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!

Friday, September 18, 2009

Color Blindness, Gene Therapy & Brain Plasticity

In my post dated May 28, 2009, I wrote about the squirrel monkey Miss Baker. She was the first primate to successfully complete a space voyage half a century ago. Yesterday, two other squirrel monkeys, Dalton and Sam, accomplished another first. That is, they were the first to successfully undergo gene therapy, repairing color blindness.

Our color vision is provided by photoreceptor cells in the eye's retina known as cones. Three types of cones have been identified. Each type is particularly sensitive to either blue, green, or red light, depending on the molecular structure of their photopigments known as opsins. While two copies of the gene encoding the blue-sensitive opsin are located on a pair of somatic chromosomes, the genes for green and red exist in only one copy on x chromosomes. If they are damaged, we are not able to distinguish between green and red. Particularly males are vulnerable because a spare is unavailable. Thus, color blindness affects 8 percent of white men, but less than 0.5 percent of white women. By contrast, most male new world monkeys, including squirrel monkeys, are color-blind.

The Therapy
Yesterday, Katherine Mancuso and her colleagues published a letter in the journal Nature online in which the authors provide evidence that two adult male squirrel monkeys, Dalton and Sam, gained color vision within about half a year after they had been treated with gene therapy (Mancuso and others, 2009). That is, their eyes had been injected with an engineered virus encoding the missing long wavelength-sensitive opsin or green fluorescent protein (GFP). The recombinant DNA was supposed to be inserted into cone DNA. The GFP served as an independent marker for successful insertion. The hope was that the cones with the engineered DNA would synthesize the missing opsin, eventually enabling the monkeys to see differences between red and green.

Dalton and Sam were experts in visual discrimination tasks. They had been trained in a modified Cambridge Colour Test before the intervention and were experienced participants. The test consisted of colored dot patterns embedded in gray dots similar to tests with numbers or letters laid out in colored dots for people. Dalton and Sam began to distinguish red and green in the sixth month after the virus injection.

The authors monitored retinal function using wide-field color multifocal electroretinography. This type of electrophysiological recording describes stimulus reception, transduction and processing in the retina. Retinal responses to red were undetectable four months after the intervention, but had risen to prominence at ten months and were further enhanced at 18 months. At that time, the monkeys could distinguish between 16 hues of red and green. GFP was expressed in roughly 15–36 percent of the cones. Taken together, the findings of this study constitute a promising prove of concept for the great potential gene therapy may hold for the restoration of retinal function.

Brain Plasticity
The question remains how Dalton and Sam could correctly interpret visual cues they had never seen before. Could the nerve cell connections in the visual system reorganize on a scale required to process the novel sensory input?

Around 1960, the Nobel Prize-laureates Torsten Wiesel and David Hubel had discovered that the cerebral cortex reorganized profoundly in response to sensory deprivation in a limited window of time during brain maturation. Primary visual cortex consists of interdigitated domains in which neurons respond mainly to input from one eye. In their pioneering study, Wiesel and Hubel occluded one eye in newborn kittens and observed that the other eye's cortical domain enlarged into the deprived territory (Wiesel and Hubel, 1963). The effect could be reversed, if the eye occlusion was reversed within a critical period. Reversal in adults had no effect. Much research ensued to uncover the underlying mechanisms.

By contrast, Dalton and Sam were adult at the time of the intervention. The authors took their findings to suggest that the visual system remains highly plastic even in maturity. However, compared with monocular deprivation color-blindness seems to pose a minor challenge to the visual system. In the gene therapy study, the neural circuitry necessary for color discrimination may have already been in place at the time of the intervention. Existing connections may have only needed strengthening to attune the neurons to the novel inputs, precipitating the monkeys' correct decision in the test. It is puzzling, however, that it took the monkeys months to improve the new skill.

To better understand the neural mechanisms involved in the monkeys' novel ability to discriminate color, it is crucial to find out precisely when after the intervention the photoreceptors become sensitive to new wavelengths of light.

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Saturday, September 12, 2009

Good News for Brain Energy Use

Brain work has enjoyed wide-spread scientific interest since Roy and Sherrington (1890) observed that sensory nerve stimulation expanded cerebral cortex as a result of an increase in blood flow. Increased brain function seemed to produce metabolites that caused the dilation of cerebral blood vessels presumably to fulfill the increased demand for nutrients. These findings sparked intense research to identify the cellular metabolic pathways involved and to uncover the mechanisms that couple blood flow to brain function.

Indeed, brain cells need plenty calories. The brain accounts for an estimated 20 percent of our body's energy consumption. The aerobic metabolism of glucose, also known as aerobic glycolysis, has been shown to constitute the most prominent source for the brain's energy needs.

Aerobic glycolysis entails the oxidation of glucose into water and carbon dioxide to produce energy-rich phosphorous compounds, mainly adenosine triphosphate (ATP). The compounds are used to fuel active transmembrane transport mechanisms of ions and molecules that are needed for nerve cell communication and information processing. However, only insufficient amounts of glucose and no oxygen can be stored in the brain. Both have to be delivered to the brain tissue on demand.

In the late 1960s, Lou Sokoloff and his colleagues at the National Institute of Mental Health succeeded in developing a procedure with which the local cerebral metabolic rates of glucose could be determined in vivo. They named it the autoradiographic deoxyglucose method (Sokoloff and others, 1977). Autoradiography involves the administration of a radioactively labeled tracer, the distribution of which in the tissue can be visualized postmortem by apposing X-ray films to tissue sections. The grain density in the autoradiograms on the developed films corresponds to the tracer concentration in the sections. The grain density can be measured with an optical densitometer and calibrated to represent  tracer concentration in the tissue.

Glucose is rapidly metabolized and the metabolites are quickly removed from the brain. Therefore, Sokoloff and colleagues could not use radioactively labeled glucose as tracer for their measurements. They used deoxyglucose instead. Deoxyglucose is a competitive analogue of glucose. It is phosphorylated to deoxyglucose-6-phosphate by the enzyme hexokinase in the first step of glycolysis, but is not metabolized further, and accumulates in the brain tissue. The tracer accumulation is proportionate to the rate of glucose metabolism.

Using this method, numerous studies have shown that glucose metabolism in the central nervous system is associated with electrical nerve cell spiking activity (e.g. Kadekaro and others, 1985). That is, nerve cell stimulation evokes electrical millivolt spikes in the nerve cell's axon by rapid fluxes of sodium ions into the cell and potassium ions out of the cell through ion-specific membrane channels. The spikes are known as action potentials. The axon is the long process that connects the nerve cells. Trains of action potentials traveling along axons essentially constitute packets of data transmitted to other nerve cells. 

In order to prepare the cell for the next spike, the ion concentrations must be restored to their pre-action potential level, known as resting potential. The sodium ions must be pumped out of the cells with an energy-consuming pump. In accord, Mata and others, (1980) observed that glucose metabolism diminishes significantly, when the sodium pump is blocked and the production of action potentials ceases, supporting the idea that cerebral energy consumption is related to the spiking activity of nerve cells.

Sokoloff encountered criticism. One critic was the Dutch biochemist Cees Van den Berg, who claimed that the deoxyglucose method did not measure glucose metabolism accurately. He had measured concentrations of pyruvate, a glycolytic key intermediate, and his results suggested a much greater glucose consumption (Van den Berg and Bruntink, 1983). I met him after he had applied for a grant to fund experiments designed to support his claim.  He was invited to present a lecture on what he thought needed to be done. Many years later, I asked Lou Sokoloff about Van den Berg's ideas. Lou swiftly and resolutely retorted, "Nonsense!" In as much as the size of a lake does not tell us how fast water is running through it, the concentration of a metabolite is no indication of its metabolic rate.

However, one of Van den Berg's claims still resonates in my mind. He noted that the rate of glucose metabolism measured with the deoxyglucose method would increase no more than two-fold from baseline in normal physiological circumstances. I have carried out hundreds of experiments with this method in rodents. On this point, Van den Berg was correct. Physiological whisker stimulation increases glucose metabolic rates in primary somatic sensory cortex less than two-fold. Indeed, the average is about 1.3-fold.

Nerve cell activity-related increases in glucose metabolism should have been greater, if they had been  tightly correlated with the energy demands of axonal sodium pump activity estimated at the time. In their pioneering work, the Noble Prize-laureates Hodgkin and Huxley were the first to describe the ion currents that produced action potentials in squid giant axons (Hodgkin and Huxley, 1952a). In a subsequent paper, the authors suggested a model quantifying these currents (Hodgkin and Huxley,1952b). According to the model, sodium influx was anticipated to rise 4-fold or greater. Consequentially, stimulus-evoked nerve cell spiking activity should have resulted in much greater increases in glucose use to pump out the ions than found with the deoxyglucose method.

The New Find
Yesterday, Science Magazine published a study the results of which may resolve this apparent inconsistency (Alle and others, 2009). The authors used intracellular patch clamp recordings in mice to quantify the action potential-related transmembrane sodium and potassium ion fluxes in mossy fibers of a phylogenetically old part of the cerebral cortex known as hippocampus. They are unmyelinated. That is, like axons of short-distance nerve cell connections in neocortex, mossy fibers lack a fatty sheeth for electrical insulation and, therefore, propagate action potentials similarly as the axons of local intracortical circuits. The authors observed sodium and potassium ion fluxes that were less overlapping and smaller than those found in squid giant axons. They suggest that these differences reduce the action potential-related increase in energy demand to 1.3-fold. This increase is in good agreement with the observed stimulus-related increases in glucose utilization rates discussed above.

In addition, supra-cellular mechanism play a crucial role in the reduction of cortical energy needs. Cortical nerve cells are able to control their gain. That is, the rate of action potentials can be reduced in response to an increase in the strength of stimulation without losing information (Melzer and others, 2006). Van den Berg erred in his critique of deoxyglucose method, because the energy demand of cerebral function is lower than he presumed.

On the other hand, it must be noted that sodium pump activity does not solely account for stimulus-related increases in cerebral glucose metabolism. Nerve cells release glutamate at their synapses, that is the contacts between nerve cells, to excite other nerve cells. Glutamate is the most prevalent neurotransmitter in the cerebral cortex. Astrocytes near the synapses are known to remove the released glutamate with active, that is energy-consuming, transport (Pellerin and Magistretti, 1994; Takahashi and others, 1995). Hence, astrocytic glutamate uptake may contribute considerably to the cerebral rates of glucose metabolism. This contribution remains to be quantified.


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