Science Fail Monday: How a dead salmon taught us about statistics

Any scientist knows the importance of a good negative control. A negative control in an experiment is a group of samples or subjects in which no response is expected to an experimental treatment. The experimental group can then be compared to the control group. Such negative controls are gold standards in science and are supposed to provide confidence in experimental results. However, occasionally, a negative control gives unexpected and hilarious results worth of an Ig-Nobel Prize, the highest honor for scientists who publish the silliest research. Such was the case in an experiment involving fMRI, human emotions, and an Atlantic salmon.

An example of an fMRI scan in a human. The red spots have higher brain activity when subjects are performing a memory task.

fMRI stands for functional magnetic resonance imaging. If you’ve ever had a knee injury or a concussion, you have likely experienced a normal MRI scan, which uses radio waves and a magnet to take a structural picture of the organ of interest. The “functional” in fMRI means that researchers can use MRI images to measure brain activity and take a snapshot of changes over time. When a strong magnet is turned on over the brain, the hydrogen atoms in all of the water molecules in the blood point in the same direction, like a compass needle next to a refrigerator magnet. When the magnet is turned off, the hydrogen atoms relax back to their original positions, which releases a signal. This signal changes based on how much oxygen is in the blood, so the end result is a picture of the brain with information about which regions have more oxygenated blood. Regions needing more oxygen are generally assumed to be more active. Researchers can even have study participants perform a task during an fMRI scan, such as viewing particular images or listening to music, and use the fMRI data to determine which areas of the brain are active during the task. These types of studies can tell us a lot about which brain regions are involved in everything from social situations to processing fear.

In the Ig-Nobel Prize-worthy experiment, researchers wanted to use fMRI to determine which parts of the brain were active in response to seeing human faces displaying different emotions. However, they needed a negative control for their human subjects just to make sure that any brain activity they saw in response to the faces wasn’t just due to chance. The ideal candidate for such a negative control? A four-pound Atlantic salmon, purchased by one of the researchers at the local fish market.

The authors of the IgNobel prize study used an Atlantic salmon like this one as their negative control.

The researchers put the dead salmon in their fMRI scanner and, for the sake of science, asked it what emotions it thought the humans were displaying in pictures flashed up on the screen in the scanner. The authors do not comment on the salmon’s responses, but it can be assumed that the salmon was not a model experimental participant and did not comply with the study directions. Expecting to see nothing, the authors analyzed the fMRI signal in the salmon’s brain before and after the salmon “saw” the photos of the faces. Imagine the shock in the room when a few spots in the salmon’s itty-bitty brain lit up like a Christmas tree, suggesting that it was thinking about the faces it saw. Duh duh duuuuuhh….zombie salmon?

Obviously, the salmon was not alive, nor was it thinking about the emotional state of humans. Luckily for the field of fMRI, instead of publishing a paper telling everyone they should use dead salmon to study human response to the emotions of others, the authors of this study delved deeper into why they were seeing “brain activity” in this very dead fish. In their original data, the researchers failed to correct for multiple comparisons: basically, because you are comparing so many brain regions to so many other brain regions, you’re much more likely to find a spot with significant activity in fMRI purely by chance (for more info on multiple comparisons, click here). The authors applied the appropriate statistical corrections to their data, and voila, no more zombie salmon. And then, because scientists have a funny sense of humor, they wrote up and published these results as a lesson to all on the importance of having a good statistician.

Peer edited by Claire Gyorke.

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How Reliable Is Our Memory?

How memories are formed, stored, and modified has been one of the key topics in neuroscience studies. It’s fascinating to realize that not only can we enhance our memory through constant practice and exercise, but also alter or eliminate existing memory in some trauma cases. Within the past few years, neuroscientists have even found ways to create fake memories or artificially manipulate memories. All of these lead to a huge question: how reliable is our memory?

Picture adapted from:

Picture adapted from:

Most of us have perhaps heard of the analogy that our brain functions like a computer. Information we perceive through our sensory organs is transduced and processed by various neurons and experiences are accumulated to create memories that are stored in different regions of the brain. As our existential evidence, memories constitute our identities and to certain extent determine who we are as human beings. Scientifically, the formation of memories is facilitated by the dynamic generation and deterioration of synapses that connect different neurons to spike different responses. Put it in a simple way, a new memory is created when neurons that didn’t connect before are wired together in the brain, forming new contacts and synapses. The memory will be strong if it is constantly revisited and the relevant synapses get strengthened by reactivating this particular group of neurons. On the other hand, the memory will gradually fade if these neurons stop firing and the synapses dissemble. This is similar to how we maintain friendships at the macro-level. Throughout our entire life, we have a lot of friends. We make new friends everyday and perhaps also lose some from time to time. Like some friends maintain close relationships because they spend time together more often, some memories stay lucid because of repetitive reminders. Understanding which neurons and synapses store which memory is thus like deciphering a codebook. By genetically labeling neurons and tracing their activities, scientists have made huge progress in decoding this mystery, revealing the possibility of Inception in real life.

A research group led by Dr. Steve Ramirez at Boston University have been the experts in memory studies for years. In 2013, they made a breakthrough of implanting fake memories into the brain of mouse. Published in Science, this study has demonstrated that when neurons in a particular region of the hippocampus get activated artificially through optogentics, a pain-related memory can be recalled without actual pain stimulus. By labeling different neurons in the brain, the researchers were able to identify neurons activated by a particular pain stimulus, a foot shock in this case. Mice exposed to foot shock once were separated into two groups: one would receive a real foot shock again to trigger this pain memory, and the other would be stimulated with light for artificial neuron activation. Using optogentics, the researchers activated the particular pain-responsive neurons with light and compared the response from mice that have received real foot shock. Interestingly, light activation of these neurons produced similar response as the normal pain stimulation, which means both groups of mice were “reminded” of the pain even though only one group actually received it.  Dr. Ramirez’s group smartly bypassed the sensory neurons for pain and created this fake memory in their mice. In other words, the researchers tricked the mice to memorize a pain that they weren’t exposed to simply by shining light on them.

Similarly in a followup study published in Nature in 2015, the same group managed to rescue behavioral disorders in mice due to stress by optogenetically activating the rewarding neurons to trigger good memory. Although human brain is a hundred times more complicated than a mouse brain, with the success in manipulating mouse memory, it’s promising that we will have the capacity of interfering with human memories soon. While advancements made in this field will help cure neurodegenerative disorders such as Alzheimer’s, memory altering technologies can also bring up ethical issues and concerns on how reliable our memories are. Without doubts, positive effects in response to rewarding memories can be extremely beneficial to the continuously growing population fighting with depression. On the other hand, if our life is composed of a mixture of fake and authentic memories for various reasons, how would we know what to trust and who we are? As fully conscious human beings, maybe it’s time to ponder on how reliable we want our memories to be.

Picture adapted from:

Picture adapted from:

Peer edited by Nicole Fleming.

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A Stimulating Treatment for Drug Addiction

Drug addiction is notoriously difficult to treat. Limited treatment options are available for those suffering from addiction, including behavioral therapy, rehabilitation programs, and medication. However, current drug addiction medications are only approved to treat opioid, tobacco, or alcohol abuse, leaving out many other drugs of abuse,such as cocaine or methamphetamine.

Yet even when patients successfully complete rehab or stick to a medication plan, there is still a risk of relapse. This can often be due to the emergence of drug cravings. For instance, a former alcoholic may see a sign for a bar they used to frequent. That sign can induce feelings of craving for alcohol, even long after the user quits or abstains from drinking. Strong cravings could lead to a relapse and a resumption of the cycle of addiction.

No pharmaceutical treatments are currently available for cocaine addiction.

However, a recent discovery may change the way we approach drug addiction treatment. Italian researchers, working alongside the National Institute on Drug Abuse (NIDA), were able to reduce drug cravings and usage in cocaine addicts for the first time using a technique called transcranial magnetic stimulation (TMS).

Long-term use of drugs change how brain cells communicate to each other. Think of a drug addict’s brain cells as speaking in gibberish, or unable to speak at all. Important messages aren’t being sent correctly, which contributes to the negative effects of addiction.

In a TMS procedure, researchers place a figure-8-shaped magnetic coil on the patient’s head. When turned on, the coil can send electrical signals into the brain. Importantly, brain cells communicate using electricity, and the “messages” between cells depend on the strength and frequency of these signals. Researchers found that the electrical signals from TMS help change the way brain cells “speak” to each other, getting rid of the gibberish and making cells communicate normally.

TMS uses a magnetic coil to send electric signals into the brain.

In the case of drug addicts, the electrical signals from the magnetic coil are focused at a brain region called the dorsolateral prefrontal cortex (dlPFC). This is a part of the brain that handles decision making and cognitive ability, and is affected by drugs of abuse. For instance, drug addicts demonstrate lower dlPFC activity compared to non-addicted individuals during cognitive tasks.

Knowing how important this brain region is, researchers performed a study where they stimulated the dlPFC of drug addicts using TMS. They had cocaine addicts undergo either the TMS procedure or take medication (as a control group). They found that the cocaine users who experienced TMS had less cocaine cravings than their control counterparts. Further, the TMS group had more cocaine-free urine samples compared to the control group.

The dorsolateral prefrontal cortex is affected by drug addiction.

Other studies support these results, focusing specifically on the prefrontal cortex, which appears to be a “sweet spot” for treating drug addiction. For instance, an earlier study found that daily TMS sessions, focused more broadly at the left prefrontal cortex, reduced cocaine craving. A later study honing in on the left dlPFC found similar reduction of craving in cocaine users.

Interestingly, the Italian TMS study was based on a rodent experiment with a very similar design. In this study, researchers allowed rats to develop a cocaine addiction and then stimulated a brain region analogous to the human dlPFC. Amazingly, the rats decreased cocaine seeking behaviors, much like their human counterparts in the TMS study. When this brain region was inhibited, or “turned off”, the rats increased their cocaine seeking.

Despite their promise, these TMS studies are just the beginning. Researchers are still a long way from developing a cure or reliable treatment for drug addiction. Like any new drug or treatment, it will be many years before TMS could be accepted as standard care for drug addicts. However, TMS has been successfully used to help patients in other ways. For instance, it has been used to help treat depression and is often used to help doctors identify damage from strokes, brain injuries, and neurodegenerative diseases. TMS holds a lot of promise and is on the cusp of being a successful drug addiction treatment. It’s only a matter of time before this stimulating idea becomes reality.

Peer edited by Robert Lee and Julia DiFiore.

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A Call to Guts

I’ve found that it is rather difficult to write an article when you are lying in the fetal position and afraid to move. No, I wasn’t trying to hide from a T-rex. I was having a migraine attack, and what really kept me curled up was its good friend and accomplice, nausea. According to a 2013 study, over half of migraineurs suffer from this additional punch to the stomach during the majority of their attacks. Is this collection of symptoms merely a coincidence, or are the brain and stomach in cahoots to make people miserable?

The brain “communicates” back and forth with the gastrointestinal tract via neuronal, immunological, and hormonal signals, forming what is now called the gut-brain axis. The link between the immune system and microbes within the body has already been well established, and has recently begun percolating into the mainstream media. For example, podcasts are discussing fecal transplants (yes, really) to combat gastrointestinal disease, and the hygienic hypothesis has parents thinking that maybe they should let their kids eat dirt every now and then to avoid developing allergies. Only recently has the contribution of the brain been thrown into the mix to form the microbiome-gut-brain axis.

The mutual communication between the gut (enteric nervous system) and brain (central nervous system) was discovered serendipitously, with a fascinating backstory. As scientists were attempting to form a link between the immune response, stress, and disease, they discovered that mice, when fed a bacterium that does not trigger the murine immune response but that makes humans sick, exhibited more anxious behavior. Meanwhile, animals raised in germ-free environments were less anxious. As interest in the link between behavior and biota grew, more associations between gut bacteria and emotional changes or changes in an animal’s ability to learn were discovered. This line of research has been taking off ever since.gutbrainaxis

According to PubMed, “gut-brain axis” publications per year recently nearly doubled from 75 (2014) to 146 (2016), and over 70 have already been published in 2017. Thanks to this fervor, preliminary evidence is popping up everywhere linking the microbiome, which is predominantly focused in the gut, to neurological disorders from anxiety to Alzheimer’s disease (reviewed recently in Cell). This is also jumping into the mainstream media, with such catchy headlines as, “A Yogurt a Day Could Relieve Depression.” Always remember to take such information with a grain of salt, as this information is preliminary.

So why did it take so long to find the link between this system? More often than not, scientists have been approaching the human body much like the Blind Men and the Elephant parable*: A neuroscientist grabbed a tusk and an immunologist grabbed a tail. While each scientist can make valid conclusions based on their own observations, neither can fully understand the elephant by themselves. Likewise, if scientists had continued studying the gut and the brain independently, without also considering behavior and learning, we would still be blind to their reciprocal relationship. Now, by investigating neurological health problems from a gastrointestinal point of view and vice versa, we can potentially double the chances of finding treatments for each. There is hope yet for my migraine.

*Special thanks to Dr. Keith Kelley, editor-in-chief of Brain, Behavior, and Immunity for using this in his talk at the 2016 Triangle Society for Neuroscience Spring Meeting!

Peer edited by Rachel Haake.

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Say It Ain’t So, Joe!

Mauer & former teammate Justin Morneau.

As a huge Minnesota Twins fan, I was sad to hear that former catcher and current first baseman Joe Mauer is still reporting concussion-related vision problems. These symptoms stem from three years ago, when multiple concussions led to his full-time conversion from behind the plate to first base. While problems with concussions are certainly more publicized in the NFL, they are also a concern in the MLB and other sports. Single concussions rarely have long-lasting, documented effects in athletes if they are allowed sufficient time to recover. However, repeated concussions seem to be a different story altogether. Despite having no recent reported concussions, Mauer is still struggling to return to the elite level of play that he displayed before his multiple head injuries in 2013. Similarly, former teammate Justin Morneau had a childhood history of concussions playing hockey, suffered a major concussion in 2010, and has since never fully recovered his form. Why do multiple concussions seem to play such an exaggerated effect on athletic performance?

        Before we can understand how repeated concussions affect the brain, it’s important to understand how energy consumption is controlled in healthy brain tissue. Despite composing only 2% of the body’s mass, the brain utilizes ~20% of its energy! This energy is typically provided by the consumption of glucose, which is carried to the brain via cerebral blood flow, a process that generates ATP. The largest portion of this energy goes towards maintaining ionic concentration gradients across neuronal membranes, which are crucial for regular brain activity. The key player is the sodium-potassium (Na+/K+) pump, which regulates ionic transport in and out of brain cells and has been shown to consume up to 2/3 of each neuron’s available energy. Ultimately, the brain is a tightly regulated, highly energy efficient organ.

Credit: Lindsay Walton

Concussions impact this energy regulation process in many ways. Blunt trauma to the brain (say, caused by a foul tip of a speedy baseball into a catcher’s mask) can cause physical stretching and distortion of neuronal membranes. This allows ions to diffuse in and out of neurons without the aid of functional ion channels. In addition, excitatory neurotransmitters, such as glutamate, begin to be released at a higher rate, leading to even further accumulation of extracellular K+ ions. In response, the Na+/K+ pump starts to frantically consume energy in an attempt to restore equilibrium. This energy is primarily provided via glucose metabolism. The rapid, exaggerated use of glucose causes a ‘cellular energy crisis’ in which sources of energy to meet the subsequent energy demand are dangerously low. During this state, neuron firing is globally suppressed, a phenomenon known as ‘spreading depression’. In addition, blood flow to the brain is reduced for a time following injury, preventing the restoration of ordinary energy consumption.

The brain can bounce back from this injury if it is given adequate time to recover, though recovery time can vary from days to months. However, a second brain injury that occurs during these ‘cellular energy crises’ can have dire consequences. Due to decreased cerebral blood flow (and thus glucose transport), the brain cannot compensate for further expenses of energy. As a result, ionic gradients are difficult to maintain and more dramatic damage can occur. In particular, excessive influx of calcium ions can cause defects in neuronal function and even trigger cell death! In fact, a progressive disease, referred to as chronic traumatic encephalopathy (CTE), has been discovered in individuals who undergo repeated brain trauma. Unfortunately, overt symptoms of CTE often do not emerge until years after the multiple concussions were suffered, making early detection difficult. Instead, definitive diagnosis of CTE is performed post-mortem. Interestingly, imaging studies in humans with CTE have revealed accumulation of the same toxic proteins seen in patients with Alzheimer disease. These proteins are thought to induce neuronal death, causing shrinking and distortion of brain tissue. Despite the increasing press that traumatic brain injury is receiving in the public and media, research on CTE is still in its infancy. As a result, no common treatment plan has yet been developed for CTE patients.

Image compared a normal healthy brain to a brain suffering from CTE.

Because concussions are notoriously difficult to treat, the MLB has taken steps to diagnose and prevent concussions that may bode well for brain health amongst professional baseball players. Furthermore, several athletes are donating their brains to aid concussion research. Hopefully, increased awareness of the consequences of repetitive brain injury will cause cases like Mauer and Morneau to become distant, but poignant, memories.

Peer edited by Lindsay Walton and Adele Musicant.