Color Blindness, Gene Therapy & Brain Plasticity

Prologue
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.

References

• Mancuso K, Hauswirth WW, Li Q, Connor TB, Kuchenbecker JA, Mauck MC, Neitz J, Neitz M (2009) Gene therapy for red-green colour blindness in adult primates. Nature:online.
• Wiesel TN, Hubel DH (1963) Single-cell responses in striate cortex of kittens deprived of vision in one eye. J Neurophysiol 26:1003-1017.

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Good News for Brain Energy Use

Prologue
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.

References

• Alle H, Roth A, Geiger JRP (2009) Energy-efficient potentials in hippocampal mossy fibers. Science 325:1405-1408.
• Hodgkin AL, Huxley AF (1952a) Propagation of electrical signals along giant nerve fibers. Proc R Soc Lond B Biol Sci 140:177-183.
• Hodgkin AL, Huxley AF (1952b) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol (Ldn) 117:500-544.
• Kadekaro M, Crane AM, Sokoloff L (1985) Differential effects of electrical stimulation of sciatic nerve on metabolic activity in spinal cord and dorsal root ganglion in the rat. Proc Natl Acad Sci U S A 82:6010-6013.
• Mata M, Fink DJ, Gainer H, Smith CB, Davidsen L, Savaki H, Schwartz WJ, Sokoloff L (1980) Activity-dependent energy metabolism in rat posterior pituitary primarily reflects sodium pump activity. J Neurochem 34:213-215.
• Melzer P, Sachdev RN, Jenkinson N, Ebner FF (2006) Stimulus frequency processing in awake rat barrel cortex. J Neurosci 26:12198-2205.
• Pellerin L, Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci U S A 91:10625-10629.
• Roy CS, Sherrington CS (1890) On the regulation of the blood-supply of the brain. J Physiol (Ldn) 11:85-108.
• Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, Pettigrew KD, Sakurada O, Shinohara M (1977) The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28:897-916.
• Takahashi S, Driscoll BF, Law MJ, Sokoloff L (1995) Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia. Proc Natl Acad Sci U S A 92:4616-4620.
• Van den Berg CJ, Bruntink R (1983) Glucose oxidation in the brain during seizures: experiments with labeled glucose and deoxyglucose. In Glutamine, Glutamate and GABA in the Central Nervous System (Hertz L, Kvamme E, McCeer EG, Schousboe A, eds), New York, Alan R. Liss:pp 619−624.

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Constantin von Economo's Spindle Cells & The Mind

Today we celebrate the birthday of Constantin von Economo. He was born in 1876. A comprehensive biography of his life entitled "Baron Constantin von Economo His Life and Work"can still be found as a used book. He was known as an avid aviator and a superb physician scientist. Elaborating on Korbinian Brodmann's ground-breaking maps of cerebral cortex, he meticulously charted his own divisions based on the composition of nerve cells seen in histological preparations. In this process, he identified a rare and special type of nerve cell that has intrigued neuroscientists to this day.

The cerebral cortex constitutes our brain's most prominent feature. It consists of a large convoluted sheet of nervous tissue separated at the mid-line into two hemispheres. Its gray matter can be divided into layers of nerve cells of varying shape and density with characteristic in- and output. Pyramidal cells are the predominant type of nerve cell providing output to nerve cells in other layers of the same area, other cortical areas as well as to nerve cells in subcortical structures and to the motor neurons in the spinal cord that innervate the musculature. The roughly triangular cell bodies of the majority of pyramidal cells are located in the deep layers of cortex near the white matter, that Ramon y Cajal defined as layers 5 and 6.

Pyramidal cells feature a distinct distribution of dendrites, that is the processes on which inputs from other nerve cells terminate. The apical dendrite points straight up. It traverses all cortical layers and ramifies in layer 1 under the cortical surface. In addition, a set of dendrites, known as basal dendrites, spreads out radially from the cell body, collecting input from the layer in which the cell body resides. The axon, that is the process conveying the nerve cell's output, emerges from the underside of the cell body. The axons of pyramidal cells are wrapped in sheaths of a fatty substance, known as myelin, for insulation and form the cortical white matter.

Clustered among the ubiquitous pyramidal cells, nerve cells of peculiar, strikingly different shape caught Constantin von Economo's eye. They were unique to only a select few areas in anterior cingulate and insular cortex. The cells possessed conspicuous large cell bodies. Like pyramidal cells, they featured a prominent straight dendrite spanning the cortical thickness and an axon projecting to distant brain structures,  but lacked basal dendrites, resulting in a distinctly bipolar appearance. Von Economo named them "Spindelzellen" or spindle cells in English.

In the past, spindle cells had only been found in humans and other great apes (Nimchinsky and others, 1999). The exclusivity has been taken to suggest that this type of nerve cell may be associated with the complex higher cognitive and affective mental functions that seem to distinguish us from the rest of animaldom, e.g. language, decision making and empathy. Spindle cells are known to be particularly prone to degenerate in people with Alzheimer's disease (Nimchinsky and others, 2004). Recently however, the cells have also been discovered in cetaceans (Marino and others, 2007) and pachyderms (Hakeem and others, 2009).

Little is known about the function of spindle cells. The distribution of their dendrites suggests that they collect input from other nerve cells along narrow radial columns across the cortical thickness, supporting the idea that the cerebral cortex is a functionally organized in discrete modules rather than in broad layers. Their long-distance projections suggest a role in a distributed neural network, processing information across sensory modalities. However, more research clearly needs to be carried out to examine their responsiveness to stimulation and the influences they exert on other nerve cells before any specific role can be assigned with certainty to Constantin von Economo's Spindelzellen.