Comments: Two molecular switches play prominent roles in all addictions, including behavioral addictions. One I emphasize, called DeltaFosB, the other is CREB, the subject of this article.
20 years ago, scientists got hooked on a single transcription factor that responds to a number of drugs of abuse. Will their work lead to treatments?
By Kerry Grens
Stephen Mague, a graduate student at the University of Pennsylvania, wheels a cart loaded with mouse cages into a room about the size of a large walk-in closet. The room resembles a photography developing lab, lit only by red light and crowded with small work benches and hanging black curtains. In the hallway a television screen displays a video camera’s view from inside the room, looking down upon a row of Plexiglas boxes.
One by one, a latex-gloved hand comes into view on the screen and plunks a brown mouse in each box. The animals scurry around exploring the corners; a few pause in the middle of the chamber and groom themselves energetically. Mague is conditioning the animals to associate a drug with one side of the chamber over another, for example, the one with striped walls instead of solid walls. Adding a drug such as cocaine to the chambers makes the job incredibly easy. Just one exposure to the drug will do it, says graduate student Jess Cleck: “One time I had a mouse sit for 13 minutes and 30 seconds on the side where he’d gotten cocaine previously.”
Over time, in both animals and humans, exposure to the drug leads to dependency, craving, and withdrawal when the drug is taken away. Anxiety and depression become more common, and all other sources of reward lose their appeal as the drug becomes a primary source of motivation. The physiologic changes in people with addiction are striking. “It’s not that they’re more or less sensitive to a drug, they’re different people,” says Eric Nestler at the University of Texas Southwestern Medical Center at Dallas. “You’re profoundly altering the nature of nerve cells.”
The downward spiral from first rush to lasting addiction involves numerous neurobiologic adaptations: long-term depression at synapses, neurodegeneration, and permanent modifications in gene expression. No two drugs are alike, however, as each has its own idiosyncrasies. For example, stimulants such as amphetamine and cocaine increase neuronal branching in the nucleus accumbens, whereas morphine has the opposite effect.
The classic description of a drug’s effect on the brain is centered on the reward system, in particular, neurons that project ventrally from the ventral tegmental area (VTA) to the nucleus accumbens. Those neurons release dopamine, and alcohol and drugs of abuse excite them directly or indirectly. When dopamine is delivered to the nucleus accumbens, it stimulates pleasure.
The trouble starts when, over time, this system starts to erode and develop tolerance. The same amount of drug induces smaller dopamine responses, VTA neurons can shrink, synaptic connections decay, receptor densities change, and the expression of certain genes increases, particularly those related to anxiety and depression. This, according to George Koob at the Salk Institute in La Jolla, Calif., is the “dark side of addiction,” when an addict continues using a drug merely to ameliorate the bad feelings of being addicted.
Julie Blendy, Mague’s principal investigator at the University of Pennsylvania, Nestler, and others are sifting through the myriad molecular changes that accompany drug exposure and addiction to nail down precisely what happens transcriptionally in the brain. For nearly two decades addiction researchers have eyed the activity of one transcription factor: cAMP-response-element-binding protein (CREB). This protein responds to a variety of drugs of abuse, including cocaine, morphine, alcohol, amphetamine, and nicotine. Despite all that time, however, says Nestler: “We’re just at the tip of the iceberg now in identifying CREB target” genes. Still, they hope to find leads for preventing or repairing those changes.
Blendy remembers precisely when her first CREB-deficient mice were born. “They’re 15 years old,” she says with a laugh, “I gave birth to my daughter [in June] and two weeks later they had the first mutant born.” At the time Blendy was a postdoc in Gunther Schutz’s lab at the German Cancer Research Center in Heidelberg, and there was a lot of enthusiasm over knockout technology. Other groups clamored to use the mice.
That all ground to a halt, however, when Schutz’s group began to characterize the mice. “We realized we hadn’t made a complete knockout,” says Blendy. The mutant animals had alternate splice forms of the CREB gene, but it turned out to be a fortuitous accident. CREB knockout animals don’t survive the perinatal period. Having just a small amount of CREB allowed these mutants to survive to adulthood, with functional changes that would allow researchers to probe CREB’s role in a number of neurologic aspects, including learning, memory, mood disorders, and addiction.
By this time, in the mid-90s, the transcription factor was already a target of addiction research. It began in the 1970s with the enzyme adenylyl cyclase in the locus ceruleus, a bluish-looking area of the brain stem that delivers norepinephrine to numerous parts of the brain. Adenylyl cyclase synthesizes cAMP, which in turn activates CREB. Nobel laureate Marshall Nirenberg and his colleagues provided evidence in locus ceruleus neurons of a “cellular tolerance” to morphine. They showed that, while the activity of adenylyl cyclase drops after exposure to morphine, when the drug is left to incubate with the cells for more than a day adenylyl cyclase activity bounces back.1 When the drug was removed, the enzyme’s activity spiked, which the authors interpret as a cellular withdrawal from dependency: “This phenomenon can be likened to the abstinence syndrome in animals.”
“You’re profoundly altering the nature of nerve cells.” -Eric Nestler
It wasn’t until more than a decade later, in the early 1990s, when Nestler, then at Yale University, and his group replicated the results in vivo and moved two steps downstream from adenylyl cyclase to the activation of CREB. They showed that a dose of morphine impairs the phosphorylation of CREB (a marker of CREB activation), but that activity returns to normal after a longer exposure to the drug.2 “Around the same time,” Nestler recalls, “we were asking: The locus ceruleus is just a model system for the opiate system, but do other neurons respond?” He turned to the nucleus accumbens, a group of neurons that receive dopaminergic inputs from the ventral tegmental area, and which are involved in the brain’s reward system. There, Nestler found similar results: Chronic use of morphine increases the activity of CREB.
The CREB-deficient mice in Schtz’s lab presented an opportunity to measure whether CREB was necessary in the addiction process. With Rafael Maldonado, who was then at the University of Paris, Blendy showed in 1996 that her mutant mice lacked the symptoms of morphine withdrawal that normal animals exhibit.3 “By definition, dependence means the presence of a withdrawal syndrome when the drug is removed,” Blendy says. “The question is, were the animals never dependent on the drug in the first place?” Blendy concluded that CREB was important in initiating addiction. But such a simple explanation was too good to be true.
Bill Carlezon, now an associate professor of psychiatry at Harvard’s McLean Hospital in Belmont, Mass., was a postdoc in Nestler’s lab in the mid-1990s, studying cocaine in the nucleus accumbens. At the time, there was no good way to target CREB directly, so Nestler’s group developed a virus vector with a mutant form of CREB that competes with endogenous CREB and blocks its activity. When mutant animals were given cocaine they showed a heightened preference for the drug, whereas when CREB was overexpressed in animals they showed an aversion to it.4
Blendy found similar results after she moved to the University of Pennsylvania in 1997 with the CREB-deficient mouse line. When these animals were given low doses of cocaine (doses small enough to make them indistinguishable from saline to wild-type animals) the animals showed a strong place-preference for the side of the box where they received cocaine.5 “CREB-deficient animals show an enhancement in cocaine reward,” Blendy says.
Though Blendy’s data agreed with Nestler and Carlezon’s results, they were findings that seemed out of line with her results for morphine. While CREB deficiency seemed to get animals more interested in cocaine, it acted oppositely with morphine. Blendy suspects the discrepancy is related to the different brain regions each drug works on. Though all drugs of abuse end up increasing dopamine in the nucleus accumbens, they act through different mechanisms: Cocaine blocks dopamine transporters in the nucleus accumbens, whereas morphine disinhibits dopamine cells in the ventral tegmental area.
The complexity of molecular changes that are involved in drug addiction doesn’t stop there. Alcohol and nicotine act differently on CREB than cocaine and morphine do. Moreover, another transcription factor, DFosB, is upregulated in similar fashion to CREB, though with opposite effects on behavior.6
The DFosB response to drugs may be just as important to addiction as CREB, particularly regarding long-term changes. According to Nestler, CREB essentially delivers negative feedback from drugs, andDFosB promotes the rewarding effects of drugs.
“DFosB can be seen in many ways as the converse of CREB,” Nestler says. Yet with bewildering complexity, both transcription factors can be upregulated in the same cell. “Some cells show CREB activation, some induce DFosB, and some overlap,” Nestler explains. “It’s a very complex process that needs to be worked out.”
Despite these gaps in knowledge, by the 1990s, scientists felt sure that CREB was important in regulating the effects of drugs of abuse. They had also realized, however, that it was apparently not a one-size-fits-all marker for all drugs in all regions of the brain, let alone a treatment. “CREB will never be a therapeutic target. It’s too important, it’s too ubiquitous,” Blendy says.
She decided to turn downstream. Her group is tracking the expression of a number of CREB target genes during the process of drug addiction, from initial exposure to dependency to withdrawal. “The hope is that some of the target [genes] that it’s responsible for will be ideal.”
One downstream target gene that has shown the most dramatic results in differential expression through these phases is corticotropin-releasing factor (CRF, also referred to as corticotrophin-releasing hormone, CRH). CRF is important in mediating stress responses, but it is also involved in Koob’s dark side of addiction. “What we’re finding,” says Koob, “and this is particularly true with alcohol and opiates and nicotine and maybe a little less so for cocaine – when animals take a lot of drug, the CRF system becomes engaged and contributes to excessive drug-taking.”
Markus Heilig, the clinical director at the National Institute on Alcohol Abuse and Alcoholism, says the upregulation of the CRF system appears to be permanent. “In the last year a series of papers nailed down in rodents that if you have a long history of dependence on alcohol, and cycles of intoxication and withdrawal, it will induce remarkably long-lasting changes in the endogenous [CRF] system,” Heilig says.
Koob has shown most recently that animals dependent on alcohol drink less during withdrawal when they’ve been given an antagonist to the CRF1 receptor.7 “One would predict [a CRF1 receptor antagonist] could be a great drug for withdrawal,” Nestler says. Finding an antagonist safe for use in humans, however, has been difficult. Clinical trials on one drug, NBI 30775, were discontinued several years ago after two patients developed elevated liver enzyme levels.8 Koob and Heilig, among others, are working to find a CRF1 receptor antagonist that can be used safely to treat addiction.
In March of this year, Heilig published promising results in alcohol-dependent rats, showing that the drug stopped dependent animals from seeking alcohol when they were exposed to a stressor.9 If a CRF1 receptor antagonist could be developed into a drug, says Koob, the most likely application would be during acute withdrawal.
“CREB will never be a therapeutic target. It’s too important, it’s too ubiquitous.”-Julie Blendy
About 300 miles north of Blendy’s Penn lab, a similarly gowned, gloved, and bootied scientist at Harvard’s McLean Hospital wheels a cart of animals down a hallway. These are Carlezon’s experimental rats, each outfitted with an antenna-like electrode surgically mounted to the top of its head. The electrode feeds pleasurable stimulation, in the form of square-wave pulses of electrical current, into the brain’s reward center. In Carlezon’s office he plays a video of a mouse incessantly spinning a wheel to receive the pleasure-giving current. “It probably feels like the best thing ever. Animals will choose it over drugs, food, sex – anything,” he says.
At a certain point, the mouse stops spinning the wheel when the current dips below a frequency where the reward is diminished, Carlezon says. When animals go through withdrawal from cocaine, the minimum amount of current they are willing to work for – their “reward threshold” – goes up.10 “Because the stimulation isn’t as rewarding during withdrawal, the mice require higher frequencies to get them to work [for the stimulation].” Carlezon’s conclusion: “We think they’re making more dynorphin.”
Dynorphin is an endogenous opioid that acts at k-opioid receptors and is a downstream target gene of CREB. Carlezon showed that when CREB or dynorphin function is elevated in the nucleus accumbens, cocaine is less rewarding, and sometimes even aversive. But when k-receptors are blocked, the effect goes away.4 Carlezon is experimenting now with giving k-opioid receptor antagonists to rats experiencing cocaine withdrawal and measuring whether these drugs can hold reward thresholds steady. If the antagonists work, Carlezon says they could be candidates for treating the symptoms of drug withdrawal.
Koob and Brendan Walker, also at Scripps, have used a k-opioid receptor antagonist, nor-binaltorphimine, in ethanol dependent rats and found they drank less. In animals who were not dependent, they’re drinking behaviors didn’t change. Walker says it supports the idea that the dynorphin-k-opioid system is involved in the “dark side” of addiction11. “In general,” Walker says, “that’s the hypothesis-when animals are dependent and you remove alcohol, the heightened negative affect makes them want to get more alcohol. It looks like if we can block that system then we can, in a sense, block the motivation of the aninal to excessively consume alcohol.”
Charles O’Brien, the vice chair of psychiatry at the University of Pennsylvania and director of the Center for Studies in Addiction, says research on dynorphin and CRF1 receptor antagonists appears promising, but to truly treat addiction the target has to be the brain’s memory center, which may or may not involve CREB.
“Addiction is compulsive drug-seeking behavior,” O’Brien says. “It’s not the changes produced by alcohol, cocaine, or heroin itself. It’s the fact that after the drug is gone, there’s a learning, a memory trace, that produces craving, that produces drug-seeking and relapse.” Nestler says those memories are life long. “As a kid we touched a hot stove and remembered it’s never worth experimenting with a hot stove…. Drug memories may be as powerful as those memories.”
Whether the learning of addiction involves CREB is uncertain. Nevertheless, Nestler says that manipulating CREB’s target genes to ease withdrawal symptoms can aid other addiction treatments, such as cognitive-behavioral therapy, which might influence memories. “In my opinion, the sooner that you get these [potential drugs] into human subjects, the better,” says O’Brien. “The animal models can point the way, but sooner or later you have to get them into human beings.”
References
1. S.K. Sharma et al., “Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance,” Proc Nat Acad Sci, 72:3092-6, 1975. [PUBMED]
2. X. Guitart et al., “Regulation of cyclic AMP response element-binding protein (CREB) phosphorylation by acute and chronic morphine in the rat locus ceruleus,” J Neurochem, 58:1168-71, 1992. [PUBMED]
3. R. Maldonado et al., “Reduction of morphine abstinence in mice with a mutation in the gene encoding CREB,” Science, 273:657-9, 1996.[PUBMED]
4. W.A. Carlezon, Jr. et al., “Regulation of cocaine reward by CREB,” Science, 282:2272-5, 1998. [PUBMED]
5. C.L. Walters, J.A. Blendy, “Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse,” J Neurosci, 21:9438-44, 2001. [PUBMED]
6. E.J. Nestler, “Is there a common molecular pathway for addiction?” Nat Neurosci, 8:1445-9, 2005. [PUBMED]
7. C.K. Funk et al., “Corticotropin-releasing factor 1 antagonists selectively reduce ethanol self-administration in ethanol-dependant rats,” Biol Psych, 61:78-86, 2007. [PUBMED]
8. C. Chen, D.E. Grigoriadis, “NBI 30775 (R121919), an orally active antagonist of the corticotropin-releasing factor (CRF) type-1 receptor for the treatment of anxiety and depression,” Drug Dev Res, 65:216-26, 2005. [PUBMED]
9. D.R. Gehlert et al., “3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine: a novel brain-penetrant, orally available corticotropin-releasing factor receptor 1 antagonist with efficacy in animal models of alcoholism,” J Neurosci, 27:2718-26, 2007. [PUBMED]
10. I. Goussakov et al., “LTP in the lateral amygdala during cocaine withdrawal,” Eur J Neurosci, 23:239-50, 2006. [PUBMED]
11. Walker BM and Koob GF, “Pharmacological evidence for a motivational role of ??-opioid systems in ethanol dependence,” Neuropsychopharmacology, online publication May 2, 2007. [PUBMED]
Read more: Addictive Research – The Scientist – Magazine of the Life Sciences http://www.the-scientist.com/article/display/53236/#ixzz17vJl152n
;