought to the U.S. at very young age, some developed cognitive and behavioral differences, including impulsive reactions as well as attention and social deficits, in the years after their arrival.

Research at the orphanages provided evidence that the children had persistently augmented levels of cortisol n their blood stream as a result of the severe stress they endured (Carlson and Earls, 1997). Cortisol is a known steroid stress hormone produced in the adrenal glands and can fundamentally affect brain maturation. The hormone suppresses the activity of glia. A type of glia, astrocytes, helps regulate the extracellular glutamate concentration. Glutamate constitutes the most prevalent excitatory neurotransmitter in the brain, playing a major role in the stabilization of connections between nerve cells during brain maturation. Elevated concentrations of extracellular glutamate can trigger pre-programmed cell death known as apoptosis, otherwise occurring only during early stages of brain development. Presumably, the orphans' excessive stress-related exposure to cortisol led to modifications of nerve cell networks, underlying the children's behavioral differences. Imaging the brain's energy consumption provided a method to uncover whether and where nerve cell activity changed in cerebral cortex as a consequence of the children's stay in the orphanages. 

Using the fluorodeoxyglucose method, Chugani and others (2001) could show that the use of glucose was drastically reduced in the cerebral cortex of the orphans enrolled in the study, particularly in temporal and prefrontal cortical areas and in structures of the limbic system, notably the amygdala. The cortical regions are involved in executive functions and short-term memory crucial for social behavior and affect. The amygdala play an important role in fearful reactions. The observed reductions in energy consumption could not have been detected with the standard fMRI or PET procedures discussed above. The fluorodeoxyglucose method, hence, constitutes the procedure of choice when the fundamental metabolic state of the brain is in question.

  • Take some time and listen to this show on National Public Radio's This American Life with the title "Unconditional Love". The first half of the show is about an orphaned Romanian boy adopted by an American couple at the age of eight. It demonstrates in great clarity the at times overwhelming difficulties the family faced to remedy important steps of personality development that were missed early in the boy's life. Finally, the challenges were overcome with passion and a professional attitude. It is reassuring to find out that success is possible (10/23/10).


Monday, October 12, 2009

The Quest for the Infrasound Acoustic Fovea

In my post dated Sep. 30, 2009, I briefly discussed the discovery of echolocation as a means for bats to navigate their environment in total darkness and identify insects to feast on. I noted that in echolocating bats a large part of the auditory system is devoted to the processing of ultrasound, that is frequencies higher than the human audible (>20 kHz).  A disproportionate number of auditory nerve cells are tuned to analyze the frequencies of the echos of the echolocation calls bats emit. I worked on research with echolocating bats in Gerhard Neuweiler's laboratory at the Institute of Zoology of the Johann Wolfgang Goethe University, Frankfurt am Main. I helped show that exposing a bat to its echolocation frequency activated prominent regions in its auditory midbrain structure known as inferior colliculus (Melzer, 1985). The proclivity in the bat auditory system for a narrow band of ultrasound used for echolocation is known as high frequency filter or acoustic fovea (Neuweiler and others, 1980).

As informative contrast to the bats' fovea for extremely high pitch, a mammal was sought with a fovea for extremely low pitch. It was hoped that a similar association between behaviorally relevant sound and its representation in the brain might exist in species whose hearing is particularly sensitive to infrasound, that is at frequencies beneath the human audible (<20 Hz). Elephants (Herbst and others, 2012) and humpbacked whales are known to produce and hear infrasound, but were considered difficult to study.

By contrast, burrowing rodents were thought to constitute suitable candidates, because they spend much of the day underground and were observed to be able to use seismic vibrations to identify and locate conspecifics and predators. Particularly, gerbils were of interest. They are essentially crepuscular, that is most active above ground at dawn and dusk. They live in semi-arid deserts where compacted gravel and sand carry low frequency sound long distances. Gerbils use hind foot drumming as means of communication. The drumming exhibits species-specific differences and has been observed to alert companions to one's own presence in as much as to approaching predators (Randall, 1997). Differences in the signature of the sound may permit the animals to distinguish between various types of predator, e.g. snakes or birds. Moreover, the sounds may convey to predators that they have been discovered (Randall, 2001).

Christian Winter recruited Jürgen Möller as a junior faculty member to investigate whether gerbils may represent the small mammals with an acoustic fovea for infrasound. He had extensive experience in acoustics, micro-electrode recordings of electrical nerve cell discharges, and animal behavior.

courtesy J. Möller
In addition to the well-known Mongolian gerbil (Meriones unguiculatus), Jürgen succeeded in bringing a number of gerbil species from Israel to the laboratory, testing their hearing for low frequency sensitivity with audiograms. Field studies on the animals' behavior were conducted in Israel.

The photograph depicts a sand rat (Psammomys obesus) in the Negev desert at dusk. Sand rats were the largest gerbils in Jürgen's collection.  Power density spectra of their drumming's acoustic frequency components were recorded to examine whether the drumming produced infrasound. Broad spectrum audiograms were constructed from recordings of small voltage changes in the inner ear (cochlear microphonics) and from nerve cell activity in the auditory midbrain (inferior colliculus) to investigate whether the animals could hear infrasound.

The results were presented at a joint symposium of Hebrew University of Jerusalem, Université de Lyon, and Johann Wolfgang von Goethe University entitled "Neurobiology and Strategies of Adaptation". The symposium was convened in Frankfurt am Main in 1981. The two figures below show representative results of the frequencies of the sound produced by drumming (A) and of the sound processed by the auditory system (B). The green band covers the frequency range between 0.5 and 1.0 kHz in which the audiograms (B) show peculiar low frequency sensitivity.

(A) Power Density Spectrum
The frequency of the drumming's acoustic components [kHz] (y-axis) is plotted versus time [ms] (x-axis; courtesy J. Möller).

(B) Audiograms
The sound pressure level [dB SPL] of the most sensitive response (y-axis) is plotted versus sound frequency [kHz] (x-axis). Cochlear microphonics: CM; nerve cell responses in the inferior colliculus: IC (courtesy J. Möller).
Analysis of the power spectra revealed that the animals' drumming contained low frequencies of significant power, infrequently dipping into infrasound. Yet, the audiograms of the animals provided no evidence of an acoustic fovea for such low frequencies. Regardless, the findings clearly suggested that the drumming produced sound to which that the animals' hearing was sensitive.

My project's aim was to visualize pitch-related nerve cell activation in the brain with a functional imaging method (Melzer, 1984). M. Müller (now private docent at the J.W. Goethe University) helped me substantially in this endeavor. The project would not have been possible without the use of the whole-body cryotome in H.-M. Kellner's division at the Hoechst AG's Radiochemical Laboratory.

Animals were exposed to sound of low, medium and high pitch. Pitch is represented tonotopically on the transverse plane through the inferior colliculus. That is, nerve cells in the upward (dorsal) aspect of the structure are particularly sensitive to low frequency sound and become progressively more sensitive to higher frequencies with increasing depth. Functional imaging would reveal the isofrequency domains, permitting us to assess the prominence of low frequency processing in the auditory system.

The figure below shows frequency-related nerve cell activation in transverse slices through the inferior colliculus. Nerve cell activation is coded in pseudo colors (blue: low; red: high). The narrow blue/purple lines near the top are the animals' scalp, that is dorsal is up. The brainstem is at the bottom, that is ventral is down. The animals' left side is on the right.

Neurofunctional Images
The slices were obtained from sand rats exposed to tone pips of (clockwise from top, left) 0.8, 2.5 and 17.0 kHz, respectively. Stimulus-unrelated nerve cell activity was obtained from unexposed animals (bottom, left). The inferior colliculus is the distinctly sound-activated, double-lobed structure at the center of the images. Unexposed animals revealed slightly elevated nerve cell activity in the top aspect of the structure on both sides (bottom, left). Low frequency stimulation produced wide-spread activation, peaking in a band at the top of the structure (top, left). This activation partially overlapped with the observed stimulus-unrelated activity (bottom, left). By contrast, the middle frequency activated a narrow band at mid-depth of the structure on both sides (top, right), whereas activity at the top was almost completely suppressed. High frequency stimulation resulted in similarly narrow bands of activation on both sides. Only in this case, the bands were located at the bottom of the inferior colliculus (bottom, right). In addition, prominent activity was distinct at the top of the structure.

The progression of bands of elevated nerve cell activation from top to bottom with increasing frequency of stimulation is consistent with the notion of a tonotopic map where frequencies are represented in logarithmic progression. Intriguingly, the low part of the sound spectrum was slightly disproportionally represented. However, such disproportionality is not uncommon and has been observed in a number of species (N. Suga, personal communication).

The narrow bands of activation observed with the middle stimulus frequency conformed most distinctly with a representation of frequencies in discrete isofrequency layers. According to the cochlear microphonics, the animals' ear is most sensitive at this frequency. By contrast, the wide-spread activation at the low frequency and the additional foci at the top of the inferior colliculus at the low and the high frequency did not precisely adhere to the principle of discrete frequency representation. We did not know how to interpret these results.

In hindsight, we were perhaps too narrowly focused on auditory responses. Parts of the inferior colliculus are known to receive somatic sensory input. Sand rats have long mystacial whiskers that are in frequent contact with the soil. Cytoarchitectonic structures known as barrels represent the mystacial whiskers topographically in a large swath of the gerbil's cerebral somatic sensory cortex (Rice and others, 1985). Distinct septa separate the barrels. Nerve cells in the septa are known to be particularly attuned to the processing of stimulus frequency (Melzer and others, 2006). Recent observations show that whiskers may resonate (Moore, 2004) to low frequency vibrations, touch receptors in the whisker follicles transduce frequencies up to 1.0 kHz and possibly greater (Gottschaldt and Vahle-Hinz, 1981), and nerve cells in the somatic sensory pathway may process this information (Kleinfeld and others, 2006). Perhaps, gerbils do possess an infrasound fovea after all! Only the processing of this sound may not be strictly auditory.

  • Watching gerbils around the time of an earthquake may prove insightful. A tiny whiskered burrowing rodent, the Northern Pocket Gopher Thomomys talpoides, survived the catastrophic eruption of Mt. St. Helens in 1980 (11/06/09).
  • Caldwell and others (2010) produced an impressive demonstration of the importance of vibrations in vertebrate sensation and behavior. The research feature on National Public Radio's Talk of the Nation (Science Friday) yesterday with the title "Rumble in The Jungle" shows red-eyed treefrogs (Agalychnis callidryas) from the rain forests of central America communicating with vibrations mediated by the twig they sit on. Listen to the podcast of Ira Flatow's conversation with Flora Lichtman entitled "Red-Eyed Treefrogs Rumble in the Jungle". The authors of the study, affiliated with Boston University, contributed excellent footage of the frogs' interactions (05/22/10).
  • On Apr. 8, 2011, National Public Radio's Talk of the Nation/Science Friday broadcast an insightful segment with the title "Seeing The World Through Whiskers" on the research of Mitra Hartman and her colleagues (Towal and others, 2011) examining whisking in rats with high-speed video (04/11/2011):
  • The magnitude 5.8 earthquake on the East Coast on Aug. 23, 2011, centered near Mineral, Virginia, was felt roughly 120 miles away by animals of the National Zoo in Washington D.C., in some instances tens of minutes before humans did. Listen to Ira Flatow's interview with Brandie Smith, Senior Curator of the National Zoological Park, Smithsonian Institution, Washington D.C., entitled "Did you feel it?" on National Public Radio's Talk of the Nation/Science Friday broadcast today (08/26/2011).
  • Listen to Dr. Tecumseh Fitch explaining to Audie Cornish of National Public Radio's All Things Considered in his interview with the title "Study: Humans, Elephants User Similar Vocalizations" broadcast today how elephants produce and may use infrasound (08/09/2012).
  • The text of this post is available for download in pdf-format from the scribd store.
Related Posts
I attached this amateur video of a pet gerbil drumming in the cage. The clip provides an idea of drumming speed and periodicity. Keep in mind that a gerbil in a burrow will sound quite different.

Friday, October 9, 2009

Water & the Mind

“All day I've faced the barren waste
Without the taste of, water.
Ole Dan and I, with throats burned dry ,
and souls that cry
for, clear water.”
We need water to live. In the developed world, we take abundant water supply for granted. However, where I live the recent past demonstrated in all harshness that we are cradling ourselves in a false sense of security. Water is in fact in short supply. In contrast to the Southwestern states of the U.S., the Southeastern states look emerald green most of the year when you look down on the beautiful land of rolling mountains from an airplane. You would never believe that there is not enough water for this land!

However, severe drought struck this land two years ago. After several years of insufficient rainfall, hardly any came. The grass turned brown already in June, the hack berry trees dropped their leaves in August. Our willow dropped a ton of whip-like branches and is half dead today. Metropolitan Atlanta was much worse off. A major source of the city's potable water, Lake Lanier, reached historic low levels (Shaila Dewan and Brenda Goodman for The New York Times, Oct. 23, 2007, "New to Being Dry the South Struggles to Adapt"). Severe restrictions for the use of water were imposed.

Legal fights ensued between users in the region. The states of Florida, Alabama and Georgia, sharing the Chattahoochee River basin, began to quarrel about water rights with the U.S. Army Corps of Engineers (Shaila Dewan for The New York Times, Aug. 15, 2009, "River Basin Fight Pits Atlanta Against Neighbors"). The Corps administers the river's flow. Georgia became so desperate that lawmakers briefly resurrected a 19th century border dispute with its neighbor Tennessee in a vain attempt to gain access to the Tennessee River (Shaila Dewan and Brenda Goodman for The New York Times, Feb. 22, 2008, "Georgia Claims a Sliver of the Tennessee River").

The state of Tennessee was not much better off (Adam Nossiter for The New York Times, Jul. 4, 2007, "Drought Saps the Southeast, and its Farmers"). Water had to be trucked in with fire engines for a number of communities where the wells were running dry. Entire counties declared water emergencies. On top of this calamity, deep cracks were discovered in the bedrock under several large dams the Tennessee Valley Authority had built in the wake of the Great Depression, e.g. Wolf Creek Dam (Ian Urbina and Bob Dreihaus for The New York Times, Mar. 4, 2007, "Fears for a Dam's Safety Put Tourist Area on Edge"). Water had to be released, leaving the intake pipes of a number of lakeside communities on dry ground. Obviously, improved water management was direly needed.

This year, by contrast, we saw record rainfall down here. Georgia and Tennessee experienced catastrophic flash floods (Robbie Brown and Liz Robbins for The New York Times, Sep. 24, 2009, "Georgians Grappling With Flood Damage"). Our house is built on an unfinished basement. In normal years, the dirt is dry for nine months. This year it never dried. The grass in the yard never turned brown. The hackberry trees have only begun to shed their leaves this month.

Unfortunately, this year's abundance of water could not be stored. The water levels behind the damaged dams had to be kept below capacity because of the repair work underway. The repairs are substantial, will cost hundreds of millions of dollars and will take several years to complete. Meanwhile, millions of gallons of precious water are flushed down the spill ways. For the first time, I saw all spill ways wide open at Percy Priest Dam. A truly majestic sight!

Clearly, we cannot afford to waste our most vital resource without paying a price. Tonight at 20 hours EDT, Guy Laliberté, the founder of Cirque du Soleil, will host a multi-media show from the International Space Station. The show entitled "Moving Stars and Earth for Water" will raise awareness to this simple, but important fact.

You may wish to tune in online here tonight:

  • Guy is back!
  • On the weekend of May 2, 2010, a 500-year flood of the Cumberland River system in Tennessee overwhelmed the U.S. Army Corps of Engineers' ability of containing the rising waters below catastrophic levels. The Corps had to release water, flooding its own facilities downstream, to protect the integrity of its dams. Eleven lives were lost. More than 2000 families suffered flood damage to their homes. Nashville's downtown was underwater to an extent unseen in 80 years. One of the two water treatment plants that supply the city with drinking water was out of order for several weeks. The estimated economic losses in the metropolitan area top two billion dollars. The Tennessean devotes a continuously updated online report entitled "Nashville Flood" on the incident and its impact. The dam and levee system in the region is underfunded and has not seen large-scale upgrades since its inception. Obviously, improved water control is direly needed (08/05/10).
  • Southeastern cities manage their drinking water on shoe-string budgets. According to an Associated Press report with the title "New Orleans Issues Boil-Water Order" published online in The Wall Street Journal today, citizens of New Orleans are advised to boil their tap water before consumption this weekend, because mechanical failure forced a water treatment plant to shut down (11/20/10).

Wednesday, September 30, 2009

Echolocation, Science & Power

The Italian scientist Lazzaro Spallanzani  attained fame beyond his country already in his life time. He studied philosophy at the University of Bologna, became member of the clergy, and taught logic, metaphysics, and Greek at the universities of Reggio, Modena and and eventually assumed the chair in natural history at Pavia. His studies contributed profoundly to the understanding of a broad range of natural phenomena spanning geology, biology and physiology. Late in his career, he became fascinated with bats, wondering how they could navigate so elegantly in full darkness complicated environments wrought with obstacles.

Spallanzani proved himself as an observant experimenter. He attacked his question methodically with a series of tedious behavioral experiments, systemically ruling out one sense after another. He observed bats flying skillfully passed nooks and crannies in a L-shaped basement. Occlusion of the eyes did not appear to degrade the bats' performance, neither did covering the skin with a paste nor occluding the nose. His findings were first published as a collection of letters by Anton-Maria Vasalli (1794). A few years later, the Swiss zoologist Charles Jurine observed that occlusion of the ears rendered bats entirely disoriented. Spallanzani confirmed this observation, but was unable to explain how bats would use hearing for navigation. He had speculated earlier that the animals perhaps possessed a sixth sense unbeknownst to human kind.

Professor Spallanzani's hypothesis was met by strong and powerful resistance in the scientific community. One of the most eminent zoologists of his time, Georges Cuvier, argued against the validity of the experiments (Cuvier, 1795).  Interestingly, Cuvier's arguments were entirely based on conjecture. He did not conduct a single experiment to disprove Spallanzani's results. Instead, he appealed to common knowledge. He reasoned that everybody knows that bats, in as much as blind people, orient themselves with the sense of touch.

History would prove Cuvier wrong on both accounts. Neither bats (Griffin, 2001) nor blind people ( Wall Emerson and Ashmead, 2008) use their sense of touch for navigation in space. However, Cuvier's reputation was domineering. His influence on science was overarching. Roughly for the next century and a half researchers devoted their attention on the bats' sense of touch, until the Americans Donald R. Griffin and Robert Galambos and the Dutchman Sven Dijkgraaf discovered echolocation. That is, they unequivocally demonstrated that bats use the echos of ultrasound they emit to navigate their environment in flight and catch prey (Griffin, 2001). Griffin wrote an informative popular book about his findings entitled "Listening in the Dark: The Acoustic Orientation of Bats and Men". Vigorous research continues to the day to elucidate the nerve cell mechanisms that underlie this fascinating behavior.

I was exposed to some aspects of bat research when I was a student. The processing of ultrasound frequencies used for echolocation constitutes a prominent feature in the auditory pathway of echolocating bats (Neuweiler and others, 1980). My pilot study helped visualize this prominence with a functional imaging method (Melzer P, 1985).

The colors in the picture show ultrasound-related activation in a transverse slice through the brain of an echolocating bat. The ear on the opposite side was exposed to sound pips of this bat's individual echolocation frequency. Two regions in the auditory midbrain known as inferior colliculus (white circle) responded to the ultrasound most prominently.

Spallanzani's observations were influenced by the fact that he used pipistrelle bats (Pipistrellus pipistrellus) for his study. They emit their echolocation calls through the mouth, and his attempts to occlude the mouth noticeably compromised navigation. Had he used the more common horseshoe bat (Rhinolophus ferrumequinum), he would have been surprised to discover that the nose was doing the job.

  • The co-discoverer of bat echolocation Robert Galambos, PhD, MD, passed away a month ago at the age of 96.  Douglas Martin provides a concise summary of his career in The New York Times today with the title "Robert Galambos, Neuroscientist Who Showed How Bats Navigate, Dies at 96".  He was a profound experimentalist. In an inseminating recent paper (Galambos, 2003), he described four elegant experiments that demonstrate the principles of empirical neuroscience in most illustrative fashion (07/16/10).
  • Cuvier G (1795) Conjectures sur le sixiéme sens qu'on a cru remarquer dans les chauve-souris. Mag. Encyclopéd 6:297-301.
  • Galambos R (2003) Four favorite experiments and why I like them. Int J Psychophysiol 48:133-140.
  • Griffin DR (2001) Return to the magic well: Echolocation behavior of bats and responses of insect prey. BioScience 51:555–556.
  • Griffin DR (1958) Listening in the dark: The acoustic orientation of Bats and men. Yale Univ Press.
  • Melzer P (1985) A deoxyglucose study on auditory responses in the bat Rhinolophus rouxi. Brain Res Bull 15:677-681.
  • Neuweiler G, Bruns V, Schuller G (1980) Ears adapted for the detection of motion, or how echolocating bats have exploited the capacities of the mammalian auditory system. J Acoust Soc Am 68:741-753.
  • Vasalli A-M (1794) Lettere sopra il Sospetto di un Nuovo Senso nei Pipistrelli . . . Con le Risposte dell’Abate. Stamperia Reale (Torino).
  • Wall Emerson R, Ashmead D (2008) Visual Experience and the concept of compensatory spatial hearing abilities. In: Blindness and brain plasticity in navigation and object perception (Rieser JJ, Ashmead DH, Ebner FF, Corn AL, eds). Taylor & Francis (New York):pp367-380.
Meet Bert the kitchen bat!