Researchers use RNA sequences to map projections from specific brain regions

From Cold Spring Harbor Laboratory News:

Revolutionary method to map the brain at single-neuron resolution is successfully demonstrated

Friday, 19 August 2016 07:00

MAPseq uses RNA sequencing to rapidly and inexpensively find the diverse destinations of thousands of neurons in a single experiment in a single animal

Cold Spring Harbor, NY — Neuroscientists today publish in Neuron details of a revolutionary new way of mapping the brain at the resolution of individual neurons, which they have successfully demonstrated in the mouse brain.

The new method, called MAPseq (Multiplexed Analysis of Projections by Sequencing), makes it possible in a single experiment to trace the long-range projections of large numbers of individual neurons from a specific region or regions to wherever they lead in the brain—in experiments that are many times less expensive, labor-intensive and time-consuming than current mapping technologies allow.

Although a number of important brain-mapping projects are now under way, all of these efforts to obtain “connectomes,” or wiring maps, rely upon microscopes and related optical equipment to trace the myriad thread-like projections that link neurons to other neurons, near and far. For the first time ever, MAPseq “converts the task of brain mapping into one of RNA sequencing,” says its inventor, Anthony Zador, M.D., Ph.D., professor at Cold Spring Harbor Laboratory.

“The RNA sequences, or ‘barcodes,’ that we deliver to individual neurons are unmistakably unique,” Zador explains, “and this enables us to determine if individual neurons, as opposed to entire regions, are tailored to specific targets.”

RNA sequences

An injection into a “source” region of the brain contains a viral library encoding a diverse collection of barcode sequences, which are hitched to an engineered protein that is designed to carry the barcode along axonal pathways. The barcode RNA is expressed at high levels and transported into the terminals of axons in the source region where the injection is made. In each neuron, it travels to the point where the axon forms a synapse with a projection from another neuron. (click to enlarge)

MAPseq approach

“Bulk” labeling methods now widely in use to map brain connections are able to determine that neurons in the “source” region (left side) project to three green-shaded regions (right side), but are not able to distinguish the specific destinations of individual neurons in the source region. MAPseq enables such distinction — in this example, showing that neurons bearing specific “barcodes” (vastly reduced in complexity here for demonstration purposes) carry those barcodes to some of the 3 “destinations” but not necessarily all of them, or the same ones as other neurons in the source region. (click to enlarge)


MAPseq differs from so-called “bulk tracing” methods now in common use, in which a marker—typically a fluorescent protein—is expressed by neurons and carried along their axons. Such markers are good at determining all of the regions where neurons in the source region project to, but they cannot tell scientists that any two neurons in the source region project to the same region, to different regions, or to some of the same regions, and some different ones. That inability to resolve a neuron’s axonal destinations, cell by cell in a given region, is what motivated Zador to come up with a new technique.

One way of explaining the advantage of MAPseq over bulk tracing methods is to imagine being at an international airport, with the intention of getting on a flight to, say, Germany. “If you go to the international terminal, you see a long line of ticket counters,” Zador explains. “If you want to go to Germany, it’s not enough to take any airline at the international terminal. If you stand in line at the counter for Air Chile, you’re probably not going to be able to buy a ticket for Germany.”

“Those many airlines whose counters are adjacent serve many destinations, some of which overlap, some of which are unique. You can print out a map showing all of the foreign countries that all of the airlines serve from your airport, but that doesn’t tell you anything at all about individual airlines and where they go. This is the difference between current labeling methods and MAPseq. The ‘individual airlines’ in my example are adjacent neurons in a part of the brain whose ‘routes’ we want to trace.”

Zador and his team, including Justus Kebschull, a graduate student in his lab who is first author on the Neuron paper introducing the new method, have spent several years working out a technology that enables them to assign unique barcode-like identifiers to large numbers of individual neurons via a single injection in any brain region of interest. Each injection consists of a deactivated virus that has been engineered to contain massive pools of individually unique RNA molecules, each of whose sequence—consisting of 30 “letters,” or nucleotides—is taken up by single neurons. Thirty letters yields many, many times more barcode sequences (1018) than there are neurons in either the mouse or human brain, so this method is especially well suited to the massive complexity problem that brain mapping presents.

An injection into a “source” region of the brain contains a viral library encoding a diverse collection of barcode sequences, which are hitched to an engineered protein that is designed to carry the barcode along axonal pathways. The barcode RNA is expressed at high levels and transported into the terminals of axons in the source region where the injection is made. In each neuron, it travels to the point where the axon forms a synapse with a projection from another neuron. Tests show that the technology works—the barcodes travel reliably and evenly throughout the brain, along the “trunklines” that are the axons, and out to the “branch points” where synapses form.

About two days after one or more injections are made in a region of interest, the brain is dissected and RNA is collected and sequenced. RNA barcodes in the “source” area are now matched with the same barcodes collected in distant parts of the brain.

RNA sequences

To demonstrate MAPseq’s capabilities, Zador’s team injected a part of the mouse brain called the locus coeruleus (LC), located in the brain stem. After nearly 2 days, the cortex was divided in 22 slices, dissected and sequenced for RNA barcodes. The sequence readouts were matched with barcodes of cells in the region of the initial injection, establishing specific paths of individual LC neurons. (click to enlarge)

“Sequencing the RNA is a highly efficient, automated process, which makes MAPseq such a potentially radical tool,” Kebschull says. “In addition to the speed and economy of RNA sequencing, it has the great advantage of making it possible for researchers to distinguish between individual neurons within the same region that project to different parts of the brain.”

To demonstrate MAPseq’s capabilities, Zador’s team injected a part of the mouse brain called the locus coeruleus (LC), located in the brain stem. It is the cortex’s sole source of noradrenaline, a hormone that signals surprise. Zador’s team used MAPseq to address an old question: does the “surprise” signal get broadcast everywhere in the cortex, or only to particular places, where, perhaps, it is most needed or relevant?

In their demonstration experiment, only RNA that ended up in the cortex or olfactory bulb was sequenced, along with that of the source region in the LC where the barcodes were originally injected. The team divided the cortex into 22 slices, each about 300 microns thick, and dissected the slices. The results were exciting to the team.

“We found that neurons in the LC have a variety of idiosyncratic projection patterns,” Zador says. “Some neurons project almost exclusively to a single preferred target in the cortex or olfactory bulb. Other neurons project more broadly, although weakly.”

These results, he adds, “are consistent with, and reconcile, previous seemingly contradictory results about LC projections.” The surprise signal can reach most parts of the brain, but there are very specific parts of the brain where the signal is especially focused.

The team showed that results could be obtained in experiments based on one injection in the LC, and also two injections, on opposite sides. Already in progress are experiments in which the entire cortex is being “tiled” with injections. It is hoped this will yield the first connectome of the entire cortex at single-neuron resolution.

“Once we automate the process of using many injections, we think this kind of experiment can be completed by a single person in just a week or two, and at a cost of only a few thousand dollars,” Zador says. “We are very keen on being able to do these kind of studies in a single animal, which will eliminate the past problem of injecting multiple animals to trace multiple neurons, a method that requires one to make a single map based on many brains, each of which is somewhat different.”

Zador’s next goal with MAPseq is to map the brains of animals that model various neurodevelopmental and neuropsychiatric illnesses, to see how gene mutations strongly associated with causality alter the structure of brain circuits, and thus, presumably, brain function.

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Scientists find area in brain that is prewired for reading

From MIT News:

Neuroscientists have long wondered why the brain has a region exclusively dedicated to reading — a skill that is unique to humans and only developed about 5,400 years ago.

Study finds brain connections key to reading

Pathways that exist before kids learn to read may determine development of brain’s word recognition area.

Anne Trafton | MIT News Office
August 8, 2016

A new study from MIT reveals that a brain region dedicated to reading has connections for that skill even before children learn to read.

By scanning the brains of children before and after they learned to read, the researchers found that they could predict the precise location where each child’s visual word form area (VWFA) would develop, based on the connections of that region to other parts of the brain.

Neuroscientists have long wondered why the brain has a region exclusively dedicated to reading — a skill that is unique to humans and only developed about 5,400 years ago, which is not enough time for evolution to have reshaped the brain for that specific task. The new study suggests that the VWFA, located in an area that receives visual input, has pre-existing connections to brain regions associated with language processing, making it ideally suited to become devoted to reading.

“Long-range connections that allow this region to talk to other areas of the brain seem to drive function,” says Zeynep Saygin, a postdoc at MIT’s McGovern Institute for Brain Research. “As far as we can tell, within this larger fusiform region of the brain, only the reading area has these particular sets of connections, and that’s how it’s distinguished from adjacent cortex.”

Saygin is the lead author of the study, which appears in the Aug. 8 issue of Nature Neuroscience. Nancy Kanwisher, the Walter A. Rosenblith Professor of Brain and Cognitive Sciences and a member of the McGovern Institute, is the paper’s senior author.

Specialized for reading

The brain’s cortex, where most cognitive functions occur, has areas specialized for reading as well as face recognition, language comprehension, and many other tasks. Neuroscientists have hypothesized that the locations of these functions may be determined by prewired connections to other parts of the brain, but they have had few good opportunities to test this hypothesis.

Reading presents a unique opportunity to study this question because it is not learned right away, giving scientists a chance to examine the brain region that will become the VWFA before children know how to read. This region, located in the fusiform gyrus, at the base of the brain, is responsible for recognizing strings of letters.

Children participating in the study were scanned twice — at 5 years of age, before learning to read, and at 8 years, after they learned to read. In the scans at age 8, the researchers precisely defined the VWFA for each child by using functional magnetic resonance imaging (fMRI) to measure brain activity as the children read. They also used a technique called diffusion-weighted imaging to trace the connections between the VWFA and other parts of the brain.

The researchers saw no indication from fMRI scans that the VWFA was responding to words at age 5. However, the region that would become the VWFA was already different from adjacent cortex in its connectivity patterns. These patterns were so distinctive that they could be used to accurately predict the precise location where each child’s VWFA would later develop.

Although the area that will become the VWFA does not respond preferentially to letters at age 5, Saygin says it is likely that the region is involved in some kind of high-level object recognition before it gets taken over for word recognition as a child learns to read. Still unknown is how and why the brain forms those connections early in life.

Pre-existing connections

Kanwisher and Saygin have found that the VWFA is connected to language regions of the brain in adults, but the new findings in children offer strong evidence that those connections exist before reading is learned, and are not the result of learning to read, according to Stanislas Dehaene, a professor and the chair of experimental cognitive psychology at the College de France, who wrote a commentary on the paper for Nature Neuroscience.

“To genuinely test the hypothesis that the VWFA owes its specialization to a pre-existing connectivity pattern, it was necessary to measure brain connectivity in children before they learned to read,” wrote Dehaene, who was not involved in the study. “Although many children, at the age of 5, did not have a VWFA yet, the connections that were already in place could be used to anticipate where the VWFA would appear once they learned to read.”

The MIT team now plans to study whether this kind of brain imaging could help identify children who are at risk of developing dyslexia and other reading difficulties.

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Increased neurogenesis from exercise does not cause forgetting of previously learned task

From Vital Record News from Texas A & M University Health Science Center:

Forget old memories

Study shows exercise won’t cause you to forget things

Contradicting earlier work, researchers confirm exercise is good for you—and your brain
August 2, 2016

Research has found that exercise causes more new neurons to be formed in a critical brain region, and contrary to an earlier study, these new neurons do not cause the individual to forget old memories, according to research by Texas A&M College of Medicine scientists, in the Journal of Neuroscience.

Exercise is well known for its cognitive benefits, thought to occur because it causes neurogenesis, or the creation of new neurons, in the hippocampus, which is a key brain region for learning, memory and mood regulation. Therefore, it was a surprise in 2014 when a research study, published in the journal Science, found that exercise caused mice to forget what they’d already learned.

“It stunned the field of hippocampal neurogenesis,” said Ashok K. Shetty, PhD, a professor in the Texas A&M College of Medicine Department of Molecular and Cellular Medicine, associate director of the Institute for Regenerative Medicine, and research career scientist at the Central Texas Veterans Health Care System. “It was a very well-done study, so it caused some concern that exercise might in some way be detrimental for memory.”

The animal models in the exercise group—in the previous study—showed far more neurogenesis than the control group, but contrary to what one might think, these additional neurons seemed to erase memories that were formed before they started the exercise regimen. To test this, the researchers removed the extra neurons, and the mice suddenly were able to remember again.

“The mice who exercised had a large number of new neurons,” Shetty said, “but somehow that seemed to break down the old connections, making them forget what they knew.”

Shetty and his team decided to replicate this earlier research, using rats instead of mice. Rats are thought to be more like humans physiologically, with more-similar neuronal workings. They found that—luckily for runners everywhere—these animal models showed no such degradation in memories.

“We had completely contradictory findings from the 2014 study,” said Maheedhar Kodali, PhD, a postdoctoral fellow at the Institute for Regenerative Medicine and the first author of this study. “Now we need to study other species to fully understand this phenomenon.”

Shetty and his team trained their animal models to complete a task over the course of four days, followed by several days of memory consolidation by performing the task over and over again. Then, half the trained animal models were put into cages with running wheels for several weeks, while the control group remained sedentary.

The rats who ran further over the course of that time had much greater neurogenesis in their hippocampus, and all rats who had access to a wheel (and therefore ran at least some), had greater neurogenesis than the sedentary group. On an average, they ran about 48 miles in four weeks, and neuron formation doubled in the hippocampus of these animals.

“This is pretty clear evidence that exercise greatly increases neurogenesis in the hippocampus, which has functional implications,” Kodali said. “Neurogenesis is important for maintaining normal mood function, as well as for learning and creating new memories.” This connection may help explain why exercise is an effective antidepressant.

Importantly, despite differing levels of increased neurogenesis, both moderate runners and brisk runners (those who ran further than average) in Shetty’s study showed the same ability as the sedentary runners to recall the task they learned before they began to exercise. This means even a large amount of running (akin to people who perform significant amount of exercise on a daily basis) doesn’t interfere with the recall of memory.

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Role of Hippocampal Sleep Spindles in Memory Consolidation

From The Scientist:

Minding the Pulse of Memory Consolidation

Studying sleep spindles could help neuroscientists better understand certain cognitive impairments.

By Richard Kemeny | July 28, 2016


Sleep is essential for memory. Mounting evidence continues to support the notion that the nocturnal brain replays, stabilizes, reorganizes, and strengthens memories while the body is at rest. Recently, one particular facet of this process has piqued the interest of a growing group of neuroscientists: sleep spindles. For years these brief bursts of brain activity have been largely ignored. Now it seems that examining these neuronal pulses could help researchers better understand—perhaps even treat—cognitive impairments.

Sleep spindles are a defining characteristic of stage 2 non-rapid eye movement (NREM) sleep. These electrical bursts between 10-16 Hz last only around a second, and are known to occur in the human brain thousands of times per night. Generated by a thin net of neurons enveloping the thalamus, spindles appear across several regions of the brain, and are thought to perform various functions, including maintaining sleep in the face of disturbances in the environment.

It appears they are also a fundamental part of the process by which the human brain consolidates memories during sleep.

A memory formed during the day is stored temporarily in the hippocampus, before being spontaneously replayed during the night. Information about the memory is distributed out and integrated into the neocortex through an orchestra of slow-waves, spindles, and rapid hippocampal ripples. Spindles, it seems, could be a guiding force—providing the plasticity and coordination needed for this delicate, interregional transfer of information.

“Spindles appear to play a central role whenever memories during sleep are undergoing transformation that might be necessary to integrate them into neocortical long-term storage networks,” Jan Born, a professor of behavioral neurobiology of the University of Tübingen, told The Scientist during a conference dedicated to sleep spindles held in Budapest in May.

Fewer spindles, therefore, would be expected to coincide with a breakdown in memory consolidation.

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High dose of resveratrol helps reduce neuronal inflammation in Alzheimer’s

From EurekAlert!:

Public Release: 27-Jul-2016

Resveratrol appears to restore blood-brain barrier integrity in Alzheimer’s disease

Georgetown University Medical Center

WASHINGTON — Resveratrol, given to Alzheimer’s patients, appears to restore the integrity of the blood-brain barrier, reducing the ability of harmful immune molecules secreted by immune cells to infiltrate from the body into brain tissues, say researchers at Georgetown University Medical Center. The reduction in neuronal inflammation slowed the cognitive decline of patients, compared to a matching group of placebo-treated patients with the disorder.

The laboratory data provide a more complete picture of results from a clinical trial studying resveratrol in Alzheimer’s disease that was first reported in 2015. The new findings will be presented at the Alzheimer’s Association International Conference 2016 in Toronto on July 27th.

The Alzheimer’s disease brain is damaged by inflammation, thought to be due to a reaction to the buildup of abnormal proteins, including Abeta40 and Abeta42, linked to destruction of neurons. Researchers believe that heightened inflammation — which was historically thought to come only from “resident” brain immune cells — worsens the disease. According to the researchers, this study suggests that some of the immune molecules that can cause inflammation in the blood can enter the brain through a leaky blood-brain barrier.

“These findings suggest that resveratrol imposes a kind of crowd control at the border of the brain. The agent seems to shut out unwanted immune molecules that can exacerbate brain inflammation and kill neurons,” says neurologist Charbel Moussa, MD, PhD, scientific and clinical research director of the GUMC Translational Neurotherapeutics Program. “These are very exciting findings because it shows that resveratrol engages the brain in a measurable way, and that the immune response to Alzheimer’s disease comes, in part, from outside the brain.”

Resveratrol is a naturally occurring compound found in foods such as red grapes, red wine, raspberries and dark chocolate. GUMC researchers, led by R. Scott Turner, MD, PhD, tested the substance in 119 patients, the largest nationwide phase II clinical trial to study high-dose pure synthetic (pharmaceutical-grade) resveratrol in individuals with mild to moderate Alzheimer’s. The study was published Sept. 11, 2015 in Neurology.

The new part of the resveratrol study examines specific molecules in the cerebrospinal fluid (CSF) taken from participants with biomarker-confirmed Alzheimer’s disease — 19 were given a placebo, and 19 treated daily for a year with resveratrol, equivalent to the amount found in about 1,000 bottles of red wine.

Previous studies with animals found that age-related diseases–including Alzheimer’s — can be prevented or delayed by long-term caloric restriction (consuming two-thirds the normal caloric intake). The researchers studied resveratrol because it mimics the effects of caloric restriction by also activating proteins called sirtuins.

In this new study, Moussa and Turner found that treated patients had a 50 percent reduction in matrix metalloproteinase-9 (MMP-9) levels in the cerebrospinal fluid. MMP-9 is decreased when sirtuin1 (SIRT1) is activated. High levels of MMP-9 cause a breakdown in the blood-brain barrier, allowing proteins and molecules from the body to enter the brain. Normally low MMP-9 levels maintain the barrier, say the researchers.

“These new findings are exciting because they increase our understanding of how resveratrol may be clinically beneficial to individuals with Alzheimer’s disease. In particular, they point to the important role of inflammation in the disease, and the potent anti-inflammatory effects of resveratrol,” says Turner, director of GUMC’s Memory Disorders Program and co-director of the Translational Neurotherapeutics Program.

They also found that resveratrol increased the level of molecules linked to a long-term beneficial or “adaptive” immune reaction, suggesting involvement of inflammatory cells that are resident in the brain, says Moussa. “This is the kind of immune response you want — it is there to remove and degrade neurotoxic proteins.”

“A puzzling finding from the resveratrol study (as well as immunotherapy strategies for Alzheimer’s under investigation) is the greater shrinkage of the brain found with treatment. These new findings support the notion that resveratrol decreases swelling that results from inflammation in Alzheimer’s brain,” says Turner. “This seemingly paradoxical effect is also found with many of the drugs that are beneficial for patients with multiple sclerosis — another brain disease characterized by excessive inflammation.”

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Scientists activate and distinguish glutamate receptor subtypes

From Kyoto University research news:

Flipping a protein switch to illuminate brain functions

28 Jun 2016

Pacman-shaped receptor’s “bite” sheds light on learning and memory processes

Japan — What goes on inside the brain when we learn new things? Much still remains wrapped in mystery, but scientists have found a way to examine this at the molecular level.

Researchers in Japan have engineered an artificial switch that could let scientists turn individual neurotransmitter receptors on and off. Shedding light on these receptors’ role in memory formation could contribute to the development of new drugs for neurological diseases, including Alzheimer’s, Parkinson’s, and ALS.

Neurotransmitter receptors help relay information from neuron to neuron. “Investigating the functions of various neurotransmitter receptors could be immensely useful, because a majority of drugs on the market target them,” says lead author Ryou Kubota of Kyoto University. “But with so many similarly-structured proteins in the membrane, it’s been extremely difficult to determine which receptors do what.

“Discovering the functions of each neurotransmitter receptor in the brain could help us understand how we learn and acquire memory; to do that, it’s crucial to be able to activate them selectively.”

In the study, published in Nature Chemistry, the team succeeded in selectively activating glutamate receptors, which are pacman-shaped neutrotransmitter receptors known to be involved in memory formation.

Membrane proteins change in shape when they become active. For pacman-shaped glutamate receptors, activation happens when they “bite”. The team genetically engineered glutamate receptors to include switches forcing activation and deactivation. “The switch comes in the shape of two ‘clips’ on what would be the upper and lower lips of pacman,” explains Kubota. “When we tell the clips to bind together, we force the glutamate receptor to activate.”

The current study only reports outcomes with glutamate receptors, but the authors say that their method also shows promise with other kinds of membrane receptors. “Even within glutamate receptors there are subtypes, and within those subtypes there are further distinctions. This time we were able to distinguish and selectively activate each subtype,” remarks Kubota.



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N-Acetylcysteine (NAC) treatment may improve Parkinson’s

From Newswise:

Natural Molecule Could Improve Parkinson’s

A natural molecule shows benefit in a preliminary clinical trial for Parkinson’s Disease

Released: 16-Jun-2016 2:00 PM EDT
Brain scans of a representative patient showing Dopamine transporter binding (red) before and after 3-month NAC treatment.

Thomas Jefferson University Brain scans of a representative patient showing Dopamine transporter binding (red) before and after 3-month NAC treatment.

Newswise — (PHILADELPHIA) — The natural molecule, n-acetylcysteine (NAC), with strong antioxidant effects, shows potential benefit as part of the management for patients with Parkinson’s disease, according to a study published today in the journal PLOS ONE. Combining clinical evaluations of a patient’s mental and physical abilities with brain imaging studies that tracked the levels of dopamine, the lack of which is thought to cause Parkinson’s, doctors from the Departments of Integrative Medicine, Neurology, and Radiology, at Thomas Jefferson University showed that patients receiving NAC improved on both measures.

Current treatments for Parkinson’s disease are generally limited to temporarily replacing dopamine in the brain as well as some medications designed to slow the progression of the disease process. Recently, researchers have shown that oxidative stress in the brain may play a critical role in the Parkinson’s disease process, and that this stress also lowers levels of glutathione, a chemical produced by the brain to counteract oxidative stress. Studies in brain cells showed that NAC helps reduce oxidative damage to neurons by helping restore the levels of the antioxidant glutathione. NAC is an oral supplement that can be obtained at most nutrition stores, and interestingly also comes in an intravenous form which is used to protect the liver in acetaminophen overdose.

“This study reveals a potentially new avenue for managing Parkinson’s patients and shows that n-acetylcysteine may have a unique physiological effect that alters the disease process and enables dopamine neurons to recover some function,” said senior author on the paper Daniel Monti, M.D., M.B.A., Director of the Myrna Brind Center of Integrative Medicine, and the Brind-Marcus Center of Integrative Medicine at Thomas Jefferson University.

In this study, Parkinson’s patients who continued their current standard of care treatment, were placed into two groups. The first group received a combination of oral and intravenous (IV) NAC for three months. These patients received 50mg/kg NAC intravenously once per week and 600mg NAC orally 2x per day on the non IV days. The second group, the control patients, received only their standard of care for Parkinson’s treatment. Patients were evaluated initially, before starting the NAC and then after three months of receiving the NAC while the control patients were simply evaluated initially and three months later. The evaluation consisted of standard clinical measures such as the Unified Parkinson’s Disease Rating Scale (UPDRS), a survey administered by doctors to help determine the stage of disease, and a brain scan via DaTscan SPECT imaging, which measures the amount of dopamine transporter in the basal ganglia, the area most affected by the Parkinson’s disease process. Compared to controls, the patients receiving NAC had improvements of 4-9 percent in dopamine transporter binding and also had improvements in their UPDRS score of about 13 percent.

“We have not previously seen an intervention for Parkinson’s disease have this kind of effect on the brain,” said first author and neuro-imaging expert Andrew Newberg, M.D., Professor at the Sidney Kimmel Medical College at Jefferson and Director of Research at the Myrna Brind Center of Integrative Medicine. The investigators hope that this research will open up new avenues of treatment for Parkinson’s disease patients.

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