Robots Strike Fear in the Hearts of Fish

Anxious Zebrafish Help NYU-Poly Researchers Understand How Alcohol Affects Fear

The latest in a series of experiments testing the ability of robots to influence live animals shows that bio-inspired robots can not only elicit fear in zebrafish, but that this reaction can be modulated by alcohol. These findings may pave the way for new methodologies for understanding anxiety and other emotions, as well as substances that alter them.

Maurizio Porfiri, associate professor of mechanical and aerospace engineering at the Polytechnic Institute of New York University (NYU-Poly) and Simone Macrì, a collaborator at the Istituto Superiore di Sanità in Rome, Italy, published their findings in PLOS ONE, an international, peer-reviewed, open-access, online publication.

This latest study expands Porfiri and Macrì’s efforts to determine how bio-inspired robots can be employed as reliable stimuli to elicit reactions from live zebrafish. Previous studies have established that zebrafish show a strong affinity for robotic members designed to swim and appear as one of their own and that this preference can be abolished by exposing the fish to ethanol.

Porfiri and Macri, along with students Valentina Cianca and Tiziana Bartolini, hypothesized that robots could be used to induce fear as well as affinity and designed a robot mimicking the morphology and locomotion pattern of the Indian leaf fish, a natural predator of the zebrafish. In the lab, they simulated a harmless predatory scenario, placing the zebrafish and the robotic Indian leaf fish in separate compartments of a three-section tank. The other compartment was left empty. The control group uniformly avoided the robotic predator, showing a preference for the empty section.

To determine whether alcohol would affect fear responses, the researchers exposed separate groups of fish to different doses of ethanol in water. Ethanol has been shown to influence anxiety-related responses in humans, rodents and some species of fish. The zebrafish exposed to the highest concentrations of ethanol showed remarkable changes in behavior, failing to avoid the predatory robot. Acute administration of ethanol causes no harm and has no lasting effect on zebrafish.

“These results are further evidence that robots may represent an exciting new approach in evaluating and understanding emotional responses and behavior,” said Porfiri. “Robots are ideal replacements as independent variables in tests involving social stimuli—they are fully controllable, stimuli can be reproduced precisely each time, and robots can never be influenced by the behavior of the test subjects.”

To validate their findings and ensure that the zebrafish behavior being modulated was, in fact, a fear-based response, Porfiri and his collaborators conducted two traditional anxiety tests and evaluated whether the results obtained therein were sensitive to ethanol administration.

They placed test subjects in a two-chamber tank with one well-lit side and one darkened side, to establish which conditions were preferable. In a separate tank, they simulated a heron attack from the water’s surface—herons also prey on zebrafish—and measured how quickly and how many fish took shelter from the attack. As expected, the fish strongly avoided the dark compartment, and most sought shelter very quickly from the heron attack. Ethanol exposure significantly modulated these fear responses as well, abolishing the preference for the light compartment and significantly slowing the fishes’ retreat to shelter during the simulated attack.

“We hoped to see a correlation between the robotic Indian leaf fish test results and the results of the other anxiety tests, and the data support that,” Porfiri explained. “The majority of control group fish avoided the robotic predator, preferred the light compartment and sought shelter quickly after the heron attack. Among ethanol-exposed fish, there were many more who were unaffected by the robotic predator, preferred the dark compartment and were slow to swim to shelter when attacked.”

Porfiri and his colleagues believe zebrafish may be a suitable replacement for higher-order animals in tests to evaluate emotional responses. This novel robotic approach would also reduce the number of live test subjects needed for experiments and may inform other areas of inquiry, from collective behavior to animal protection.

Scientists Identify Key Brain Circuits that Control Compulsive Drinking in Rats

Gallo Center Research Could Have Direct Application For Treating Human Drinking Problems

A research team led by scientists from the Ernest Gallo Clinic and Research Center at UC San Francisco has identified circuitry in the brain that drives compulsive drinking in rats, and likely plays a similar role in humans.

The scientists found they could reduce compulsive drinking in rats by inhibiting key neural pathways that run between the prefrontal cortex, which is involved with higher functions such as critical thinking and risk assessment, and the nucleus accumbens, a critical area for reward and motivation.

The authors noted that there are already several FDA-approved medications that target activity in these pathways, thus potentially opening an accelerated track to new treatments for compulsive drinking.

The study describing their finding was published online on June 30 in Nature Neuroscience.

The study was conducted on rats that regularly drank 20 percent alcohol. The rats drank both unmixed alcohol and alcohol mixed with extremely bitter quinine, said senior investigator F. Woodward Hopf, PhD, an assistant adjunct professor of neurology at UCSF.

Hopf explained that this alcohol-quinine solution, which he described as “like a vodka tonic without the sugar,” is often used as a rodent model of compulsive drinking, or “drinking in the face of negative consequences.” In rats, he said, the negative consequence is the bitter taste, while in humans who drink compulsively, “the negative consequences are profound: people continue to drink despite the potential loss of jobs, marriages, freedom, even their lives.”

The drinking rats showed a notable increase in the NMDA receptor (NMDAR), which lead author Taban Seif, PhD, a Gallo Center researcher, called “a molecule that excites the brain.” When the rats were injected with an NMDAR blocker, their consumption of quinine-laced alcohol dropped significantly, while regular alcohol use was unaffected. “In other words, only the compulsive drinking was affected,” said Seif.

Focus on Two Regions of the Prefrontal Cortex

The team then focused its research on connections from two specific regions of the rats’ prefrontal cortex where they had discovered the presence of unusual types of NMDARs: the medial prefrontal cortex, which mediates conflict during decision-making, and the insula, which is critical for self-awareness and feelings.

“In a non-addict, these brain areas tell you when something is potentially harmful and bad, and to run away as fast as possible,” said Hopf. “But if you’re a compulsive drinker, it seems instead that they give you a comforting pat on the back, in effect telling you it’s OK to have another drink, nothing to worry about.”

Using a technique called optogenetics, the scientists inserted halorhodopsin, a light-sensitive protein, into these areas. They then used fiber-optic cables implanted in the rats’ brains to send pulses of laser light that activated the halorhodopsin, which in turn inhibited the regions’ connections to the nucleus accumbens. The researchers found that rats inhibited in this way drank significantly less quinine-laced alcohol, while their intake of regular alcohol solution remained unaffected.

“The fact that we reduced the rats’ compulsive drinking using two different methods – an NMDAR blocker and direct inhibition of connections – tells us that we have probably identified the right areas,” said Hopf.

Potential Treatments for Humans

The next logical step for the research team, said Hopf, would be to work with clinical researchers on an NMDAR blocker trial in human subjects.

“What is interesting is that we have a new drug which could perhaps treat compulsive aspects of drinking,” said Hopf, “but only if you are in conflict about your drinking – if you care. Any therapy with NMDAR blockers would need a strong behavioral and cognitive component to make sure the patient stayed mentally engaged.”

Seif and Hopf also plan further experimental studies focusing on how the insula drives behavior and connects to other areas of the brain.

Drinking alcohol during pregnancy affects learning and memory function in offspring?

Maternal alcohol consumption during pregnancy has detrimental effects on fetal central nervous system development. Maternal alcohol consumption prior to and during pregnancy significantly affects cognitive functions in offspring, which may be related to changes in cyclin-dependent kinase 5 because it is associated with modulation of synaptic plasticity and impaired learning and memory. Prof. Ruiling Zhang and team from Xinxiang Medical University explored the correlation between cyclin-dependent kinase 5 expression in the hippocampus and neurological impairments following prenatal ethanol exposure, and found that prenatal ethanol exposure could affect cyclin-dependent kinase 5 and its activator p35 in the hippocampus of offspring rats. These findings, which reported in the Neural Regeneration Research (Vol. 8, No. 18, 2013), propose new insights into the mechanisms underlying the role of ethanol exposure in central nervous system injuries, and provide a new strategy for treating the consequences of prenatal ethanol exposure.

Thanks to Rare Alpine Bacteria, Researchers Identify One of Alcohol’s Key Gateways to the Brain

Thanks to a rare bacteria that grows only on rocks in the Swiss Alps, researchers at The University of Texas at Austin and the Pasteur Institute in France have been the first to identify how alcohol might affect key brain proteins.

It’s a major step on the road to eventually developing drugs that could disrupt the interaction between alcohol and the brain.

“Now that we’ve identified this key brain protein and understand its structure, it’s possible to imagine developing a drug that could block the binding site,” said Adron Harris, professor of biology and director of the Waggoner Center for Alcohol and Addiction at The University of Texas at Austin.

Harris and his former postdoctoral fellow Rebecca Howard, now an assistant professor at Skidmore College, are co-authors on the paper that was recently published in Nature Communications. It describes the structure of the brain protein, called a ligand-gated ion channel, that is a key enabler of many of the primary physiological and behavioral effects of alcohol.

Harris said that for some time there has been suggestive evidence that these ion channels are important binding sites for alcohol. Researchers couldn’t prove it, however, because they couldn’t crystallize the brain protein well enough, and therefore couldn’t use X-ray crystallography to determine the structure of the protein with and without alcohol present.

“For many of us in the alcohol field, this has been a Holy Grail, actually finding a binding site for alcohol on the brain proteins and showing it with X-ray crystallography,” said Harris. “But it hasn’t been possible because it is not possible to get a nice crystal.”

The breakthrough came when Marc Delarue and his colleagues at the Pasteur Institute sequenced the genome of cyanobacteria Gloeobacter violaceus. They noted a protein sequence on the bacteria that is remarkably similar to the sequence of a group of ligand-gated ion channels in the human brain. They were able to crystallize this protein. Harris saw the results and immediately got in touch.

“This is something you never would have found with any sort of logical approach,” he said. “You never would have guessed that this obscure bacterium would have something that looks like a brain protein in it. But the institute, because of Pasteur’s fascination with bacteria, has this huge collection of obscure bacteria, and over the last few years they’ve been sequencing the genomes, keeping an eye out for interesting properties.”

Harris and Howard asked their French colleagues to collaborate, got the cyanobacteria, changed one amino acid to make it sensitive to alcohol, and then crystallized both the original bacteria and the mutated one. They compared the two to see whether they could identify where the alcohol bound to the mutant. With further tests they confirmed that it was a meaningful site.

“Everything validated that the cavity in which the alcohol bound is important,” said Harris. “It doesn’t account for all the things that alcohol does, but it appears to be important for a lot of them, including some of the ‘rewarding’ effects and some of the negative, aversive effects.”

Going forward, Harris and his lab plan to use mice to observe how changes to the key protein affect behavior when the mice consume alcohol.

They’re also hoping to identify other important proteins from this family of ligand-gated ion channels. In the long term, he hopes to be involved in developing drugs that act on these proteins in ways that help people diminish or cease their drinking.

“So why do some people drink moderately and some excessively?” he said. “One reason lies in that the balance between the rewarding and the aversive effects, and that balance is different for different people, and it can change within an individual depending on their drinking patterns. Some of those effects are determined by the interactions of alcohol and these channels, so the hope is that we can alter the balance. Maybe we can diminish the reward or increase the aversive effects.”

Taste of beer, without effect from alcohol, triggers dopamine release in the brain

The taste of beer, without any effect from alcohol itself, can trigger dopamine release in the brain, which is associated with drinking and other drugs of abuse, according to Indiana University School of Medicine researchers.

Using positron emission tomography (PET), the researchers tested 49 men with two scans, one in which they tasted beer, and the second in which they tasted Gatorade, looking for evidence of increased levels of dopamine, a brain neurotransmitter long associated with alcohol and other drugs of abuse. The scans showed significantly more dopamine activity following the taste of beer than the sports drink. Moreover, the effect was significantly greater among participants with a family history of alcoholism.

Results of the study were published online Monday by the journal Neuropsychopharmacology.

"We believe this is the first experiment in humans to show that the taste of an alcoholic drink alone, without any intoxicating effect from the alcohol, can elicit this dopamine activity in the brain’s reward centers," said David A. Kareken, Ph.D., professor of neurology at the IU School of Medicine and the deputy director of the Indiana Alcohol Research Center.

The stronger effect in participants with close alcoholic relatives suggests that the release of dopamine in response to such alcohol-related cues may be an inherited risk factor for alcoholism, said Dr. Kareken.

Research for several decades has linked dopamine to the consumption of various drugs of abuse, although researchers have differing interpretations of the neurotransmitter’s role. Sensory cues that are closely associated with drug intoxication (ranging from tastes and smells to the sight of a tavern) have long been known to spark cravings and induce treatment relapse in recovering alcoholics. Many neuroscientists believe that dopamine plays a critical role in such cravings.

The study participants received a very small amount of their preferred beer — 15 milliliters — over a 15-minute time period, enabling them to taste the beer without resulting in any detectable blood alcohol level or intoxicating effect.

Using a PET scanning compound that targets dopamine receptors in the brain, the researchers were able to assess changes in dopamine levels occurring after the participants tasted the liquids.

In addition to the PET scan results, participants reported an increased beer craving after tasting beer, without similar responses after tasting the sports drink — even though many thought the Gatorade actually tasted better, said Brandon G. Oberlin, Ph.D., post-doctoral fellow and first author of the paper.

(Image: iStockphoto)

Research Provides Clues to Alcohol Addiction Vulnerability

A Wake Forest Baptist Medical Center team studying alcohol addiction has new research that might shed light on why some drinkers are more susceptible to addiction than others.

Jeff Weiner, Ph.D., professor of physiology and pharmacology at Wake Forest Baptist, and colleagues used an animal model to look at the early stages of the addiction process and focused on how individual animals responded to alcohol. Their findings may lead not only to a better understanding of addiction, but to the development of better drugs to treat the disease as well, Weiner said.

"We know that some people are much more vulnerable to alcoholism than others, just like some people have a vulnerability to cancer or heart disease," Weiner said. "We don’t have a good understanding of what causes this vulnerability, and that’s a big question. But if we can figure it out, we may be able to better identify people at risk, as well as gain important clues to help develop better drugs to treat the disease."

The findings are published in the March 13 issue of the Journal of Neuroscience. Weiner, who directs the Translational Studies on Early-Life Stress and Vulnerability to Alcohol Addiction project at Wake Forest Baptist, said the study protocol was developed by the first author of the paper, Karina Abrahao, a graduate student visiting from the collaborative lab of Sougza-Formigoni, Ph.D, of the Department of Psychobiology at the Federal University of Sao Paulo, Brazil.

Weiner said the study model focused on how individual animals responded to alcohol. Typically, when a drug like alcohol is given to a mouse every day, the way the animals respond increases - they become more stimulated and run around more. “In high doses, alcohol is a depressant, but in low doses, it can have a mellowing effect that results in greater activity,” he said. “Those low dose effects tend to increase over time and this increase in activity in response to repeated alcohol exposure is called locomotor sensitization.”

Prior studies with other drugs, such as cocaine and amphetamine, have suggested that animals that show the greatest increases in locomotor sensitization are also the animals most likely to seek out or consume these drugs. However, the relationship between locomotor sensitization and vulnerability to high levels of alcohol drinking is not as well established, Weiner said.

Usually when researchers are studying a drug, they give it to one test group while the other group gets a control solution, and then they look for behavioral differences between the two, Weiner said. But in this study, the researchers focused on individual differences in how each animal responded to the alcohol. A control group received a saline injection while another was injected with the same amount of alcohol every day for three weeks. Weiner said they used mice bred to be genetically variable like humans to make the research more relevant.

"We found large variations in the development of locomotor sensitization to alcohol in these mice, with some showing robust sensitization and others showing no more of a change in locomotor activity than control mice given daily saline injections," Weiner said. "Surprisingly, when all of the alcohol-exposed mice were given an opportunity to voluntarily drink alcohol, those that had developed sensitization drank more than those that did not. In fact, the alcohol-treated mice that failed to develop sensitization drank no more alcohol than the saline-treated control group."

The authors also conducted a series of neurobiological studies and discovered that mice that showed robust locomotor sensitization had deficits in a form of brain neuroplasticity - how experiences reorganize neural pathways in the brain - that has been linked with cocaine addiction in other animal models.

"We found that this loss of the ability of brain cells to change the way that they communicate with each other only occurred in the animals that showed the behavioral response to alcohol," he said. "What this suggests for the first time in the alcohol addiction field is that this particular deficit may represent an important brain correlate of vulnerability to alcoholism. It’s a testable hypothesis. That’s why I think it’s an important finding."

Brain-mapping increases understanding of alcohol’s effects on college freshmen

A research team that includes several Penn State scientists has completed a first-of-its-kind longitudinal pilot study aimed at better understanding how the neural processes that underlie responses to alcohol-related cues change during students’ first year of college.

Anecdotal evidence abounds attesting to the many negative social and physical effects of the dramatic increase in alcohol use that often comes with many students’ first year of college. The behavioral changes that accompany those effects indicate underlying changes in the brain. Yet in contrast to alcohol’s numerous other effects, its effect on the brain’s continuing development from adolescence into early adulthood — which includes the transition from high school to college — is not well known.

Penn State psychology graduate student Adriene Beltz, with a team of additional researchers, investigated the changes that occurred to alcohol-related neural processes in the brains of a small group of first-year students.

Using functional magnetic resonance imaging (fMRI) and a data analysis technique known as effective connectivity mapping, the researchers collected and analyzed data from 11 students, who participated in a series of three fMRI sessions beginning just before the start of classes and concluding part-way through the second semester.

"We wanted to know if and how brain responses to alcohol cues — pictures of alcoholic beverages in this case — changed across the first year of college," said Beltz, "and how these potential changes related to alcohol use. Moreover, we wanted our analysis approach to take advantage of the richness of fMRI data."

Analysis of the data collected from the study participants revealed signs in their brains’ emotion processing networks of habituation to alcohol-related stimuli, and noticeable alterations in their cognitive control networks.

Recent studies have indicated that young adults’ cognitive development continues through the ages of the mid-20s, particularly in those regions of the brain responsible for decision-making or judgment-related activity — the sort of cognitive “fine tuning” that potentially makes us, in some senses, as much who we are (and will be) as any other stage of our overall development.

Other recent studies suggest that binge drinking during late adolescence may damage the brain in ways that could last into adulthood.

Beltz’s study indicates that connections among brain regions involved in emotion processing and cognitive control may change with increased exposure to alcohol and alcohol-related cues. Those connections also may influence other parts of the brain, such as those still-developing regions responsible for students’ decision-making and judgment abilities.

"The brain is a complex network," Beltz said. "We know that connections among different brain regions are important for behavior, and we know that many of these connections are still developing into early adulthood. Thus, alcohol could have far-reaching consequences on a maturing brain, directly influencing some brain regions and indirectly influencing others by disrupting neural connectivity."

While in an fMRI scanner at the Penn State Social, Life and Engineering Sciences Imaging Center, students participating in the study completed a task: responding as quickly as possible, by pressing a button on a grip device, to an image of either an alcoholic beverage or a non-alcoholic beverage when both types of images were displayed consecutively on a screen. From the resulting data, effective connectivity maps were created for each individual and for the group.

Examining the final maps, the researchers found that brain regions involved in emotion-processing showed less connectivity when the students responded to alcohol cues than when they responded to non-alcohol cues, and that brain regions involved in cognitive control showed the most connectivity during the first semester of college. The findings suggest that the students needed to heavily recruit brain regions involved in cognitive control in order to overcome the alcohol-associated stimuli when instructed to respond to the non-alcohol cues.

"Connectivity among brain regions implicated in cognitive control spiked from the summer before college to the first semester of college," said Beltz. "This was particularly interesting because the spike coincided with increases in the participants’ alcohol use and increases in their exposure to alcohol cues in the college environment. From the first semester to the second semester, levels of alcohol use and cue exposure remained steady, but connectivity among cognitive control brain regions decreased. From this, we concluded that changes in alcohol use and cue exposure — not absolute levels — were reflected by the underlying neural processes."

Although the immediate implications of the pilot study for first-year students are fairly clear, there are still a number of unanswered questions related to alcohol’s longer-term effects on development, for college students after their first year and for those same individuals later in life.

To begin exploring those potential long-term effects, Beltz has planned a follow-up study to track a larger number of participants over a greater length of time.

Researchers find that alcohol consumption damages brain’s support cells

Alcohol consumption affects the brain in multiple ways, ranging from acute changes in behavior to permanent molecular and functional alterations. The general consensus is that in the brain, alcohol targets mainly neurons. However, recent research suggests that other cells of the brain known as astrocytic glial cells or astrocytes are necessary for the rewarding effects of alcohol and the development of alcohol tolerance. The study, first-authored by Dr. Leonardo Pignataro, was published in the February 6th issue of the scientific journal Brain and Behavior.

"This is a fascinating result that we could have never anticipated. We know that astrocytes are the most abundant cell type in the central nervous system and that they are crucial for neuronal growth and survival, but so far, these cells had been thought to be involved only in brain’s support functions. Our results, however, show that astrocytes have an active role in alcohol tolerance and dependence," explains Dr. Pignataro.

The team of researchers from Columbia and Yale Universities analyzed how alcohol exposure changes gene expression in astrocyte cells and identified gene sets associated with stress, immune response, cell death, and lipid metabolism, which may have profound implications for normal neuronal activity in the brain. “Our findings may explain many of the long-term inflammatory and degenerative effects observed in the brain of alcoholics,” says Dr. Pignataro. “The change in gene expression observed in alcohol-exposed astrocytes supports the idea that some of the alcohol consumed reaches the brain and that ethanol (the active component of alcoholic beverages) is locally metabolized, increasing the production free radicals that react with cell components to affect the normal function of cells. This activates a cellular stress response in the cells in an attempt to defend from this chemical damage. On the other hand, the body recognizes these oxidized molecules as “foreign objects” generating an immune response against them that leads to the death of damage cells. This mechanism can explain the inflammatory degenerative process observed in the brain of chronic alcoholics, allowing for the development of different and novel therapeutically approaches to treat this disease” added Dr. Pignataro.

The consequences of alcohol on astrocytes revealed in this study go far beyond what happens to this particular cell type. Astrocytes play a crucial role in the CNS, supporting normal neuronal activity by maintaining homeostasis. Therefore, alcohol changes in gene expression in astrocytes may have profound implications for neuronal activity in the brain.

These findings will help scientists better understand alcohol-associated disorders, such as the brain neurodegenerative damage associated with chronic alcoholism and alcohol tolerance and dependence. “We hope that this newly discovered role of astrocytes will give scientists new targets other than neurons to develop novel therapies to treat alcoholism,” Leonardo Pignataro concluded.