Developing Our Sense of Smell

When our noses pick up a scent, whether the aroma of a sweet rose or the sweat of a stranger at the gym, two types of sensory neurons are at work in sensing that odor or pheromone. These sensory neurons are particularly interesting because they are the only neurons in our bodies that regenerate throughout adult life—as some of our olfactory neurons die, they are soon replaced by newborns. Just where those neurons come from in the first place has long perplexed developmental biologists.

Previous hypotheses about the origin of these olfactory nerve cells have given credit to embryonic cells that develop into skin or the central nervous system, where ear and eye sensory neurons, respectively, are thought to originate. But biologists at the California Institute of Technology (Caltech) have now found that neural-crest stem cells—multipotent, migratory cells unique to vertebrates that give rise to many structures in the body such as facial bones and smooth muscle—also play a key role in building olfactory sensory neurons in the nose.

"Olfactory neurons have long been thought to be solely derived from a thickened portion of the ectoderm; our results directly refute that concept," says Marianne Bronner, the Albert Billings Ruddock Professor of Biology at Caltech and corresponding author of a paper published in the journal eLIFE on March 19 that outlines the findings.

The two main types of sensory neurons in the olfactory system are ciliated neurons, which detect volatile scents, and microvillous neurons, which usually sense pheromones. Both of these types are found in the tissue lining the inside of the nasal cavity and transmit sensory information to the central nervous system for processing.

In the new study, the researchers showed that during embryonic development, neural-crest stem cells differentiate into the microvillous neurons, which had long been assumed to arise from the same source as the odor-sensing ciliated neurons. Moreover, they demonstrated that different factors are necessary for the development of these two types of neurons. By eliminating a gene called Sox10, they were able to show that formation of microvillous neurons is blocked whereas ciliated neurons are unaffected.

They made this discovery by studying the development of the olfactory system in zebrafish—a useful model organism for developmental biology studies due to the optical clarity of the free-swimming embryo. Understanding the origins of olfactory neurons and the process of neuron formation is important for developing therapeutic applications for conditions like anosmia, or the inability to smell, says Bronner.

"A key question in developmental biology—the extent of neural-crest stem cell contribution to the olfactory system—has been addressed in our paper by multiple lines of experimentation," says Ankur Saxena, a postdoctoral scholar in Bronner’s laboratory and lead author of the study. "Olfactory neurons are unique in their renewal capacity across species, so by learning how they form, we may gain insights into how neurons in general can be induced to differentiate or regenerate. That knowledge, in turn, may provide new avenues for pursuing treatment of neurological disorders or injury in humans."

Next, the researchers will examine what other genes, in addition to Sox10, play a role in the process by which neural-crest stem cells differentiate into microvillous neurons. They also plan to look at whether or not neural-crest cells give rise to new microvillous neurons during olfactory regeneration that happens after the embryonic stage of development.

DNA damage occurs as part of normal brain activity

Scientists at the Gladstone Institutes have discovered that a certain type of DNA damage long thought to be particularly detrimental to brain cells can actually be part of a regular, non-harmful process. The team further found that disruptions to this process occur in mouse models of Alzheimer’s disease—and identified two therapeutic strategies that reduce these disruptions.

Scientists have long known that DNA damage occurs in every cell, accumulating as we age. But a particular type of DNA damage, known as a double-strand break, or DSB, has long been considered a major force behind age-related illnesses such as Alzheimer’s. Today, researchers in the laboratory of Gladstone Senior Investigator Lennart Mucke, MD, report in Nature Neuroscience that DSBs in neuronal cells in the brain can also be part of normal brain functions such as learning—as long as the DSBs are tightly controlled and repaired in good time. Further, the accumulation of the amyloid-beta protein in the brain—widely thought to be a major cause of Alzheimer’s disease—increases the number of neurons with DSBs and delays their repair.

"It is both novel and intriguing team’s finding that the accumulation and repair of DSBs may be part of normal learning," said Fred H. Gage, PhD, of the Salk Institute who was not involved in this study. "Their discovery that the Alzheimer’s-like mice exhibited higher baseline DSBs, which weren’t repaired, increases these findings’ relevance and provides new understanding of this deadly disease’s underlying mechanisms."

In laboratory experiments, two groups of mice explored a new environment filled with unfamiliar sights, smells and textures. One group was genetically modified to simulate key aspects of Alzheimer’s, and the other was a healthy, control group. As the mice explored, their neurons became stimulated as they processed new information. After two hours, the mice were returned to their familiar, home environment.

The investigators then examined the neurons of the mice for markers of DSBs. The control group showed an increase in DSBs right after they explored the new environment—but after being returned to their home environment, DSB levels dropped.

"We were initially surprised to find neuronal DSBs in the brains of healthy mice," said Elsa Suberbielle, DVM, PhD, Gladstone postdoctoral fellow and the paper’s lead author. "But the close link between neuronal stimulation and DSBs, and the finding that these DSBs were repaired after the mice returned to their home environment, suggest that DSBs are an integral part of normal brain activity. We think that this damage-and-repair pattern might help the animals learn by facilitating rapid changes in the conversion of neuronal DNA into proteins that are involved in forming memories."

The group of mice modified to simulate Alzheimer’s had higher DSB levels at the start—levels that rose even higher during neuronal stimulation. In addition, the team noticed a substantial delay in the DNA-repair process.

To counteract the accumulation of DSBs, the team first used a therapeutic approach built on two recent studies—one of which was led by Dr. Mucke and his team—that showed the widely used anti-epileptic drug levetiracetam could improve neuronal communication and memory in both mouse models of Alzheimer’s and in humans in the disease’s earliest stages. The mice they treated with the FDA-approved drug had fewer DSBs. In their second strategy, they genetically modified mice to lack the brain protein called tau—another protein implicated in Alzheimer’s. This manipulation, which they had previously found to prevent abnormal brain activity, also prevented the excessive accumulation of DSBs.

The team’s findings suggest that restoring proper neuronal communication is important for staving off the effects of Alzheimer’s—perhaps by maintaining the delicate balance between DNA damage and repair.

"Currently, we have no effective treatments to slow, prevent or halt Alzheimer’s, from which more than 5 million people suffer in the United States alone," said Dr. Mucke, who directs neurological research at Gladstone and is a professor of neuroscience and neurology at the University of California, San Francisco, with which Gladstone is affiliated. "The need to decipher the causes of Alzheimer’s and to find better therapeutic solutions has never been more important—or urgent. Our results suggest that readily available drugs could help protect neurons against some of the damages inflicted by this illness. In the future, we will further explore these therapeutic strategies. We also hope to gain a deeper understanding of the role that DSBs play in learning and memory—and in the disruption of these important brain functions by Alzheimer’s disease."

(Image courtesy: Lulu Qian, Erik Winfree & Jehoshua Bruck | California Institute of Technology)

Neuronal Morphology Goes Digital: A Research Hub for Cellular and System Neuroscience

The importance of neuronal morphology in brain function has been recognized for over a century. The broad applicability of ‘‘digital reconstructions’’ of neuron morphology across neuroscience subdisciplines has stimulated the rapid development of numerous synergistic tools for data acquisition, anatomical analysis, three-dimensional rendering, electrophysiological simulation, growth models, and data sharing. Here we discuss the processes of histological labeling, microscopic imaging, and semiautomated tracing. Moreover, we provide an annotated compilation of currently available resources in this rich research ‘‘ecosystem’’ as a central reference for experimental and computational neuroscience.

Researchers image most of vertebrae brain at single cell level

Misha Ahrens and Philipp Keller, researchers with the Howard Hughes Medical Institute have succeeded in making a near real-time video of most of a zebrafish’s brain showing individual neuron cells firing. To create the video, as the team reports in their paper published in the journal Nature Methods, the two developed a type of modified light-sheet microscopy and used it in on genetically modified fish.

To create the video, the researchers turned to zebrafish in their larval state—their brains are transparent and small. To cause firing neurons to be visible they genetically altered the fish’s brains, giving them a protein that glows when responding to changes in calcium ion levels, which happen when nerve cells fire. Next, they used a microscope that was able to broadcast a sheet of light through the fish’s brain allowing for the detection of the firing neurons. The system recorded images every 1.3 seconds. The final step was stitching the images together to create a video. The result is nothing short of breathtaking—looking like something out of a science fiction movie’s special effects department.

The video marks the first visual capture of most of a living vertebrae brain at the neuron level, as it works in near real-time and offers striking evidence of the complexity of the brain—even one as small as 100,000 neurons. The researchers say their video shows approximately 80 percent of the zebrafish’s brain as it operates—though what all those firing neurons represent in particular, is still unknown.

The researchers are careful to point out that what they’ve accomplished does not portend the creation of a video of a human brain in action—our brains are much larger, have billions more neurons and perhaps more importantly, are not transparent and are covered by a thick skull. Instead they suggest that studying a simpler brain in action might help to explain how biological neural networks actually work, perhaps leading to theories that can be generalized over larger animals.

But before that can happen, the procedure the team has developed needs to be improved—neurons can fire at hundreds of times per second, which means a lot of firing in the video has been missed. Capturing at a faster rate would mean generating nearly unmanageable amounts of data—at the current rate, just one hour of capture creates a terabyte of data. Thus a new way to store and process the data must be developed.

Neural “Synchrony” May be Key to Understanding How the Human Brain Perceives

Despite many remarkable discoveries in the field of neuroscience during the past several decades, researchers have not been able to fully crack the brain’s “neural code.” The neural code details how the brain’s roughly 100 billion neurons turn raw sensory inputs into information we can use to see, hear and feel things in our environment.

In a perspective article published in the journal Nature Neuroscience on Feb. 25, 2013, biomedical engineering professor Garrett Stanley detailed research progress toward “reading and writing the neural code.” This encompasses the ability to observe the spiking activity of neurons in response to outside stimuli and make clear predictions about what is being seen, heard, or felt, and the ability to artificially introduce activity within the brain that enables someone to see, hear, or feel something that is not experienced naturally through sensory organs.

Stanley also described challenges that remain to read and write the neural code and asserted that the specific timing of electrical pulses is crucial to interpreting the code. He wrote the article with support from the National Science Foundation (NSF) and the National Institutes of Health (NIH). Stanley has been developing approaches to better understand and control the neural code since 1997 and has published about 40 journal articles in this area.

“Neuroscientists have made great progress toward reading the neural code since the 1990s, but the recent development of improved tools for measuring and activating neuronal circuits has finally put us in a position to start writing the neural code and controlling neuronal circuits in a physiological and meaningful way,” said Stanley, a professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

With recent reports that the Obama administration is planning a decade-long scientific effort to examine the workings of the human brain and build a comprehensive map of its activity, progress toward breaking the neural code could begin to accelerate.

The potential rewards for cracking the neural code are immense. In addition to understanding how brains generate and manage information, neuroscientists may be able to control neurons in individuals with epilepsy and Parkinson’s disease or restore lost function following a brain injury. Researchers may also be able to supply artificial brain signals that provide tactile sensation to amputees wearing a prosthetic device.

Stanley’s paper highlighted a major challenge neuroscientists face: selecting a viable code for conveying information through neural pathways. A longstanding debate exists in the neuroscience community over whether the neural code is a “rate code,” where neurons simply spike faster than their background spiking rate when they are coding for something, or a “timing code,” where the pattern of the spikes matters. Stanley expanded the debate by suggesting the neural code is a “synchrony code,” where the synchronization of spiking across neurons is important.

A synchrony code argues the need for precise millisecond timing coordination across groups of neighboring neurons to truly control the circuit. When a neuron receives an incoming stimulus, an electric pulse travels the neuron’s length and triggers the cell to dump neurotransmitters that can spark a new impulse in a neighboring neuron. In this way, the signal gets passed around the brain and then the body, enabling individuals to see, touch, and hear things in the environment. Depending on the signals it receives, a neuron can spike with hundreds of these impulses every second.

“Eavesdropping on neurons in the brain is like listening to a bunch of people talk—a lot of the noise is just filler, but you still have to determine what the important messages are,” explained Stanley. “My perspective is that information is relevant only if it is going to propagate downstream, a process that requires the synchronization of neurons.”

Neuronal synchrony is naturally modulated by the brain. In a study published in Nature Neuroscience in 2010, Stanley reported finding that a change in the degree of synchronous firing of neurons in the thalamus altered the nature of information as it traveled through the pathway and enhanced the brain’s ability to discriminate between different sensations. The thalamus serves as a relay station between the outside world and the brain’s cortex.

Synchrony induced through artificial stimulation poses a real challenge for creating a wide range of neural representations. Recent technological advances have provided researchers with new methods of activating and silencing neurons via artificial means. Electrical microstimulation had been used for decades to activate neurons, but the technique activated a large volume of neurons at a time and could not be used to silence them or separately activate excitatory and inhibitory neurons. Stanley compared the technique with driving a car that has the gas and brake pedals welded together.

New research methods, such as optogenetics, enable activation and silencing of neurons in close proximity and provide control unavailable with electrical microstimulation. Through genetic expression or viral transfection, different cell types can be targeted to express specific proteins that can be activated with light.

“Moving forward, new technologies need to be used to stimulate neural activity in more realistic and natural scenarios and their effects on the synchronization of neurons need to be thoroughly examined,” said Stanley. “Further work also needs to be completed to determine whether synchrony is crucial in different contexts and across brain regions.”

Scientists Identify Buphenyl as a Possible Drug for Alzheimer’s disease

Buphenyl, an FDA-approved medication for hyperammonemia, may protect memory and prevent the progression of Alzheimer’s disease. Hyperammonemia is a life-threatening condition that can affect patients at any age. It is caused by abnormal, high levels of ammonia in the blood.

Studies in mice with Alzheimer’s disease (AD) have shown that sodium phenylbutyrate, known as Buphenyl, successfully increases factors for neuronal growth and protects learning and memory, according to neurological researchers at the Rush University Medical Center.

Results from the National Institutes of Health funded study, recently were published in the Journal of Biological Chemistry.

“Understanding how the disease works is important to developing effective drugs that protect the brain and stop the progression of Alzheimer’s disease,” said Kalipada Pahan, PhD, the Floyd A. Davis professor of neurology at Rush and lead investigator of this study.

A family of proteins known as neurotrophic factors help in survival and function of neurons. Past research indicates that these proteins are drastically decreased in the brain of patients with Alzheimer’s disease (AD).

“Neurotrophic factor proteins could be increased in the brain by direct injection or gene delivery,” said Pahan. “However, using an oral medication to increase the level of these protein may be the best clinical option and a cost effective way to increase the level of these proteins directly in the brain.”

“Our study found that after oral feeding, Buphenyl enters into the brain, increases these beneficial proteins in the brain, protects neurons, and improves memory and learning in mice with AD-like pathology,” said Pahan.

In the brain of a patient with AD, two abnormal structures called plaques and tangles are prime suspects in damaging and killing nerve cells. While neurons die, other brain cells like astroglia do not die.

The study findings indicate that Buphenyl increases neurotrophic factors from astroglia. Buphenyl stimulates memory-related protein CREB (cyclic AMP response element-binding protein) using another protein known as Protein Kinase C (PKC) and increases neurotrophic factors in the brain.

"Now we need to translate this finding to the clinic and test Buphenyl in Alzheimer’s disease patients,” said Pahan. “If these results are replicated in Alzheimer’s disease patients, it would open up a promising avenue of treatment of this devastating neurodegenerative disease.”