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Keyword: brain

Fluorescent labeling of KV7 channel expression in neurons


Researchers at the Medical University of South Carolina identified potassium channel genes as novel preclinical pharmacogenetic targets that show early promise for reducing heavy alcohol drinking.







Fluorescent labeling of  KV7 channel expression in neurons. Image courtesy of Dr. Patrick Mulholland.

A handful of FDA-approved drugs exist for treating individuals with alcohol use disorder but they have been largely ineffective at reducing the high rates of relapse. As such, there remains a critical need to identify and develop alternative pharmacological treatment options.

Researchers at the Medical University of South Carolina (MUSC), through collaborative efforts with the NIH-funded INIAstress Consortium, have identified novel potassium (K+) channel genes within addiction brain circuitry that are altered by alcohol dependence and correlate with drinking levels in a mouse model of alcohol drinking. Significant reduction of heavy alcohol drinking after administration of a KV7 channel–positive modulator validated Kcnq, one of the identified genes that encodes KV7 type K+ channels, as a potential pharmacogenetic target. These preclinical findings, published in the February 2017 special issue of Alcohol on mouse genetic models of alcohol-stress interactions, suggest that K+ channels could be promising therapeutic targets that may advance personalized medicine approaches for treating heavy drinking in alcoholics.

Alcohol is known to change how neurons fire, and K+ channels play a crucial role in modulating a neuron’s excitability by returning the cell membrane potential back to baseline after the neuron has fired an action potential. Although there is an old literature that links K+ channels and alcohol use disorder, the alcohol field has not actively pursued this line of research.

Recently, the MUSC research team lead by Patrick J. Mulholland, Ph.D., associate professor of Neuroscience and Psychiatry & Behavioral Sciences and senior author on the article, revisited this research area in a novel way. By applying new genomic database technologies, the team became the first to use an experimental genetic bioinformatics approach to determine the relationship between expression levels of brain K+ channel genes with alcohol consumption.

“We looked at all 79 K+ channel genes in an alcohol drinking model using genetically diverse strains of mice and were trying to find the genes that might be risk genes for drinking and the genes that are changed by alcohol dependence,” said Mulholland. “More critically, we wanted to determine how alcohol changed expression of K+ channel genes and how those changes predicted how the mice drank after they were rendered dependent. In other words, we wanted to know what the mechanisms are that facilitate enhanced drinking in alcohol dependence.”

In this preclinical study, INIAstress researchers exposed strains of mice with diverse genetic backgrounds and varied drinking behaviors (BXD recombinant inbred) to alcohol drinking bottles. Half of the mice remained on this protocol and represented non-dependent mice (i.e., mice that consumed alcohol but were not rendered dependent). Alcohol dependence was induced in the other half of mice using a chronic intermittent ethanol exposure model. After 10 weeks, microarray analyses were completed in the prefrontal cortex and nucleus accumbens. Mulholland and colleagues then performed a targeted analysis of K+ channel genes and alcohol drinking in BXD strains using the GeneNetwork software system.

In non-dependent mice, expression levels of several K+ channel genes significantly correlated with the amount of alcohol consumed. Along with identifying novel genes (e.g., Kcnd2), the findings validated genes that were previously implicated in alcohol use disorder.

In particular, low expression levels of Kcnq genes were significantly correlated with high drinking levels. As these correlations were seen prior to dependence, they may represent risk markers for heavy alcohol consumption.

In dependent mice, the expression levels of Kcnq5 were significantly dysregulated across BXD strains, and as the researchers expected, these gene adaptations correlated with the degree of escalated drinking during dependence.

Mulholland and his team were particularly excited by the findings implicating Kcnq genes and KV7 channels in non-dependent and dependent drinking behavior as these findings replicated their previous study in rats (published November 2016 in Addiction Biology). In this prior study, retigabine, an FDA-approved KV7 channel–positive modulator, significantly reduced alcohol consumption in high-drinking non-dependent rats. This study was the first to identify KV7 channels and Kcnq genes as a potential target to reduce heavy drinking.

To further validate Kcnq as a therapeutic target, the researchers induced chronic alcohol drinking in a strain of mice with high drinking behavior (C57BL/6J). After seven weeks, the mice were treated with retigabine. Consistent with the rat studies, retigabine significantly reduced alcohol consumption in high-drinking non-dependent mice. These findings were also consistent with clinical evidence in humans that mutations in KCNQ genes associate with early-onset alcohol dependence.

Together, these studies provide, both genetically and pharmacologically, strong evidence that KV7 channels and KCNQ genes are promising pharmacogenetic targets for treating alcohol use disorder.

“With all of the preclinical and clinical genetic evidence linking KV7 channels and heavy drinking, it would be great to have a precision medicine follow-up study examining the relationship of KCNQ single-nucleotide polymorphisms (i.e., mutations) with retigabine’s response at reducing heavy alcohol drinking and alcohol relapse,” said Mulholland.

Given that retigabine is an FDA-approved drug, its use in a clinical trial on alcohol use disorder is theoretically feasible. However, there is a roadblock to clinical trial development since retigabine’s manufacturer recently announced they will stop making the drug due to commercial reasons.

Fortunately, the path to translating these promising preclinical findings to humans does not end here.  

“There are better drugs that target KV7 channels that are available on the preclinical side,” said Jennifer A. Rinker, Ph.D., postdoctoral fellow in the Department of Neuroscience and first author on the Alcohol paper. “For example, retigabine hits most of the KV7 channel subtypes. There are selective drugs that target just two of the subunits instead of all of them. That’s where we are headed, to figure out which of the subunits are critical for the effects of retigabine to reduce drinking.”

In an article published online ahead of print on May 25, 2016 in Nature (doi: 10.1038/ nature17965), MUSC investigators report that, during sensory stimulation, increases in blood flow are not precisely “tuned” to local neural activity, challenging the long-held view that vascular and local neural responses are tightly coupled.

Many brain-imaging techniques that rely on changes in the flow and oxygenation of blood—including functional magnetic resonance imaging (fMRI)—assume that vascular changes reflect a proportional change in local neural activity.

“Because there isn’t enough blood to send everywhere in the brain at the same time with the optimal levels of oxygen and glucose needed to support neural activity, it is widely accepted that the brain has a built-in Communication between neurons and blood vessels in the brainauto-regulatory mechanism for increasing blood flow to regions with increased activity,” says Prakash Kara, Ph.D., Associate Professor in the Department of Neurosciences at MUSC and senior author on the Nature article.

But how precise is this auto-regulation? With resolution typically at about one millimeter, the fMRI signal represents the blood flow averaged across many blood vessels. Using micron-scale resolution two-photon imaging in an animal model, the MUSC team studied blood flow in single vessels simultaneously with neural activity.

In higher mammals, the neurons in the visual cortex are organized into columns, each of which specializes in responding to a specific stimulus orientation. For example, neurons responding to a “horizontal” stimulus reside in one column and those to “vertical” in another. When the specialized neurons in one of these columns respond to a horizontal stimulus, for example, it would be expected that the blood vessels in the vicinity would likewise respond by dilating and increasing blood flow locally if vascular and neural responses are indeed congruent.

Instead, Kara and colleagues showed that, while blood flow did increase with neural activity, it also increased in response to certain sensory stimuli that did not evoke local neural activity.

To account for this “surplus dilation” and the resultant increase in blood flow, Kara and colleagues have devised a hypothesis. “The blood vessel dilation triggered by local, selective neural activity does not remain entirely local,” says Kara. “From a vessel deep within the brain, the dilation propagates up along the vessel walls into a surface vessel and then down into other vessels that enter neighboring columns.”

Thus, there appears to be no tight correlation between blood flow and local neural activity, and so hemodynamic imaging techniques such as fMRI may only reveal a “blurred” representation of the underlying neural activity.

The news for fMRI could then be mixed. The good news is that the strongest vascular response matched the strongest nearby neural activity, suggesting that fMRI has much to tell us about the general function of an area of the brain. The bad news is that precisely mapping neuronal circuitry could be forever out of fMRI’s reach.

But Kara cautions that much more work is needed, particularly on the generalization of this principle of "surplus dilation” and blood flow occurring in response to other forms of sensory stimuli. "Our team has just taken the first step, albeit an important one, in untangling the spatial precision of neurovascular coupling using very high-resolution imaging," says Kara.

Image Caption: Communication between neurons and blood vessels in the brain. Illustration by Emma Vought of the Medical University of South Carolina.

brain optics graphicIn 2015, the National Science Foundation's (NSF) Experimental Program to Stimulate Competitive Research (EPSCoR) focused on three priority areas: the complexity of the brain, clean energy, and food security. After reviewing nearly sixty applications, the NSF awarded eight awards to the tune of $42 million in early August. Only three of these awards were for brain research, and MUSC was a recipient. A $4 million grant to MUSC will be used to develop and implement new optical technologies to image brain function at a very high resolution. The Principal Investigators of the grant (Peter W. Kalivas, Ph.D., and Prakash Kara, Ph.D., both from the Department of Neurosciences at MUSC) felt that partnering with the University of Alabama at Birmingham (UAB) played an important role in securing this grant. UAB and MUSC will bring complementary technologies to develop a parallel pipeline of state-of-the-art brain scanners in both states. Optical technologies previously used to look at the stars in the sky will now be miniaturized to look inside the brain. Additional partners in this grant include Furman University, the University of South Carolina Beaufort, and Clemson University.

The NSF recognizes the importance of funding new research projects that merge science, technology, engineering, and mathematics (STEM). Grant recipients must also develop a STEM workforce in order to grow their research programs. While the NSF funds basic science research, this infrastructure often leads to new discoveries that improve health care. For example, this grant thematically focuses on developing tools to determine the precise mechanisms (on the microcircuit scale) by which neurons normally communicate with blood vessels in the brain. However, this “neurovascular” communication breaks down in many neurological diseases. Thus, this grant will have far-reaching impacts in the fields of neuroimaging, neurology, and neuroscience education.

Scientists and engineers at the Institute for Applied Neurosciences at MUSC Health have developed a device that detects mild traumatic brain injury, also known as concussion.  Previously, there was no objective way to identify concussion, leaving clinicians and athletic trainers with only subjective measures of altered behavior or cognitive function.  The Blink Reflexometer uses stimuli to trigger a blink and a high-speed camera to collect data on the body’s response to these stimuli.  When a blow to the head occurs on the athletic field or battlefield, for example, trained personnel can use this device to stimulate and record a blink, then compare the person’s data to his or her baseline measurement (recorded previously in the Blink Reflexometer). The technology is now being refined into a hand-held device and the research team is collecting baseline measurements on football players from The Citadel and Charleston Southern University. Clinical trials will continue through Fall 2015. This device is expected to be commercially available by approximately 2017. 

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